WO2025022367A2 - Nucléases guidées par arn et fragments actifs, variants associés et méthodes d'utilisation - Google Patents

Nucléases guidées par arn et fragments actifs, variants associés et méthodes d'utilisation Download PDF

Info

Publication number
WO2025022367A2
WO2025022367A2 PCT/IB2024/057272 IB2024057272W WO2025022367A2 WO 2025022367 A2 WO2025022367 A2 WO 2025022367A2 IB 2024057272 W IB2024057272 W IB 2024057272W WO 2025022367 A2 WO2025022367 A2 WO 2025022367A2
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
seq
set forth
sequence set
acid position
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/IB2024/057272
Other languages
English (en)
Other versions
WO2025022367A3 (fr
Inventor
Shaorong Chong
Michael Coyle
Alexandra Briner CRAWLEY
Salem FAHAM
Drew KELSO
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Life Edit Therapeutics Inc
Original Assignee
Life Edit Therapeutics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Life Edit Therapeutics Inc filed Critical Life Edit Therapeutics Inc
Priority to EP24758895.7A priority Critical patent/EP4750890A2/fr
Publication of WO2025022367A2 publication Critical patent/WO2025022367A2/fr
Publication of WO2025022367A3 publication Critical patent/WO2025022367A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to the field of molecular biology and gene editing.
  • RNA-guided nucleases such as the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (Cas) proteins of the CRISPR-Cas bacterial system, allow for the targeting of specific sequences by complexing the nucleases with guide RNA that specifically hybridizes with a particular target sequence.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • RNA-guided nucleases can be used to edit genomes optionally through the introduction of a sequencespecific, double-stranded break that is repaired via error-prone non-homologous end-joining (NHEJ) to introduce a mutation at a specific genomic location.
  • NHEJ error-prone non-homologous end-joining
  • heterologous DNA may be introduced into the genomic site via homology-directed repair.
  • RNA-guided nucleases RGNs
  • RGNs can also be used for base editing when fused with a deaminase or prime editing when fused with reverse transcriptase.
  • Prime editing is a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using an RNA-guided DNA binding protein (e.g., RGN) working in association with a reverse transcriptase (described in, e.g., US 11,447,770BI; WO2021072328; WO2021226558; WO2020156575; W02021042047; US11193123; each incorporated by reference in its entirety herein).
  • the prime editing system uses an RGN that is a nickase and a polymerase, and the system is programmed with a prime editing guide RNA that comprises a primer binding site (PBS) and a DNA synthesis template that serves as the template for the replacement strand comprising the edit.
  • PBS primer binding site
  • the prime editor nicks the non-target strand upstream of the sequence to be edited and upstream of the PAM, creating a 3' flap on the non-target strand.
  • the PBS is complementary to the 3' flap of the non-target strand and hybridrization of the PBS and 3' flap of the non-target strand allows for the polymerization of the replacement strand containing the edit using the DNA synthesis template.
  • compositions and methods for binding a target sequence of interest find use in cleaving or modifying a target sequence of interest, detection of a target sequence of interest, and modifying the expression of a sequence of interest.
  • Compositions comprise: RNA-guided nuclease (RGN) polypeptide variants that have increased editing efficiency as compared to their counterpart wild-type RGN polypeptide; base editors; polymerase editors (PEs); CRISPR RNAs (crRNAs); trans-activating CRISPR RNAs (tracrRNAs); guide RNAs (gRNAs); nucleic acid molecules encoding the same; vectors and host cells comprising the nucleic acid molecules; and pharmaceutical compositions comprising the same.
  • RGN RNA-guided nuclease
  • PEs polymerase editors
  • CRISPR RNAs CRISPR RNAs
  • tracrRNAs trans-activating CRISPR RNAs
  • gRNAs guide RNAs
  • RGN systems and ribonucleoprotein complexes for binding a target sequence of interest, wherein the RGN system comprises an RNA-guided nuclease polypeptide and one or more guide RNAs.
  • PE polymerase editor
  • PgRNAs polymerase editing guide RNAs
  • RGN polypeptide an RGN polypeptide
  • methods disclosed herein are drawn to binding a target sequence of interest in a target polynucleotide, and in some embodiments, cleaving or modifying the target polynucleotide of interest.
  • the target polynucleotide of interest can be modified, for example, as a result of non-homologous end joining, homology-directed repair with an introduced donor sequence, or base editing, or polymerase editing.
  • FIG. 1 shows percent gene editing efficiency for 581 constructs in an initial screen.
  • Each construct encoding a variant LPG10145 nuclease was delivered, along with a plasmid encoding a guide RNA, to HEK293T cells by plasmid lipofection.
  • Tracking of Indels by DEcomposition (TIDE) analysis was performed to determine % gene editing efficiency 2 days post-transfection.
  • the gene editing efficiency of wild-type LPG10145 nuclease was normalized to 1.
  • FIG. 2 shows that 123 variant LPG10145 nucleases having an increase in gene editing activity > 15% were obtained from the initial screen.
  • the 123 variant LPG10145 nucleases were subjected to next generation sequencing (NGS) for confirmation of editing activity, which narrowed the 123 hits to 71 variants.
  • the 71 variants were further tested with more guide RNAs.
  • the gene editing efficiency of wildtype LPG10145 nuclease was normalized to 1.
  • FIG. 3 shows gene editing activity (% Indel) for variant LPG10145 nucleases tested with 3 additional guide RNAs (for a total of 4 guide RNAs tested: guides A, B, C, D).
  • Each construct encoding a variant LPG10145 nuclease was delivered, along with a plasmid encoding a guide RNA, to HEK293T cells by plasmid lipofection.
  • the numbers on the x-axis for each guide from left to right are: 778, 969, 856, 822, 974, 55, 643, 653, 780, 954, 472, 968, 52, 86, 745, 973, 795, 541, 911, 533, 975, 774, and 872, and represent the amino acid position in LPG10145 nuclease of SEQ ID NO: 1 that is mutated.
  • FIG. 4 shows confirmation of gene editing activity by NGS of variant LPG10145 nucleases having an increase in gene editing activity from the initial screen.
  • Twenty R variants increased gene editing > 20%.
  • Each construct encoding a variant LPG10145 nuclease was delivered, along with a plasmid encoding a guide RNA, to HEK293T cells by plasmid lipofection.
  • Three additional guide RNAs were tested (for a total of 4 guide RNAs tested). All guides were normalized and averaged.
  • the gene editing efficiency of wild-type LPG10145 nuclease was normalized to 1.
  • FIG. 5 shows ranking of the top single variant LPG10145 nucleases with increased activity by statistical analysis.
  • Left graph Variant E778R at top to variant K872R, p ⁇ 0.001 ; variant K843R to variant K871R, p ⁇ 0.01.
  • the right graph shows the statistical ranking of variants according to their position.
  • FIG. 6 shows a structural alignment of LPG10145 with a guide RNA. Many mutations, but not all, are at the interface with DNA/RNA. Set2 residues are labeled (unless disordered).
  • the LPG10145 homology model is based on the structure of the RGN from .S', thermophilus (6M0W).
  • FIG. 7 shows that 6 variant LPG10145 nucleases were selected based on statistical analysis and structural modeling for a combinatorial library.
  • the single variants (lx variant), double variant combinations (2x variants), triple variant combinations (3x variants), quadruple variant combinations (4x variants), quintuple variant combinations (5x variant), and the sextuple combination (6x variant) are shown.
  • Sixty- three constructs encoding the 63 variant/combinatorial variant were generated and were tested with multiple different guide RNAs.
  • Each construct encoding a variant/combinatorial variant LPG10145 nuclease was delivered, along with a plasmid encoding a guide RNA, to HEK293T cells by plasmid lipofection.
  • FIG. 8 shows increased gene editing by combinatorial variant LPG10145 nucleases with multiple different guide RNAs.
  • Each construct encoding a combinatorial variant LPG10145 nuclease was delivered, along with a plasmid encoding a guide RNA, to HEK293T cells by plasmid lipofection.
  • a total of seven guide RNAs shown as A, Bl, B2, Cl, C2, DI, and D2 were tested.
  • Percent Indel insertions/deletions
  • FIG. 9 shows combinatorial editing efficiency by combinatorial variant LPG10145 nucleases.
  • the highest gene editing was obtained with 3x, 4x, and 5x variants.
  • E778R and E969R were the common variants in the high editing populations.
  • the gene editing efficiency of wild-type LPG10145 nuclease was normalized to 1.
  • FIG. 10 shows the top combinatorial variants with the highest significant editing. Fifteen combinatorial variant LPG10145 nucleases demonstrate the highest editing.
  • RNA-guided nucleases allow for the targeted manipulation of specific site(s) within a genome and are useful in the context of gene targeting for therapeutic and research applications.
  • RNA-guided nucleases In a variety of organisms, including mammals, RNA-guided nucleases have been used for genome engineering by stimulating non-homologous end joining and homologous recombination, for example.
  • the compositions and methods described herein are useful for creating single- or double-stranded breaks in polynucleotides, modifying polynucleotides, detecting a particular site within a polynucleotide, or modifying the expression of a particular gene.
  • the engineered variant LPG10145 RGNs are directed to the target sequence by a guide RNA (gRNA) as part of a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) RNA-guided nuclease system.
  • gRNA guide RNA
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • the RGNs are considered “RNA-guided” because guide RNAs form a complex with the RNA-guided nucleases to direct the RNA-guided nuclease to bind to a target sequence and in some embodiments, introduce a single-stranded or double -stranded break at the target sequence.
  • the engineered variant LPG10145 RGNs, or active variants or fragments thereof, disclosed herein can increase gene editing efficiency as compared to the corresponding wild-type LPG10145 RGN. In some embodiments, the increase in gene editing efficiency is at least 15%.
  • the break can be repaired such that the DNA sequence of the target sequence is modified during the repair process.
  • RNA-guided nucleases can be used to modify a target sequence at a genomic locus of eukaryotic cells or prokaryotic cells.
  • the variant LPG10145 RGNs can alter gene expression by modifying a target sequence.
  • base editors polymerase editors
  • base editing systems polymerase editor systems
  • methods of using the same for editing a target DNA molecule wherein the editing systems comprise a DNA polymerase and an engineered variant LPG10145 RGN polypeptide as described herein, or an active variant or fragment thereof.
  • RNA-guided nucleases refers to a polypeptide that binds to a particular target nucleotide sequence in a sequence-specific manner and is directed to the target nucleotide sequence by a guide RNA molecule that is complexed with the polypeptide and hybridizes with the target sequence.
  • RGN RNA-guided nuclease
  • an RNA-guided nuclease can be capable of cleaving the target sequence upon binding
  • the term RNA-guided nuclease also encompasses nuclease-dead RNA-guided nucleases that are capable of binding to, but not cleaving, a target sequence.
  • RNA-guided nucleases only capable of cleaving a single strand of a double -stranded nucleic acid molecule are referred to herein as nickases.
  • RNA-guided nucleases disclosed herein include engineered variants of the LPG10145 RNA- guided nuclease described in International Publ. No. WO 2023/139557, filed January 23, 2023, which is incorporated herein in its entirety.
  • the variant LPG10145 RGNs comprise an amino acid sequence set forth as SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions differs from the corresponding amino acid residue in SEQ ID NO: 1: 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975, and active fragments or variants thereof that retain the ability to bind to a target nucleotide sequence in an RNA-guided sequence-specific manner.
  • variant LPG10145 RGN that “comprises an amino acid sequence set forth as SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions differs”, it is intended to mean that the variant LPG10145 RGN comprises an amino acid sequence that is identical to the amino acid sequence set forth in SEQ ID NO: 1, except that amino acid residues at the recited positions differ from the corresponding amino acid residues in SEQ ID NO: 1.
  • amino acid position the number of the amino acid position is counted from the amino-terminus of a given polypeptide.
  • the first amino acid residue at the amino-terminus of a polypeptide is denoted as position 1.
  • the first amino acid residue is a Methionine.
  • the variant LPG10145 RGNs comprise an amino acid sequence that is identical to the amino acid sequence set forth in SEQ ID NO: 1, except that the amino acid residue at one or more of the following amino acid positions is a positively charged amino acid residue: 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975, and active fragments or variants thereof that retain the ability to bind to a target nucleotide sequence in an RNA-guided sequence-specific manner.
  • the variant LPG10145 RGNs comprise an amino acid sequence that is identical to the amino acid sequence set forth in SEQ ID NO: 1, except that the amino acid residue at one or more of the following amino acid positions is an R: 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975, and active fragments or variants thereof that retain the ability to bind to a target nucleotide sequence in an RNA-guided sequence -specific manner.
  • variant LPG10145 RGN comprising an amino acid sequence set forth as SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions is a positively charged amino acid residue
  • a variant LPG10145 RGN comprising an amino acid sequence having at least x% (e.g., 85%) sequence identity to SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions is a positively charged amino acid residue
  • the positively charged amino acid residue in the variant LPG10145 RGN is a different positively charged amino acid residue if the amino acid residue at a particular position of SEQ ID NO: 1 already comprises a positively charged amino acid residue.
  • the variant LPG10145 RGNs comprise an amino acid sequence that is identical to the amino acid sequence set forth in SEQ ID NO: 1, except that the amino acid residue at amino acid position 778 and/or 969 differs from the corresponding amino acid residue in SEQ ID NO: 1, and active fragments or variants thereof that retain the ability to bind to a target nucleotide sequence in an RNA-guided sequence -specific manner.
  • the variant LPG10145 RGNs comprise an amino acid sequence that is identical to the amino acid sequence set forth in SEQ ID NO: 1, except that the amino acid residue at amino acid position 778 and/or 969 is a positively charged amino acid residue, and active fragments or variants thereof that retain the ability to bind to a target nucleotide sequence in an RNA-guided sequence -specific manner.
  • the variant LPG10145 RGNs comprise an amino acid sequence that is identical to the amino acid sequence set forth in SEQ ID NO: 1, except that the amino acid residue at amino acid position 778 and/or 969 is an R, and active fragments or variants thereof that retain the ability to bind to a target nucleotide sequence in an RNA-guided sequence-specific manner.
  • the variant LPG10145 RGNs comprise an amino acid sequence that is identical to the amino acid sequence set forth in SEQ ID NO: 1, except that amino acid residues at positions 778 and 856 differ from the corresponding amino acid residues in SEQ ID NO: 1, and active fragments or variants thereof that retain the ability to bind to a target nucleotide sequence in an RNA-guided sequencespecific manner.
  • the variant LPG10145 RGNs comprise an amino acid sequence that is identical to the amino acid sequence set forth in SEQ ID NO: 1, except that amino acid residues at positions 778 and 856 are positively charged amino acid residues, and active fragments or variants thereof that retain the ability to bind to a target nucleotide sequence in an RNA-guided sequence -specific manner.
  • the variant LPG10145 RGNs comprise an amino acid sequence that is identical to the amino acid sequence set forth in SEQ ID NO: 1, except that amino acid residues at positions 55, 647, 778, and 969 differ from the corresponding amino acid residues in SEQ ID NO: 1, and active fragments or variants thereof that retain the ability to bind to a target nucleotide sequence in an RNA-guided sequence -specific manner.
  • the variant LPG10145 RGNs comprise an amino acid sequence that is identical to the amino acid sequence set forth in SEQ ID NO: 1, except that amino acid residues at positions 55, 647, 778, and 969 are positively charged amino acid residues, and active fragments or variants thereof that retain the ability to bind to a target nucleotide sequence in an RNA-guided sequencespecific manner.
  • the variant LPG10145 RGNs comprise: (a) the amino acid sequence set forth as SEQ ID NO: 1, wherein E at amino acid position 52 is an R; (b) the amino acid sequence set forth as SEQ ID NO: 1, wherein S at amino acid position 55 is an R; (c) the amino acid sequence set forth as SEQ ID NO: 1, wherein G at amino acid position 86 is an R; (d) the amino acid sequence set forth as SEQ ID NO: 1, wherein Y at amino acid position 472 is an R; (e) the amino acid sequence set forth as SEQ ID NO: 1, wherein K at amino acid position 533 is an R; (f) the amino acid sequence set forth as SEQ ID NO: 1, wherein K at amino acid position 541 is an R; (g) the amino acid sequence set forth as SEQ ID NO: 1, wherein Y at amino acid position 643 is substituted by an R; (h) the amino acid sequence set forth as SEQ ID NO: 1, wherein S at amino acid position 647 is an R; (i) the amino acid sequence set
  • the variant LPG10145 RGNs comprise: (a) the amino acid sequence set forth as SEQ ID NO: 2; (b) the amino acid sequence set forth as SEQ ID NO: 3; (c) the amino acid sequence set forth as SEQ ID NO: 4; (d) the amino acid sequence set forth as SEQ ID NO: 5; (e) the amino acid sequence set forth as SEQ ID NO: 6; (f) the amino acid sequence set forth as SEQ ID NO: 7; (g) the amino acid sequence set forth as SEQ ID NO: 8; (h) the amino acid sequence set forth as SEQ ID NO: 9; (i) the amino acid sequence set forth as SEQ ID NO: 10; (j) the amino acid sequence set forth as SEQ ID NO: 11; (k) the amino acid sequence set forth as SEQ ID NO: 12; (1) the amino acid sequence set forth as SEQ ID NO: 13; (m) the amino acid sequence set forth as SEQ ID NO: 14; (n) the amino acid sequence set forth as SEQ ID NO: 15; and (o
  • the active fragment or variant of a variant LPG10145 RGN is capable of cleaving a single- or double -stranded target sequence.
  • a variant LPGI0145 RGN that “comprises an amino acid sequence having at least x% (e.g., 85%) sequence identity to SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions differs”
  • the variant LPGI0145 RGN comprises an amino acid sequence that has at least x% (e.g., 85%) sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, and the amino acid residues at the recited positions differ from the corresponding amino acid residues in SEQ ID NO: 1.
  • an active variant of a variant LPG10145 RGN comprises an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions differs from the corresponding amino acid residue in SEQ ID NO: 1: 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975.
  • an active variant of a variant LPG10145 RGN comprises an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions is a positively charged amino acid residue: 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975.
  • an active variant of a variant LPG10145 RGN comprises an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions is an R: 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975.
  • the active fragment or variant of a variant LPG10145 RGN is capable of cleaving a single- or double -stranded target sequence.
  • the binding and/or cleaving of a target sequence by an active variant LPG10145 RGN can be dependent upon the active variant LPG10145 RGN recognizing a protospacer adjacent motif (PAM) adjacent and 3’ to the target sequence, and the PAM comprises a consensus sequence of NNGG.
  • PAM protospacer adjacent motif
  • an active variant of a variant LPG10145 RGN comprises an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1, wherein the amino acid residue at amino acid position 778 and/or 969 differs from the corresponding amino acid residue in SEQ ID NO: 1.
  • an active variant of a variant LPG10145 RGN comprises an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1, wherein the amino acid residue at amino acid position 778 and/or 969 is a positively charged amino acid residue.
  • an active variant of a variant LPG10145 RGN comprises an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1, wherein the amino acid residue at amino acid position 778 and/or 969 is an R.
  • an active variant of a variant LPG10145 RGN comprises an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1, wherein amino acid residues at positions 778 and 856 differ from the corresponding amino acid residues in SEQ ID NO: 1.
  • an active variant of a variant LPG10145 RGN comprises an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1, wherein the amino acid residues at positions
  • an active variant of a variant LPG10145 RGN comprises an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1, wherein amino acid residues at positions 55, 647, 778, and 969 differ from the corresponding amino acid residues in SEQ ID NO: 1.
  • an active variant of a variant LPG10145 RGN comprises an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1, wherein amino acid residues at positions 55, 647, 778, and 969 are positively charged amino acid residues.
  • an active variant of a variant LPG10145 RGN comprises an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to: (a) the amino acid sequence set forth as SEQ ID NO: 1, wherein E at amino acid position 52 is an R; (b) the amino acid sequence set forth as SEQ ID NO: 1, wherein S at amino acid position 55 is an R; (c) the amino acid sequence set forth as SEQ ID NO: 1, wherein G at amino acid position 86 is an R; (d) the amino acid sequence set forth as SEQ ID NO: 1, wherein Y at amino acid position 472 is an R; (e) the amino acid sequence set forth as SEQ ID NO: 1, wherein K at amino acid position 533 is an R; (f) the amino acid sequence set forth as SEQ ID NO: 1, wherein K at amino acid position 541
  • an active variant of a variant LPG10145 RGN comprises an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,
  • an active fragment of a variant LPG10145 RGN comprises at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050 or more contiguous amino acid residues of an amino acid sequence set forth as SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions differs from the corresponding amino acid residue in SEQ ID NO: 1: 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975.
  • an active fragment of a variant LPG10145 RGN comprises at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050 or more contiguous amino acid residues of an amino acid sequence set forth as SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions is a positively charged amino acid residue: 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975.
  • an active fragment of a variant LPG10145 RGN comprises at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050 or more contiguous amino acid residues of an amino acid sequence set forth as SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions is an R: 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975.
  • an active fragment of a variant LPG10145 RGN comprises at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050 or more contiguous amino acid residues of an amino acid sequence selected from: (a) the amino acid sequence set forth as SEQ ID NO: 1, wherein E at amino acid position 52 is an R; (b) the amino acid sequence set forth as SEQ ID NO: 1, wherein S at amino acid position 55 is an R; (c) the amino acid sequence set forth as SEQ ID NO: 1, wherein G at amino acid position 86 is an R; (d) the amino acid sequence set forth as SEQ ID NO: 1, wherein Y at amino acid position 472 is an R; (e) the amino acid sequence set forth as SEQ ID NO: 1, wherein K at amino acid position 533 is an R; (f) the amino acid sequence set forth as SEQ ID NO: 1, wherein K at amino acid position 533 is an R
  • an active fragment of a variant LPG10145 RGN comprises at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050 or more contiguous amino acid residues of an amino acid sequence set forth as SEQ ID NO: 1, wherein the amino acid residue at amino acid position 778 and/or 969 differs from the corresponding amino acid residue in SEQ ID NO: 1.
  • an active fragment of a variant LPG10145 RGN comprises at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050 or more contiguous amino acid residues of an amino acid sequence set forth as SEQ ID NO: 1, wherein the amino acid residue at amino acid position 778 and/or 969 is a positively charged amino acid residue.
  • an active fragment of a variant LPG10145 RGN comprises at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050 or more contiguous amino acid residues of an amino acid sequence set forth as SEQ ID NO: 1, wherein the amino acid residue at amino acid position 778 and/or 969 is an R.
  • an active fragment of a variant LPG10145 RGN comprises at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050 or more contiguous amino acid residues of an amino acid sequence set forth as SEQ ID NO: 1, wherein amino acid residues at positions 778 and 856 differ from the corresponding amino acid residues in SEQ ID NO: 1.
  • an active fragment of a variant LPG10145 RGN comprises at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050 or more contiguous amino acid residues of an amino acid sequence set forth as SEQ ID NO: 1, wherein the amino acid residues at positions 778 and 856 are positively charged amino acid residues.
  • an active fragment of a variant LPG10145 RGN comprises at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050 or more contiguous amino acid residues of an amino acid sequence set forth as SEQ ID NO: 1, wherein amino acid residues at positions 55, 647, 778, and 969 differ from the corresponding amino acid residues in SEQ ID NO: 1.
  • an active fragment of a variant LPG10145 RGN comprises at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050 or more contiguous amino acid residues of an amino acid sequence set forth as SEQ ID NO: 1, wherein the amino acid residues at positions 55, 647, 778, and 969 are positively charged amino acid residues.
  • an active fragment of a variant LPG10145 RGN comprises at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050 or more contiguous amino acid residues of an amino acid sequence selected from: (a) the amino acid sequence set forth as SEQ ID NO: 2; (b) the amino acid sequence set forth as SEQ ID NO: 3; (c) the amino acid sequence set forth as SEQ ID NO: 4; (d) the amino acid sequence set forth as SEQ ID NO: 5; (e) the amino acid sequence set forth as SEQ ID NO: 6; (f) the amino acid sequence set forth as SEQ ID NO: 7; (g) the amino acid sequence set forth as SEQ ID NO: 8; (h) the amino acid sequence set forth as SEQ ID NO: 9; (i) the amino acid sequence set forth as SEQ ID NO: 10; (j) the amino acid sequence set forth as SEQ ID NO:
  • An RGN polypeptide of the disclosure can comprise one or more of the following domains: a linker domain (linker domain 1, linker domain 2), a wedge (WED) domain, a RuvC nuclease domain, an HNH nuclease domain, a Rec domain, or a PAM-interacting (PI) domain.
  • the RuvC domain can include a RuvCI, a RuvCII, a RuvCIII domain, or a combination thereof.
  • a Rec or recognition lobe mediates nucleic acid binding through multiple Rec domains (e.g., Recl-3) by sensing nucleic acids, regulating the HNH conformational transition, and locking the catalytic HNH domain at the cleavage site.
  • a wedge domain is responsible for the recognition of guide RNA scaffolds.
  • a PAM-interacting domain is the domain of an RGN polypeptide that binds to a PAM site.
  • Non-limiting examples of domains within LPG10145 RGN (SEQ ID NO: 1) or a variant LPG10145 RGN (SEQ ID NOs: 2-16, 182-196, and 271-285) include: RuvC-I from amino acid residues 1 to 42; BH from amino acid residues 43 to 79; RECI from amino acid residues 80 to 236; REC2 from amino acid residues 237 to 476; RuvC-II from amino acid residues 477 to 524; LI from amino acid residues 525 to 560; HNH from amino acid residues 561 to 676; L2 from amino acid residues 677 to 690; RuvC-III from amino acid residues 691 to 828; WED from amino acid residues 829 to 976; and PI from amino acid residues 977 to 1130.
  • An active variant or fragment of a variant LPG10145 RGN disclosed herein can comprise a RuvCI domain comprising amino acid residues 1 to 42 or that differs from amino acid residues 1 to 42 by 1, 2, or 3 amino acid residues, wherein the amino acid positions are in reference to SEQ ID NO: 1 or to a variant LPG10145 RGN, or an active variant or fragment thereof, disclosed herein (e.g., any one of SEQ ID NOs: 2- 16, 182-196, and 271-285).
  • an active variant or fragment of a variant LPG10145 RGN disclosed herein can comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NOs: 2-16, 182-196, and 271-285 and can comprise a RuvCI domain comprising amino acid residues 1 to 42 or that differs from amino acid residues 1 to 42 of the RGN sequence (i.e. SEQ ID NOs: 2-16, 182-196, and 271-285) by 1, 2, or 3 amino acid residues.
  • An active variant or fragment of a variant LPG10145 RGN disclosed herein can comprise a BH domain comprising amino acid residues 43 to 79 or that differs from amino acid residues 43 to 79 by 1, 2, or 3 amino acid residues, wherein the amino acid positions are in reference to SEQ ID NO: 1 or to a variant LPG10145 RGN, or an active variant or fragment thereof, disclosed herein (e.g., any one of SEQ ID NOs: 2- 16, 182-196, and 271-285).
  • an active variant or fragment of a variant LPG10145 RGN disclosed herein can comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NOs: 2-16, 182-196, and 271-285 and can comprise a BH domain comprising amino acid residues 43 to 79 or that differs from amino acid residues 43 to 79 of the RGN sequence (i.e. SEQ ID NOs: 2-16, 182-196, and 271-285) by 1, 2, or 3 amino acid residues.
  • An active variant or fragment of a variant LPG10145 RGN disclosed herein can comprise a RECI domain comprising amino acid residues 80 to 236 or that differs from amino acid residues 80 to 236 by 1, 2, or 3 amino acid residues, wherein the amino acid positions are in reference to SEQ ID NO: 1 or to a variant LPG10145 RGN, or an active variant or fragment thereof, disclosed herein (e.g., any one of SEQ ID NOs: 2- 16, 182-196, and 271-285).
  • an active variant or fragment of a variant LPG10145 RGN disclosed herein can comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NOs: 2-16, 182-196, and 271-285 and can comprise a RECI domain comprising amino acid residues 80 to 236 or that differs from amino acid residues 80 to 236 of the RGN sequence (i.e. SEQ ID NOs: 2-16, 182-196, and 271-285) by 1, 2, or 3 amino acid residues.
  • An active variant or fragment of a variant LPG10145 RGN disclosed herein can comprise a REC2 domain comprising amino acid residues 237 to 476 or that differs from amino acid residues 237 to 476 by 1, 2, or 3 amino acid residues, wherein the amino acid positions are in reference to SEQ ID NO: 1 or to a variant LPG10145 RGN, or an active variant or fragment thereof, disclosed herein (e.g., any one of SEQ ID NOs: 2-16, 182-196, and 271-285).
  • an active variant or fragment of a variant LPG10145 RGN disclosed herein can comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NOs: 2-16, 182-196, and 271-285 and can comprise a REC2 domain comprising amino acid residues 237 to 476 or that differs from amino acid residues 237 to 476 of the RGN sequence (i.e. SEQ ID NOs: 2-16, 182-196, and 271-285) by 1, 2, or 3 amino acid residues.
  • An active variant or fragment of a variant LPG10145 RGN disclosed herein can comprise a RuvCII domain comprising amino acid residues 477 to 524 or that differs from amino acid residues 477 to 524 by 1, 2, or 3 amino acid residues, wherein the amino acid positions are in reference to SEQ ID NO: 1 or to a variant LPG10145 RGN, or an active variant or fragment thereof, disclosed herein (e.g., any one of SEQ ID NOs: 2-16, 182-196, and 271-285).
  • an active variant or fragment of a variant LPG10145 RGN disclosed herein can comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NOs: 2-16, 182-196, and 271-285 and can comprise a RuvCII domain comprising amino acid residues 477 to 524 or that differs from amino acid residues 477 to 524 of the RGN sequence (i.e. SEQ ID NOs: 2-16, 182-196, and 271-285) by 1, 2, or 3 amino acid residues.
  • An active variant or fragment of a variant LPG10145 RGN disclosed herein can comprise an LI domain comprising amino acid residues 525 to 560 or that differs from amino acid residues 525 to 560 by 1, 2, or 3 amino acid residues, wherein the amino acid positions are in reference to SEQ ID NO: 1 or to a variant LPG10145 RGN, or an active variant or fragment thereof, disclosed herein (e.g., any one of SEQ ID NOs: 2-16, 182-196, and 271-285).
  • an active variant or fragment of a variant LPG10145 RGN disclosed herein can comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NOs: 2-16, 182-196, and 271-285 and can comprise an LI domain comprising amino acid residues 525 to 560 or that differs from amino acid residues 525 to 560 of the RGN sequence (i.e. SEQ ID NOs: 2-16, 182-196, and 271-285) by 1, 2, or 3 amino acid residues.
  • An active variant or fragment of a variant LPG10145 RGN disclosed herein can comprise an HNH domain comprising amino acid residues 561 to 676 or that differs from amino acid residues 561 to 676 by 1, 2, or 3 amino acid residues, wherein the amino acid positions are in reference to SEQ ID NO: 1 or to a variant LPG10145 RGN, or an active variant or fragment thereof, disclosed herein (e.g., any one of SEQ ID NOs: 2-16, 182-196, and 271-285).
  • an active variant or fragment of a variant LPG10145 RGN disclosed herein can comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NOs: 2-16, 182-196, and 271-285 and can comprise an HNH domain comprising amino acid residues 561 to 676 or that differs from amino acid residues 561 to 676 of the RGN sequence (i.e. SEQ ID NOs: 2-16, 182-196, and 271-285) by 1, 2, or 3 amino acid residues.
  • An active variant or fragment of a variant LPG10145 RGN disclosed herein can comprise an L2 domain comprising amino acid residues 677 to 690 or that differs from amino acid residues 677 to 690 by 1, 2, or 3 amino acid residues, wherein the amino acid positions are in reference to SEQ ID NO: 1 or to a variant LPG10145 RGN, or an active variant or fragment thereof, disclosed herein (e.g., any one of SEQ ID NOs: 2-16, 182-196, and 271-285).
  • an active variant or fragment of a variant LPG10145 RGN disclosed herein can comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NOs: 2-16, 182-196, and 271-285 and can comprise an L2 domain comprising amino acid residues 677 to 690 or that differs from amino acid residues 677 to 690 of the RGN sequence (i.e. SEQ ID NOs: 2-16, 182-196, and 271-285) by 1, 2, or 3 amino acid residues.
  • An active variant or fragment of a variant LPG10145 RGN disclosed herein can comprise a RuvCIII domain comprising amino acid residues 691 to 828 or that differs from amino acid residues 691 to 828 by 1, 2, or 3 amino acid residues, wherein the amino acid positions are in reference to SEQ ID NO: 1 or to a variant LPG10145 RGN, or an active variant or fragment thereof, disclosed herein (e.g., any one of SEQ ID NOs: 2-16, 182-196, and 271-285).
  • an active variant or fragment of a variant LPG10145 RGN disclosed herein can comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NOs: 2-16, 182-196, and 271-285 and can comprise a RuvCIII domain comprising amino acid residues 691 to 828 or that differs from amino acid residues 691 to 828 of the RGN sequence (i.e. SEQ ID NOs: 2-16, 182-196, and 271-285) by 1, 2, or 3 amino acid residues.
  • An active variant or fragment of a variant LPG10145 RGN disclosed herein can comprise a WED domain comprising amino acid residues 829 to 976 or that differs from amino acid residues 829 to 976 by 1, 2, or 3 amino acid residues, wherein the amino acid positions are in reference to SEQ ID NO: 1 or to a variant LPG10145 RGN, or an active variant or fragment thereof, disclosed herein (e.g., any one of SEQ ID NOs: 2-16, 182-196, and 271-285).
  • an active variant or fragment of a variant LPG10145 RGN disclosed herein can comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NOs: 2-16, 182-196, and 271-285 and can comprise a WED domain comprising amino acid residues 829 to 976 or that differs from amino acid residues 829 to 976 of the RGN sequence (i.e. SEQ ID NOs: 2-16, 182-196, and 271-285) by 1, 2, or 3 amino acid residues.
  • An active variant or fragment of a variant LPG10145 RGN disclosed herein can comprise a PI domain comprising amino acid residues 977 to 1130 or that differs from amino acid residues 977 to 1130 by
  • amino acid residues 1, 2, or 3 amino acid residues, wherein the amino acid positions are in reference to SEQ ID NO: 1 or to a variant LPG10145 RGN, or an active variant or fragment thereof, disclosed herein (e.g., any one of SEQ ID NOs: 2-16, 182-196, and 271-285).
  • an active variant or fragment of a variant LPG10145 RGN disclosed herein can comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NOs: 2-16, 182-196, and 271-285 and can comprise a PI domain comprising amino acid residues 977 to 1130 or that differs from amino acid residues 977 to 1130 of the RGN sequence (i.e. SEQ ID NOs: 2-16, 182-196, and 271-285) by 1,
  • the present disclosure provides an active variant or fragment of a variant LPG10145 RGN that has increased nuclease activity as compared to the RGN of SEQ ID NO: 1.
  • an active variant or fragment of a variant LPG10145 RGN disclosed herein comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NOs: 2-16, 182-196, and 271-285 has increased nuclease activity as compared to the RGN of SEQ ID NO: 1.
  • an active variant or fragment of a variant LPG10145 RGN disclosed herein comprising the amino acid sequence of any one of SEQ ID NOs: 2-16, 182-196, and 271-285 has increased nuclease activity as compared to the RGN of SEQ ID NO: 1.
  • the present disclosure provides an active variant or fragment of a variant LPG10145 RGN that has nuclease activity that is from about 80% to about 500%, from about 80% to about 200%, or from about 90% to about 150%, or from about 95% to about 120%, or is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%
  • a reference LPG10145 RGN has the amino acid sequence set forth as SEQ ID NO: 1.
  • a reference LPG10145 RGN is a variant of SEQ ID NO: 1 that lacks the corresponding mutations of the active variant or fragment thereof that has nuclease activity, e.g., as described above.
  • a reference LPG10145 RGN is a non-identical variant LPG10145 RGN.
  • the active variant of a variant LPG10145 RGN can comprise an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions differs from the corresponding amino acid residue in SEQ ID NO: 1: 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975.
  • an active variant of a variant LPG10145 RGN can comprise an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions is a positively charged amino acid residue: 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975.
  • an active variant of a variant LPG10145 RGN can comprise an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions is an R: 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975.
  • the present disclosure provides an active variant or fragment of a variant LPG10145 RGN that has increased nuclease activity as compared to the RGN of SEQ ID NO: 1.
  • an active variant or fragment of a variant LPG10145 RGN disclosed herein comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NOs: 2-16, 182-196, and 271-285 has increased nuclease activity as compared to the RGN of SEQ ID NO: 1.
  • an active variant or fragment of a variant LPG10145 RGN disclosed herein comprising the amino acid sequence of any one of SEQ ID NOs: 2-16, 182-196, and 271-285 has increased nuclease activity as compared to the RGN of SEQ ID NO: 1.
  • an active variant or fragment of a variant LPG10145 RGN disclosed herein can have nuclease activity that is from about 80% to about 500%, from about 80% to about 200%, or from about 90% to about 150%, or from about 95% to about 120%, or is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least
  • the active variant of a variant LPG10145 RGN can comprise an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1, wherein the amino acid residue at amino acid position 778 and/or 969 differs from the corresponding amino acid residue in SEQ ID NO: 1.
  • an active variant of a variant LPG10145 RGN can comprise an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1, wherein the amino acid residue at amino acid position 778 and/or 969 is a positively charged amino acid residue.
  • an active variant of a variant LPG10145 RGN can comprise an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1, wherein the amino acid residue at amino acid position 778 and/or 969 is an R.
  • an active variant of a variant LPG10145 RGN comprises an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1, wherein amino acid residues at positions 778 and 856 differ from the corresponding amino acid residues in SEQ ID NO: 1.
  • an active variant of a variant LPG10145 RGN comprises an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1, wherein the amino acid residues at positions 778 and 856 are positively charged amino acid residues.
  • an active variant of a variant LPG10145 RGN comprises an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1, wherein amino acid residues at positions 55, 647, 778, and 969 differ from the corresponding amino acid residues in SEQ ID NO: 1.
  • an active variant of a variant LPG10145 RGN comprises an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1, wherein amino acid residues at positions 55, 647, 778, and 969 are positively charged amino acid residues.
  • the present disclosure provides an active variant or fragment of a variant LPG10145 RGN that has increased nuclease activity as compared to the RGN of SEQ ID NO: 1.
  • an active variant or fragment of a variant LPG10145 RGN disclosed herein comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NOs: 2-16, 182-196, and 271-285 has increased nuclease activity as compared to the RGN of SEQ ID NO: 1.
  • an active variant or fragment of a variant LPG10145 RGN disclosed herein comprising the amino acid sequence of any one of SEQ ID NOs: 2-16, 182-196, and 271-285 has increased nuclease activity as compared to the RGN of SEQ ID NO: 1.
  • an active variant or fragment of a variant LPG10145 RGN disclosed herein can have nuclease activity that is from about 80% to about 500%, from about 80% to about 200%, or from about 90% to about 150%, or from about 95% to about 120%, or is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least
  • the active variant of a variant LPG10145 RGN can comprise an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to: (a) the amino acid sequence set forth as SEQ ID NO: 1, wherein E at amino acid position 52 is an R; (b) the amino acid sequence set forth as SEQ ID NO: 1, wherein S at amino acid position 55 is an R; (c) the amino acid sequence set forth as SEQ ID NO: 1, wherein G at amino acid position 86 is an R; (d) the amino acid sequence set forth as SEQ ID NO: 1, wherein Y at amino acid position 472 is an R; (e) the amino acid sequence set forth as SEQ ID NO:
  • the present disclosure provides an active variant or fragment of a variant LPG10145 RGN that has increased nuclease activity as compared to the RGN of SEQ ID NO: 1.
  • an active variant or fragment of a variant LPG10145 RGN disclosed herein comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NOs: 2-16, 182-196, and 271-285 has increased nuclease activity as compared to the RGN of SEQ ID NO: 1.
  • an active variant or fragment of a variant LPG10145 RGN disclosed herein comprising the amino acid sequence of any one of SEQ ID NOs: 2-16, 182-196, and 271-285 has increased nuclease activity as compared to the RGN of SEQ ID NO: 1.
  • an active variant or fragment of a variant LPG10145 RGN disclosed herein can have nuclease activity that is from about 80% to about 500%, from about 80% to about 200%, or from about 90% to about 150%, or from about 95% to about 120%, or is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least
  • the active variant of a variant LPG10145 RGN can comprise an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to: (a) the amino acid sequence set forth as SEQ ID NO: 2; (b) the amino acid sequence set forth as SEQ ID NO: 3; (c) the amino acid sequence set forth as SEQ ID NO: 4; (d) the amino acid sequence set forth as SEQ ID NO: 5; (e) the amino acid sequence set forth as SEQ ID NO: 6; (f) the amino acid sequence set forth as SEQ ID NO: 7; (g) the amino acid sequence set forth as SEQ ID NO: 8SEQ ID NO: 8; (h)
  • RNA-guided nucleases can in embodiments comprise at least one nuclease domain (e.g., DNase, RNase domain) and at least one RNA recognition and/or RNA binding domain to interact with guide RNAs.
  • the RGN comprises only one active nuclease domain and thus functions as a nickase.
  • the RGN nuclease domain that is active in an RGN nickase can be a RuvC domain or an HNH domain.
  • An RGN nickase can comprise an inactivated HNH nuclease domain or can lack an HNH domain.
  • RNA-guided nucleases include, but are not limited to: DNA binding domains, helicase domains, protein-protein interaction domains, and dimerization domains.
  • the RNA-guided nucleases provided herein can comprise at least 70%, 75%, 80%,
  • variant LPG10145 RGNs of the disclosure do not comprise amino acid residues in the PAM recognition domain, the REC 1 domain, the REC2 domain, and/or the Wing domain that differ from the corresponding residues in the wild-type LPG10145 RGN (e.g., SEQ ID NO: 1).
  • a target nucleotide sequence is bound by an RNA-guided nuclease provided herein and hybridizes with the guide RNA associated with the RNA-guided nuclease.
  • the target sequence can then be subsequently cleaved by the RNA-guided nuclease if the polypeptide possesses nuclease activity.
  • cleave or cleavage refer to the hydrolysis of at least one phosphodiester bond within the backbone of a target nucleotide sequence that can result in either single-stranded or double-stranded breaks within the target sequence.
  • the presently disclosed RGNs can cleave nucleotides within a polynucleotide, functioning as an endonuclease or can be an exonuclease, removing successive nucleotides from the end (the 5' and/or the 3' end) of a polynucleotide.
  • the disclosed RGNs can cleave nucleotides of a target sequence within any position of a polynucleotide and thus function as both an endonuclease and exonuclease.
  • the cleavage of a target polynucleotide by the presently disclosed RGNs can result in staggered breaks or blunt ends.
  • a staggered cut in a polynucleotide leads to two sticky ends or overhanging ends, and is formed when the nuclease cuts each strand of a polynucleotide such that the cuts are not directly opposite each other.
  • one strand i.e. the overhanging strand
  • the longer strand has bases which are left unpaired.
  • the longer strand of an overhanging end of a cleaved polynucleotide can have one unpaired nucleotide, two unpaired nucleotides, 3 unpaired nucleotides, 4 unpaired nucleotides, 5 unpaired nucleotides, or more unpaired nucleotides. In some embodiments, the longer strand of an overhanging end of a cleaved polynucleotide can have one unpaired nucleotide.
  • the overhanging end of a cleaved polynucleotide can be a 3' overhang or a 5' overhang. In some embodiments, the overhanging end of a cleaved polynucleotide is a 3' overhang.
  • the overhanging end of a cleaved polynucleotide is a 5' overhang.
  • a variant LPG10145 RGN, or an active variant or fragment thereof, of the disclosure cleaves a target polynucleotide to form a staggered cut, wherein the staggered cut creates a 3' overhang with one unpaired nucleotide.
  • a blunt cut generates two blunt ends, such that each blunt end of the cut polynucleotide has both strands that are of equal length - i.e. there are no unpaired bases on either strand of a blunt end.
  • RNA-guided nucleases are variants of wild-type polypeptides.
  • the wildtype RGN can be modified to alter nuclease activity or alter PAM specificity, for example.
  • the RNA-guided nuclease is not naturally-occurring.
  • the RNA-guided nuclease functions as a nickase, only cleaving a single strand of the target nucleotide sequence.
  • Such RNA-guided nucleases have a single functioning nuclease domain.
  • the nickase is capable of cleaving the positive strand or negative strand.
  • additional nuclease domains have been mutated such that the nuclease activity is reduced or eliminated.
  • the RNA-guided nuclease lacks nuclease activity altogether and is referred to herein as nuclease-dead or nuclease inactive.
  • Any method known in the art for introducing mutations into an amino acid sequence such as PCR-mediated mutagenesis and site-directed mutagenesis, can be used for generating nickases or nuclease-dead RGNs. See, e.g., U.S. Publ. No. 2014/0068797 and U.S. Pat. No. 9,790,490; each of which is incorporated by reference in its entirety.
  • a non-limiting example of a nickase is the UPG10145 D16A nickase, which is set forth herein as SEQ ID NO: 124.
  • Nickases which comprise a mutation in the RuvC domain and have a functional UNH domain are useful in base editing wherein the nickase is fused to a base editing polypeptide such as a deaminase.
  • Another non-limiting example of a nickase is the EPG10145 H611A nickase, which is set forth herein as SEQ ID NO: 125.
  • Nickases which comprise a mutation in the HNH domain and have a functional RuvC domain are useful in polymerase (i.e.
  • a nuclease dead RGN is the LPG10145 D16A H611A sequence set forth as SEQ ID NO: 126.
  • Variant LPG10145 RGNs of the disclosure can thus additionally comprise mutations in one or more nuclease domains that confer nickase function to the variant LPG10145 RGNs.
  • a variant LPG10145 RGN of the disclosure comprises an alanine (or another nonconserved amino acid residue) at a position corresponding to 634 of SEQ ID NO: 1.
  • variant LPG10145 RGNs of the disclosure that comprise the H611A mutation in the HNH domain comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to any one of SEQ ID NOs: 182- 196.
  • variant LPG10145 RGNs of the disclosure that comprise the D16A mutation comprises the amino acid sequence set forth as any one of SEQ ID NOs: 271-285.
  • variant LPG10145 RGNs of the disclosure that comprise the D16A mutation comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to any one of SEQ ID NOs: 271-285.
  • variant LPG10145 RGNs comprises an amino acid sequence having at least 80%
  • RNA-guided nucleases that lack nuclease activity can be used to deliver a fused polypeptide, polynucleotide, or small molecule payload to a particular genomic location.
  • the RGN polypeptide or guide RNA can be fused to a detectable label to allow for detection of a particular sequence.
  • a nuclease-dead RGN can be fused to a detectable label (e.g., fluorescent protein) and targeted to a particular sequence associated with a disease to allow for detection of the disease-associated sequence.
  • nuclease-dead RGNs can be targeted to particular genomic locations to alter the expression of a desired sequence.
  • the binding of a nuclease-dead RNA-guided nuclease to a target sequence results in the reduction in expression of the target sequence or a gene under transcriptional control by the target sequence by interfering with the binding of RNA polymerase or transcription factors within the targeted genomic region.
  • the RGN e.g., a nuclease- dead RGN
  • its complexed guide RNA further comprises an expression modulator that, upon binding to a target sequence, serves to either repress or activate the expression of the target sequence or a gene under transcriptional control by the target sequence.
  • the expression modulator modulates the expression of the target sequence or regulated gene through epigenetic mechanisms.
  • the nuclease-dead RGNs or an RGN with nickase activity can be targeted to particular genomic locations to modify the sequence of a target polynucleotide through fusion to a baseediting polypeptide, for example a deaminase polypeptide or active variant or fragment thereof, that directly chemically modifies (e.g., deaminates) a nucleobase, resulting in conversion from one nucleobase to another.
  • the base-editing polypeptide can be fused to the RGN at its N-terminal or C-terminal end. Additionally, the base-editing polypeptide may be fused to the RGN via a peptide linker.
  • a non-limiting example of a deaminase polypeptide that is useful for such compositions and methods includes a cytosine deaminase or an adenine deaminase (such as the adenine deaminase base editor described in Gaudelli et al. (2017) Nature 551:464-471, U.S. Publ. Nos. 2017/0121693 and 2018/0073012, and International Publ. No. WO 2018/027078, or any of the deaminases disclosed in International Publ. No. WO 2020/139783, International Publ. No. WO 2022/056254, International Publ. No. WO 2022/204093, and International Publ. No.
  • the deaminase polypeptide that is useful for such compositions and methods is a cytosine deaminase or an adenine deaminase comprising an amino acid sequence selected from any one of SEQ ID NOs: 42-113, and 257.
  • the deaminase polypeptide that is useful for such compositions and methods is a cytosine deaminase or an adenine deaminase having a sequence that is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater identity to any one of the amino acid sequences set forth as SEQ ID NOs: 42-113, and 257.
  • the deaminase polypeptide that is useful for such the presently disclosed compositions and methods is a deaminase disclosed in Table 17 of International Publ. No. WO 2020/139783, which is incorporated herein by reference in its entirety.
  • certain fusion proteins between an RGN and a base-editing enzyme e.g., cytosine deaminase
  • Non-limiting examples of uracil stabilizing polypeptides include those disclosed in International Publ. No. WO 2022/015969, which is herein incorporated by reference in its entirety, including USP2 (SEQ ID NO: 40), and a uracil glycosylase inhibitor (UGI) domain (SEQ ID NO: 35), which may increase base editing efficiency. Therefore, a fusion protein may comprise an RGN described herein or variant thereof, a deaminase, and optionally at least one uracil stabilizing polypeptide, such as UGI or USP2.
  • the RGN that is fused to the base-editing polypeptide is a nickase that cleaves the DNA strand that is not acted upon by the base-editing polypeptide (e.g., deaminase).
  • a variant LPG10145 RGN, or active variant or fragment thereof, of the disclosure can comprise a protospacer adjacent motif (PAM)-interacting (PI) domain that contributes to recognition of a PAM site in a target polynucleotide.
  • PI protospacer adjacent motif
  • a variant LPG10145 RGN, or active variant or fragment thereof recognizes and binds a consensus nucleotide sequence set forth as NNGG adjacent to the target polynucleotide.
  • the PI domain can comprise 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 or more amino acid residues.
  • the PI domain of an RGN, or an active variant or fragment thereof, of the disclosure is located within the carboxy (C)-terminal region of the RGN.
  • the C-terminal region comprising the PI domain of an RGN, or an active variant or fragment thereof, of the disclosure can include the C-terminal 151 amino acid residues, the C-terminal 150 amino acid residues, the C-terminal 140 amino acid residues, the C-terminal 135 amino acid residues, the C-terminal 132 amino acid residues, the C-terminal 130 amino acid residues, the C-terminal 125 amino acid residues, the C-terminal 120 amino acid residues, the C-terminal 110 amino acid residues, the C-terminal 100 amino acid residues, the C-terminal 90 amino acid residues, the C-terminal 80 amino acid residues, the C-terminal 70 amino acid residues, the C-terminal 60 amino acid residues, the C-terminal 50 amino acid residues, the C-terminal 40 amino acid residues, the C-termin
  • the PI domain of an RGN, or an active variant or fragment thereof, of the disclosure is within or includes amino acid residues 977-1130 of the RGN.
  • the PI domain of a variant LPG10145 RGN polypeptide, or an active variant or fragment thereof, of the disclosure has the amino acid sequence set forth as SEQ ID NO: 253.
  • fusion proteins comprising the presently disclosed engineered variant LPG10145 RGN polypeptides, or active variants or fragments thereof, operably fused to at least one heterologous polypeptide, as well as polynucleotides encoding the fusion proteins.
  • “operably linked” or “operably fused” is intended that the coding regions are in the same reading frame, even if one is inserted into another.
  • polypeptides that are “operably fused” or “operably linked” means that the structure and/or biological activity of each individual peptide is also present in the fusion.
  • heterologous in reference to a polypeptide that is heterologous to another polypeptide (e.g., a variant LPG10145 RGN), is a polypeptide that is not operably fused to the presently described variant LPG10145 RGN in nature.
  • the heterologous polypeptide can originate from a foreign species or from the same species.
  • the heterologous polypeptide can be in its native form or is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.
  • the heterologous polypeptide can be any polypeptide, including but not limited to a localization signal, a cellpenetrating domain, a polymerase (e.g., reverse transcriptase), a base editing polypeptide, an effector domain (e.g., a cleavage domain, a deaminase domain, or an expression modulator domain), a detectable label (e.g., fluorescent protein), or a purification tag, or active variant or fragment thereof.
  • the heterologous polypeptide can be operably fused to the amino (N)-terminus, to the carboxy (C)-terminus, or to an internal location of a variant LPG10145 RGN polypeptide, or active variant or fragment thereof, described herein.
  • the heterologous polypeptide is operably fused to a variant LPG10145 RGN polypeptide by a peptide linker as described herein.
  • a variant LPG10145 RGN polypeptide, or active variant or fragment thereof, of the disclosure may be fused to a polymerase (i.e. prime) editing polypeptide (e.g., DNA polymerase or reverse transcriptase) to generate a polymerase editor (PE).
  • the polymerase editing polypeptide e.g., DNA polymerase or reverse transcriptase
  • the polymerase editing polypeptide can be operably fused to the amino (N)-terminus, to the carboxy (C)- terminus, or to an internal location of a variant LPG10145 RGN polypeptide, or active variant or fragment thereof, described herein.
  • the polymerase editing polypeptide (e.g., DNA polymerase or reverse transcriptase) is operably fused to a variant LPG10145 RGN polypeptide by a peptide linker as described herein.
  • Polymerase (i.e.p) editing is a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a nucleic acid programmable DNA binding protein working in association with a polymerase.
  • the polymerase (i.e. prime) editing system uses an RGN that is a nickase, and the system is programmed with a polymerase (i.e. prime) editing (PE) guide RNA (“PEgRNA”).
  • the PEgRNA is a guide RNA that both specifies the target sequence and provides the template for polymerization of the replacement strand containing the edit by way of an extension engineered onto the guide RNA (e.g., at the 5' or 3' end, or at an internal portion of the guide RNA).
  • the RGN nickase/polymerase (i.e. prime) editing polypeptide fusion is guided to the target sequence by the PEgRNA and nicks the target strand upstream of sequence to be edited and upstream of the PAM, creating a 3' flap on the target strand.
  • the PEgRNA includes a primer binding site (PBS) that is complementary to the 3' flap of the target strand.
  • PBS primer binding site
  • a PBS is at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length.
  • the pegRNA comprises a PBS that is at least 5 (e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 28, 19, or 20) nucleotides in length.
  • the pegRNA may comprise a PBS that is at least 8 nucleotides in length.
  • Hybridrization of the PBS and 3' flap of the target strand allows polymerization of the replacement strand containing the edit using the extension of the PEgRNA as template.
  • the extension of the PEgRNA can be formed from RNA or DNA.
  • the polymerase of the polymerase (i.e. prime) editor can be an RNA-dependent DNA polymerase (such as a reverse transcriptase).
  • the polymerase of the polymerase (i.e. prime) editor may be a DNA-dependent DNA polymerase.
  • the replacement strand containing the desired edit (e.g., a single nucleobase substitution) shares the same sequence as the target strand of the target sequence to be edited (with the exception that it includes the desired edit).
  • the target strand of the target sequence is replaced by the newly synthesized replacement strand containing the desired edit.
  • polymerase (i.e. prime) editing may be thought of as a “search-and-replace” genome editing technology since the polymerase (i.e. prime) editors not only search and locate the desired target sequence to be edited, but at the same time, encode a replacement strand containing a desired edit which is installed in place of the corresponding target strand of the target sequence.
  • a guide RNA of the disclosure comprises an extension comprising an edit template for polymerase (i.e. prime) editing.
  • a polymerase (i.e. prime) editing polypeptide that can be fused to an RGN includes a DNA polymerase.
  • the DNA polymerase is a reverse transcriptase.
  • the RGN is a nickase.
  • a fusion protein comprising a variant LPG10145 RGN polypeptide, or active variant or fragment thereof, disclosed herein and a reverse transcriptase can comprise an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to any one of SEQ ID NOs: 135, and 162-181.
  • a fusion protein comprising a variant LPG10145 RGN polypeptide, or active variant or fragment thereof, disclosed herein and a reverse transcriptase comprises the amino acid sequence set forth as any one of SEQ ID NOs: 135, and 162-181.
  • a variant LPG10145 RGN polypeptide, or active variant or fragment thereof, of the disclosure may be operably fused to a base-editing polypeptide as described herein.
  • the base-editing polypeptide e.g., deaminase
  • the base-editing polypeptide can be operably fused to the amino (N)-terminus, to the carboxy (C)-terminus, or to an internal location of a variant LPG10145 RGN polypeptide, or active variant or fragment thereof, described herein.
  • the base-editing polypeptide e.g., deaminase
  • the base editing polypeptide in a fusion protein of the disclosure is a cytosine deaminase or an adenine deaminase comprising an amino acid sequence set forth as any one of SEQ ID NOs: 42-113, and 257.
  • the base editing polypeptide in a fusion protein of the disclosure is a cytosine deaminase or an adenine deaminase having an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to the amino acid sequence set forth as any one of SEQ ID NOs: 42-113, and 257.
  • the base editing polypeptide in a fusion protein of the disclosure is a deaminase described in Gaudelli et al. (2017) Nature 551:464-471, U.S. Publ. Nos. 2017/0121693 and 2018/0073012, and International Publ. No. WO 2018/027078, or any of the deaminases disclosed in International Publ. No. WO 2020/139783, International Publ. No. WO 2022/056254, International Publ. No. WO 2022/204093, and International Publ. No. WO 2024/095245, each of which is herein incorporated by reference in its entirety.
  • the fusion protein comprising an engineered variant LPG10145 RGN polypeptide and a base-editing polypeptide may further comprise at least one uracil stabilizing polypeptide that increases the mutation rate of a nucleobase (e.g., cytidine, deoxycytidine, or cytosine to a thymidine, deoxythymidine, or thymine) in a nucleic acid molecule by a deaminase.
  • a nucleobase e.g., cytidine, deoxycytidine, or cytosine to a thymidine, deoxythymidine, or thymine
  • a USP comprises USP2 (SEQ ID NO: 40) or a uracil glycosylase inhibitor (UGI) domain (SEQ ID NO: 35), which may increase base editing efficiency.
  • the variant LPG10145 RGN polypeptide that is fused to the base-editing polypeptide is a nickase that cleaves the DNA strand that is not acted upon by the base-editing polypeptide (e.g., deaminase).
  • the variant LPG10145 RGN polypeptide that is fused to the base-editing polypeptide is a nuclease-dead RGN polypeptide.
  • a fusion protein comprising a variant LPG10145 RGN polypeptide, or active variant or fragment thereof, disclosed herein and a deaminase can comprise an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to any one of SEQ ID NOs: 258-270.
  • a fusion protein comprising a variant LPG10145 RGN polypeptide, or active variant or fragment thereof, disclosed herein and a deaminase comprises the amino acid sequence set forth as any one of SEQ ID NOs: 258-270.
  • the presently disclosed variant LPG10145 RNA-guided nucleases, or active variants or fragments thereof, can comprise at least one nuclear localization signal (NLS) to enhance transport of the RGN to the nucleus of a cell.
  • Nuclear localization signals are known in the art and generally comprise a stretch of basic amino acids (see, e.g., Lange et al., J. Biol. Chem. (2007) 282:5101-5105).
  • the RGN comprises 2, 3, 4, 5, 6 or more nuclear localization signals.
  • the nuclear localization signal(s) can be a heterologous NLS.
  • Non-limiting examples of nuclear localization signals useful for the presently disclosed RGNs are the nuclear localization signals of SV40 Large T-antigen, nucleoplasmin, and c-Myc (see, e.g., Ray et al. (2015) Bioconjug Chem 26(6): 1004-7).
  • the NLS has an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to any one of SEQ ID NOs: 36, 37, 234, or 235.
  • the RGN comprises the NLS sequence set forth as SEQ ID NO: 36, 37, 234, or 235.
  • the RGN can comprise one or more NLS sequences operably fused at its N-terminus, C- terminus, or both the N-terminus and C-terminus.
  • the RGN can comprise two NLS sequences at the N-terminal region and four NLS sequences at the C-terminal region.
  • LPG10145 RGNs Other localization signal sequences known in the art that localize polypeptides to particular subcellular location(s) can also be used to target the presently disclosed variant LPG10145 RGNs, or active variants or fragments thereof, including, but not limited to, plastid localization sequences, mitochondrial localization sequences, and dual-targeting signal sequences that target to both the plastid and mitochondria (see, e.g., Nassoury and Morse (2005) Biochim Biophys Acta 1743:5-19; Kunze and Berger (2015) Front Physiol dx.doi.org/10.3389/fphys.2015.00259; Herrmann and Neupert (2003) IUBMB Life 55:219-225; Soil (2002) Curr Opin Plant Biol 5:529-535; Carrie and Small (2013) Biochim Biophys Acta 1833:253-259; Carrie et al.
  • the presently disclosed variant LPG10145 RNA-guided nucleases, or active variants or fragments thereof comprise at least one cell-penetrating domain that facilitates cellular uptake of the RGN.
  • Cell-penetrating domains are known in the art and generally comprise stretches of positively charged amino acid residues (i.e., polycationic cell -penetrating domains), alternating polar amino acid residues and non-polar amino acid residues (i.e., amphipathic cell-penetrating domains), or hydrophobic amino acid residues (i.e., hydrophobic cell-penetrating domains) (see, e.g., Milletti F. (2012) Drug Discov Today 17:850-860).
  • a non-limiting example of a cell -penetrating domain is the trans-activating transcriptional activator (TAT) from the human immunodeficiency virus 1.
  • TAT trans-activating transcriptional activator
  • the nuclear localization signal, plastid localization signal, mitochondrial localization signal, dualtargeting localization signal, and/or cell-penetrating domain can be located at the amino-terminus (N- terminus), the carboxyl -terminus (C-terminus), or in an internal location of the RNA-guided nuclease.
  • the presently disclosed variant LPG10145 RGNs, or active variants or fragments thereof, can be fused to an effector domain, such as a cleavage domain, a deaminase domain, or an expression modulator domain, either directly or indirectly via a linker peptide.
  • an effector domain such as a cleavage domain, a deaminase domain, or an expression modulator domain, either directly or indirectly via a linker peptide.
  • a domain can be located at the N-terminus, the C-terminus, or an internal location of the RNA-guided nuclease.
  • the RGN component of the fusion protein is a nuclease-dead RGN or a nickase.
  • the RGN fusion protein comprises a cleavage domain, which is any domain that is capable of cleaving a polynucleotide (i.e., RNA, DNA, or RNA/DNA hybrid) and includes, but is not limited to, restriction endonucleases and homing endonucleases, such as Type IIS endonucleases (e.g., Fokl) (see, e.g., Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388; Linn et al. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993).
  • a cleavage domain is any domain that is capable of cleaving a polynucleotide (i.e., RNA, DNA, or RNA/DNA hybrid) and includes, but is not limited to, restriction endonucleases and homing endonucleases, such as Type IIS endonucleases (e.g., Fo
  • the RGN fusion protein comprises a deaminase domain that deaminates a nucleobase, resulting in conversion from one nucleobase to another, and includes, but is not limited to, a cytosine deaminase or an adenine deaminase (see, e.g., Gaudelli et al. (2017) Nature 551:464-471, U.S. Publ. Nos. 2017/0121693 and 2018/0073012, U.S. Patent No. 9,840,699, and International Publ. No. WO/2018/027078, or any of the deaminases disclosed in International Publ. No. WO 2020/139783, International Publ. No. WO 2022/056254, and International Publ. No. WO 2022/204093, each of which is herein incorporated by reference in its entirety).
  • the effector domain of the RGN fusion protein can be an expression modulator domain, which is a domain that either serves to upregulate or downregulate transcription.
  • the expression modulator domain can be an epigenetic modification domain, a transcriptional repressor domain or a transcriptional activation domain.
  • the expression modulator of the RGN fusion protein comprises an epigenetic modification domain that covalently modifies DNA or histone proteins to alter histone structure and/or chromosomal structure without altering the DNA sequence, leading to changes in gene expression (z.e., upregulation or downregulation).
  • epigenetic modifications include acetylation or methylation of lysine residues, arginine methylation, serine and threonine phosphorylation, and lysine ubiquitination and sumoylation of histone proteins, and methylation and hydroxymethylation of cytosine residues in DNA.
  • Non-limiting examples of epigenetic modification domains include histone acetyltransferase domains, histone deacetylase domains, histone methyltransferase domains, histone demethylase domains, DNA methyltransferase domains, and DNA demethylase domains.
  • the expression modulator of the fusion protein comprises a transcriptional repressor domain, which interacts with transcriptional control elements and/or transcriptional regulatory proteins, such as RNA polymerases and transcription factors, to reduce or terminate transcription of at least one gene.
  • Transcriptional repressor domains are known in the art and include, but are not limited to, Spl- like repressors, IKB, and Kriippel associated box (KRAB) domains.
  • the expression modulator of the fusion protein comprises a transcriptional activation domain, which interacts with transcriptional control elements and/or transcriptional regulatory proteins, such as RNA polymerases and transcription factors, to increase or activate transcription of at least one gene.
  • Transcriptional activation domains are known in the art and include, but are not limited to, a herpes simplex virus VP 16 activation domain and an NF AT activation domain.
  • the presently disclosed variant LPG10145 RGN polypeptides, or active variants or fragments thereof, can comprise a detectable label or a purification tag.
  • the detectable label or purification tag can be located at the N-terminus, the C-terminus, or an internal location of the RNA-guided nuclease, either directly or indirectly via a linker peptide.
  • the RGN component of the fusion protein is a nuclease-dead RGN. In other embodiments, the RGN component of the fusion protein is an RGN with nickase activity.
  • a detectable label is a molecule that can be visualized or otherwise observed.
  • the detectable label may be fused to the RGN as a fusion protein (e.g., fluorescent protein) or may be a small molecule conjugated to the RGN polypeptide that can be detected visually or by other means.
  • Detectable labels that can be fused to the presently disclosed RGNs as a fusion protein include any detectable protein domain, including but not limited to, a fluorescent protein or a protein domain that can be detected with a specific antibody.
  • Non-limiting examples of fluorescent proteins include green fluorescent proteins (e.g., GFP, EGFP, ZsGreenl) and yellow fluorescent proteins (e.g., YFP, EYFP, ZsYellowl).
  • Non-limiting examples of small molecule detectable labels include radioactive labels, such as 3 H and 35 S.
  • Variant LPG10145 RGN polypeptides, or active variants or fragments thereof, of the disclosure can also comprise a purification tag, which is any molecule that can be utilized to isolate a protein or fused protein from a mixture (e.g., biological sample, culture medium).
  • purification tags include biotin, myc, maltose binding protein (MBP), glutathione-S-transferase (GST), and 3X FLAG tag.
  • Linker refers to a chemical group or a molecule linking two molecules or moieties, e.g. , a binding domain and a cleavage domain of a nuclease.
  • a linker joins a gRNA binding domain of an RNA guided nuclease and a base-editing polypeptide, such as a deaminase.
  • a linker joins a nuclease-dead RGN and a deaminase.
  • the linker is positioned between, or flanked by, two groups, molecules, or other moieties and connected to each one via a covalent bond, thus connecting the two.
  • the linker is an organic molecule, group, polymer, or chemical moiety.
  • the linker is an amino acid or a plurality of amino acids (e.g., a peptide or protein).
  • a peptide linker of the disclosure can connect one polypeptide to another in a fusion protein.
  • a peptide linker can connect a variant LPGI0145 RGN polypeptide and a heterologous polypeptide (e.g., a polymerase, a base editing polypeptide, an effector domain).
  • a fusion protein comprising 3 polypeptides e.g., a variant LPGI0145 RGN polypeptide, a polymerase, and a detectable label
  • a peptide linker comprises at least one NLS.
  • a peptide linker comprises 2 NLSs.
  • the peptide linker can be operably fused at the N- terminus, the C-terminus, or both the N-terminus and C-terminus of the variant LPGI0145 RGN polypeptide or the heterologous polypeptide (e.g., a polymerase, a base editing polypeptide, an effector domain).
  • a peptide linker has a formula of -(SGGS) x -NLSm-(SGGS)y-NLSn-(SGGS) z -, wherein each of x, y, or z is 0, 1, 2, 3, or 4; and wherein each of m or n is 0 or 1.
  • a peptide linker has a formula of -(SGGS) x -NLSm-(SGGS)y-NLSn-(SGGS) z -, wherein each of x or y is 0, 1, 2, 3, or 4, and y is 0; and wherein one of m or n is 0, and the other is 1.
  • the peptide linker has a formula of -(SGGS) x -NLSm-(SGGS) y -NLSn-(SGGS) z -, wherein each of x, y, or z is 0, 1, 2, 3, or 4; and wherein each of m or n is 1.
  • a peptide linker comprises one or more copies of amino acid sequence SGGS (SEQ ID NO: 241).
  • the peptide linker is 4-100 amino acids in length, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30- 35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated.
  • a peptide linker has a length of at least 13 amino acids, including but not limited to about 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, or more amino acids.
  • the peptide linker has an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to any one of SEQ ID NOs: 236-241. In some embodiments, the peptide linker has the amino acid sequence of any one of SEQ ID NOs: 236-241.
  • the heterologous polypeptide comprises a polymerase (e.g., reverse transcriptase), and the peptide linker between the variant LPG10145 RGN polypeptide and the polymerase (e.g., reverse transcriptase) comprises at least 2 amino acids, at least 3 amino acids, at least 4 amino acids, at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids, at least 10 amino acids, at least 11 amino acids, at least 12 amino acids, or at least 13 amino acids.
  • a polymerase e.g., reverse transcriptase
  • the peptide linker between the variant LPG10145 RGN polypeptide and the polymerase comprises at least 13 amino acids, including but not limited to about 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, or more amino acids.
  • the fusion protein comprises a variant LPG10145 RGN polypeptide connected to a polymerase (e.g., reverse transcriptase) by a peptide linker having an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to any one of SEQ ID NOs: 236-241.
  • a polymerase e.g., reverse transcriptase
  • the fusion protein comprises a variant LPG10145 RGN polypeptide connected to a polymerase (e.g., reverse transcriptase) by a peptide linker having the amino acid sequence set forth as any one of SEQ ID NOs: 236-241.
  • a polymerase e.g., reverse transcriptase
  • a fusion protein of the disclosure in some embodiments comprises a presently disclosed variant LPG10145 RGN polypeptide and a presently disclosed heterologous polypeptide (e.g., a polymerase, a base editing polypeptide, an effector domain).
  • a fusion protein of the disclosure comprises from amino terminus to carboxy terminus: a variant LPG10145 RGN polypeptide and a heterologous polypeptide (e.g., a polymerase, a base editing polypeptide, an effector domain).
  • a fusion protein of the disclosure comprises from amino terminus to carboxy terminus: a heterologous polypeptide (e.g., a polymerase, a base editing polypeptide, an effector domain) and a variant LPG10145 RGN polypeptide.
  • a heterologous polypeptide e.g., a polymerase, a base editing polypeptide, an effector domain
  • the fusion protein can in some embodiments comprises a heterologous polypeptide (e.g., a polymerase, a base editing polypeptide, an effector domain) inserted within a variant LPG10145 RGN polypeptide.
  • a heterologous polypeptide e.g., a polymerase, a base editing polypeptide, an effector domain
  • the heterologous polypeptide is inserted between surface amino acid residues.
  • the heterologous polypeptide e.g., a polymerase, a base editing polypeptide, an effector domain
  • a linker domain 2 e.g., a linker domain 2
  • WED wedge
  • RuvC e.g., a RuvC domain
  • HNH e.g., HNH domain
  • Rec-2 domain e.g., Rec-2 domain
  • PAM-interacting (PI) domain e.g., PI
  • the heterologous polypeptide in some embodiments is inserted within a variant LPG10145 RGN polypeptide immediately after the amino acid position selected from the group consisting of: i) amino acid position corresponding to position 347 of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, or 285; ii) amino acid position corresponding to position 524 of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282,
  • guide RNA refers to a nucleotide sequence having sufficient complementarity with a target nucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of an associated RNA- guided nuclease to the target nucleotide sequence. More specifically, when the target nucleotide sequence is double-stranded as is the case with DNA, the target nucleotide sequence comprises a target strand and a nontarget strand (which comprises the PAM sequence).
  • the guide RNA has sufficient complementarity with the target strand of a double -stranded target sequence (e.g., target DNA sequence) such that the guide RNA hybridizes with the target strand and directs sequence-specific binding of an associated RGN to the target sequence (e.g., target DNA sequence). Therefore, in some embodiments, a guide RNA includes a spacer that is identical to the sequence of the non-target strand except that uracil (U) replaces thymidine (T) in the guide RNA.
  • U uracil
  • T thymidine
  • each of the one or more guide RNA has sufficient complementarity with the target strand of a particular target sequence and is capable of hybridizing to the target strand of that target sequence.
  • a corresponding target sequence for a guide RNA refers to the target sequence that the guide RNA has sufficient complementarity with and is capable of hybridizing to.
  • an RGN’s respective guide RNA is one or more RNA molecules (generally, one or two), that can bind to the RGN and guide the RGN to bind to a particular target nucleotide sequence, and in those embodiments wherein the RGN has nickase or nuclease activity, also cleave the target strand and/or the nontarget strand.
  • a guide RNA comprises a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA).
  • Native guide RNAs that comprise both a crRNA and a tracrRNA generally comprise two separate RNA molecules that hybridize to each other through the repeat sequence of the crRNA and the antirepeat sequence of the tracrRNA.
  • a guide RNA can encompass a polymerase editing (PE) guide RNA.
  • PE polymerase editing
  • Native direct repeat sequences within a CRISPR array generally range in length from 28 to 37 base pairs, although the length can vary between about 23 bp to about 55 bp.
  • Spacer sequences within a CRISPR array generally range from about 32 to about 38 bp in length, although the length can be between about 21 bp to about 72 bp.
  • Each CRISPR array generally comprises less than 50 units of the CRISPR repeat-spacer sequence.
  • the CRISPRs are transcribed as part of a long transcript termed the primary CRISPR transcript, which comprises much of the CRISPR array.
  • the primary CRISPR transcript is cleaved by Cas proteins to produce crRNAs or in some cases, to produce pre-crRNAs that are further processed by additional Cas proteins into mature crRNAs.
  • Mature crRNAs comprise a spacer sequence and a CRISPR repeat sequence.
  • maturation involves the removal of about one to about six or more 5', 3', or 5' and 3' nucleotides.
  • these nucleotides that are removed during maturation of the pre-crRNA molecule are not necessary for generating or designing a guide RNA.
  • a guide RNA of the disclosure can comprise at least one chemical modification.
  • the at least one chemical modification includes: a bridged nucleic acid (BNA) modification; 2'-O-methyl (2'-O-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca-OMe modification; 2'-O-methyl 3'phosphorothioate (MS) modification; 2'-O- methyl 3'thiophosphonoacetate (MSP) modification; 2'-O-methyl 3'phosphonoacetate (MP) modification; and phosphorothioate (PS) modification; or a combination thereof.
  • BNA bridged nucleic acid
  • 2'-O-methyl (2'-O-Me) modification 2'-O-methoxy-ethyl (2'MOE) modification
  • 2'-fluoro (2'-F) modification
  • the BNA comprises a 2', 4' BNA modification.
  • the 2', 4' BNA modification is selected from the group consisting of: locked nucleic acid (LNA) modification, BNA NC [N-Me] modification, 2'-O,4'-C- ethylene bridged nucleic acid (2',4'-ENA) modification, and S-constrained ethyl (cEt) modification.
  • the 2', 4' BNA is a LNA modification.
  • the 2', 4' BNA is a cEt modification.
  • the at least one chemical modification comprises a BNA modification, 2'-0-Me modification, or PS modification. Chemical modifications of spacers, crRNA repeats, crRNAs, tracrRNAs, and guide RNAs are described in International Application Publication No. WO 2024/042489, which is hereby incorporated by reference in its entirety herein.
  • compositions comprising guide RNAs comprising CRISPR RNAs (crRNAs).
  • CRISPR RNA comprises a spacer and a CRISPR repeat.
  • the “spacer” is the nucleotide sequence that directly hybridizes with the target nucleotide sequence of interest.
  • the spacer is engineered to be fully or partially complementary with the target sequence of interest.
  • the spacer can comprise from about 8 nucleotides to about 30 nucleotides, or more.
  • the spacer sequence can be about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or more nucleotides in length.
  • the spacer is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotides in length.
  • the spacer is about 10 to about 26 nucleotides in length, or about 12 to about 30 nucleotides in length.
  • the spacer is 19-27 nucleotides in length. In particular embodiments, the spacer is about 30 nucleotides in length.
  • the spacer is 30 nucleotides in length. In some embodiments, the spacer is about 20-25 nucleotides in length. In some embodiments, the degree of complementarity between a spacer and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is between 50% and 99% or more, including but not limited to about or more than about 50%, about 60%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more.
  • the degree of complementarity between a spacer and its corresponding target sequence when optimally aligned using a suitable alignment algorithm, is 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more.
  • the spacer can be identical in sequence to the non-target strand of a target sequence.
  • the spacer can be identical in sequence to the non-target strand of the target DNA sequence, with the exception of the thymidines (Ts) in the non-target strand being replaced by uracils (Us) in the spacer.
  • the spacer is free of secondary structure, which can be predicted using any suitable polynucleotide folding algorithm known in the art, including but not limited to mFold (see, e.g., Zuker and Stiegler (1981) Nucleic Acids Res. 9: 133-148) and RNAfold (see, e.g., Gruber et al. (2008) Cell 106(l):23- 24).
  • a crRNA further comprises a CRISPR RNA (crRNA) repeat.
  • a CRISPR RNA repeat comprises a nucleotide sequence that forms a structure, either on its own or in concert with a hybridized tracrRNA, that is recognized by the RGN polypeptide.
  • the spacer is 5' of the crRNA repeat.
  • the CRISPR RNA repeat can comprise from about 8 nucleotides to about 30 nucleotides, or more.
  • the CRISPR repeat can be about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or more nucleotides in length.
  • the CRISPR repeat is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotides in length.
  • a crRNA repeat comprises a total length of 19 to 40 nucleotides (nt).
  • a crRNA repeat comprises a total length of at most 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, or 30 nt. In some embodiments, the crRNA repeat is about 19 nt or 21 nt.
  • the degree of complementarity between a CRISPR repeat and its corresponding tracrRNA when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, about 60%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more.
  • the degree of complementarity between a CRISPR repeat and its corresponding tracrRNA when optimally aligned using a suitable alignment algorithm, is 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more.
  • the CRISPR repeat comprises the nucleotide sequence of SEQ ID NO: 33, 244, or 245, or an active variant or fragment thereof that when comprised within a guide RNA, is capable of directing the sequence-specific binding of an associated variant LPG10145 RGN provided herein to a target sequence of interest.
  • an active CRISPR repeat sequence variant of a wild-type sequence comprises a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the nucleotide sequence set forth as SEQ ID NO: 33, 244, or 245.
  • an active CRISPR repeat fragment of a wild-type sequence comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides of the nucleotide sequence set forth as SEQ ID NO: 33, 244, or 245.
  • the CRISPR repeat can comprise a nucleotide sequence set forth as SEQ ID NO: 33, 244, or 245, or that differs from SEQ ID NO: 33, 244, or 245 by 1 to 5 nucleotides. In some embodiments, the CRISPR repeat comprises a nucleotide sequence that differs from SEQ ID NO: 33, 244, or 245 by 5 nucleotides. In some embodiments, the CRISPR repeat comprises a nucleotide sequence that differs from SEQ ID NO: 33, 244, or 245 by 4 nucleotides. In some embodiments, the CRISPR repeat comprises a nucleotide sequence that differs from SEQ ID NO: 33, 244, or 245 by 3 nucleotides.
  • the CRISPR repeat comprises a nucleotide sequence that differs from SEQ ID NO: 33, 244, or 245 by 2 nucleotides. In some embodiments, the CRISPR repeat comprises a nucleotide sequence that differs from SEQ ID NO: 33, 244, or 245 by 1 nucleotide. In some embodiments, the CRISPR repeat comprises the nucleotide sequence set forth as SEQ ID NO: 33, 244, or 245.
  • the crRNA repeat of the associated gRNA can have the nucleotide sequence set forth as SEQ ID NO: 33, 244, or 245, or an active variant or fragment thereof.
  • the crRNA is not naturally-occurring.
  • the specific CRISPR repeat is not linked to the engineered spacer in nature and the CRISPR repeat is considered heterologous to the spacer.
  • the spacer is an engineered sequence that is not naturally occurring.
  • the guide RNA can further comprise a trans-activating CRISPR RNA (tracrRNA).
  • a tracrRNA molecule comprises a nucleotide sequence comprising a region that has sufficient complementarity to hybridize to a CRISPR repeat of a crRNA, which is referred to herein as the anti-repeat region.
  • the tracrRNA molecule further comprises a region with secondary structure (e.g., stem-loop) or forms secondary structure upon hybridizing with its corresponding crRNA.
  • the region of the tracrRNA that is fully or partially complementary to a CRISPR repeat sequence is at the 5' end of the molecule and the 3' end of the tracrRNA comprises secondary structure.
  • This region of secondary structure generally comprises several hairpin structures, including the nexus hairpin, which is found adjacent to the anti -repeat sequence.
  • the nexus forms the core of the interactions between the guide RNA and the RGN, and is at the intersection between the guide RNA, the RGN, and the target DNA.
  • the nexus hairpin often has a conserved nucleotide sequence in the base of the hairpin stem, with the motif UNANNC found in many nexus hairpins in tracrRNAs.
  • a tracrRNA comprises a non-canonical sequence in the base of the hairpin stem of its nexus hairpin, including UNANNA, UNANNG, and CNANNC.
  • terminal hairpins at the 3' end of the tracrRNA that can vary in structure and number, but often comprise a GC-rich Rho-independent transcriptional terminator hairpin followed by a string of U’s at the 3' end. See, for example, Briner et al. (2014) Molecular Cell 56:333-339, Briner and Barrangou (2016) Cold Spring Harb Pro ct doi: 10. 1101/pdb.top090902, and U.S. Publication No. 2017/0275648, each of which is herein incorporated by reference in its entirety.
  • the anti-repeat region of the tracrRNA that is fully or partially complementary to the CRISPR repeat comprises from about 8 nucleotides to about 30 nucleotides, or more.
  • the region of base pairing between the tracrRNA anti-repeat and the CRISPR repeat can be about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or more nucleotides in length.
  • the region of base pairing between the tracrRNA anti-repeat and the CRISPR repeat is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotides in length.
  • the degree of complementarity between a CRISPR repeat and its corresponding tracrRNA anti-repeat, when optimally aligned using a suitable alignment algorithm is about or more than about 50%, about 60%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more.
  • the degree of complementarity between a CRISPR repeat and its corresponding tracrRNA anti-repeat when optimally aligned using a suitable alignment algorithm, is 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more.
  • the entire tracrRNA can comprise from about 60 nucleotides to more than about 210 nucleotides.
  • the tracrRNA can be about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, or more nucleotides in length.
  • the tracrRNA is 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 150, 160, 170, 180, 190, 200, 210 or more nucleotides in length.
  • the tracrRNA is about 70 to about 105 nucleotides in length, including about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102, about
  • the tracrRNA is 70 to 105 nucleotides in length, including 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, and 105 nucleotides in length.
  • the tracrRNA comprises the nucleotide sequence of SEQ ID NO: 34, 246, 247, or 248, or an active variant or fragment thereof that when comprised within a guide RNA is capable of directing the sequence -specific binding of an associated RNA-guided nuclease provided herein to a target sequence of interest.
  • an active tracrRNA sequence variant of a wild-type sequence comprises a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the nucleotide sequences set forth as SEQ ID NO: 34, 246, 247, or 248.
  • an active tracrRNA sequence fragment of a wild-type sequence comprises at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more contiguous nucleotides of the nucleotide sequence set forth as SEQ ID NO: 34, 246, 247, or 248
  • the tracrRNA of the associated gRNA can have the nucleotide sequence set forth as SEQ ID NO: 34, 246, 247, or 248, or an active variant or fragment thereof.
  • An active variant or fragment of a variant LPG10145 RGN disclosed herein can bind a tracrRNA comprising the nucleotide sequence set forth as SEQ ID NO: 34, 246, 247, or 248, or an active variant or fragment thereof.
  • Two polynucleotide sequences can be considered to be substantially complementary when the two sequences hybridize to each other under stringent conditions.
  • an RGN is considered to bind to a particular target sequence within a sequence-specific manner if the guide RNA bound to the RGN binds to the target sequence under stringent conditions.
  • stringent conditions or “stringent hybridization conditions” is intended conditions under which the two polynucleotide sequences will hybridize to each other to a detectably greater degree than to other sequences (e.g. , at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances.
  • stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is at least about 30°C for short sequences (e.g., 10 to 50 nucleotides) and at least about 60°C for long sequences (e.g., greater than 50 nucleotides).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37°C, and a wash in 0.5X to IX SSC at 55 to 60°C.
  • Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in 0. IX SSC at 60 to 65°C.
  • wash buffers may comprise about 0.1% to about 1% SDS. Duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours. The duration of the wash time will be at least a length of time sufficient to reach equilibrium.
  • the Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched sequence.
  • stringent conditions are selected to be about 5 °C lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH.
  • severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4°C lower than the thermal melting point (Tm); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10°C lower than the thermal melting point (Tm); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20°C lower than the thermal melting point (Tm).
  • sequence specific can also refer to the binding of a RGN polypeptide or RGN polypeptide fusion to a target sequence at a greater affinity than binding to a randomized background sequence.
  • the guide RNA can be a single guide RNA (sgRNA) or a dual -guide RNA (dgRNA).
  • sgRNA single guide RNA
  • dgRNA dual -guide RNA
  • a single guide RNA comprises the crRNA and tracrRNA on a single molecule of RNA
  • a dual -guide RNA system comprises a crRNA and a tracrRNA present on two distinct RNA molecules, hybridized to one another through at least a portion of the CRISPR repeat sequence of the crRNA and at least a portion of the tracrRNA, which may be fully or partially complementary to the CRISPR repeat sequence of the crRNA.
  • the crRNA and tracrRNA are separated by a linker nucleotide sequence.
  • the linker nucleotide sequence is one that does not include complementary bases in order to avoid the formation of secondary structure within or comprising nucleotides of the linker nucleotide sequence.
  • the linker nucleotide sequence between the crRNA and tracrRNA is at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or more nucleotides in length.
  • the linker nucleotide sequence of a single guide RNA is at least 4 nucleotides in length.
  • the linker nucleotide sequence is the nucleotide sequence AAAG.
  • the guide RNA is a single guide RNA (sgRNA) having the backbone sequence (comprising a crRNA repeat, an optional linker nucleotide sequence, and a tracrRNA) of any one of SEQ ID NOs: 249-252, or an active variant or fragment thereof.
  • an active sgRNA backbone sequence variant comprises a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the nucleotide sequence set forth as any one of SEQ ID NOs: 249-252.
  • an active sgRNA backbone sequence fragment comprises at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more contiguous nucleotides of the nucleotide sequence set forth as any one of SEQ ID NOs: 249-252.
  • the sgRNA backbone can have the nucleotide sequence set forth as any one of SEQ ID NOs: 249-252, or an active variant or fragment thereof.
  • the single guide RNA or dual-guide RNA can be synthesized chemically or via in vitro transcription.
  • Assays for determining sequence-specific binding between an RGN and a guide RNA include, but are not limited to, in vitro binding assays between an expressed RGN and the guide RNA, which can be tagged with a detectable label (e.g., biotin) and used in a pull-down detection assay in which the guide RNA:RGN complex is captured via the detectable label (e.g., with streptavidin beads).
  • a detectable label e.g., biotin
  • streptavidin beads e.g., streptavidin beads
  • the guide RNA can be introduced into a target cell, organelle, or embryo as an RNA molecule.
  • the guide RNA can be transcribed in vitro or chemically synthesized.
  • a nucleotide sequence encoding the guide RNA is introduced into the cell, organelle, or embryo.
  • the nucleotide sequence encoding the guide RNA is operably linked to a promoter (e.g., an RNA polymerase III promoter).
  • the promoter can be a native promoter or heterologous to the guide RNA-encoding nucleotide sequence.
  • the guide RNA can be introduced into a target cell, organelle, or embryo as a ribonucleoprotein complex, as described herein, wherein the guide RNA is bound to an RNA-guided nuclease polypeptide.
  • the guide RNA directs an associated RNA-guided nuclease to a particular target nucleotide sequence of interest through hybridization of the guide RNA to the target nucleotide sequence.
  • a target nucleotide sequence can comprise DNA, RNA, or a combination of both and can be single-stranded or double -stranded.
  • a target nucleotide sequence can be genomic DNA (z.e., chromosomal DNA), plasmid DNA, or an RNA molecule (e.g., messenger RNA, ribosomal RNA, transfer RNA, micro RNA, small interfering RNA).
  • the target nucleotide sequence can be bound (and in some embodiments, cleaved) by an RNA-guided nuclease in vitro or in a cell.
  • the chromosomal sequence targeted by the RGN can be a nuclear, plastid or mitochondrial chromosomal sequence.
  • the target nucleotide sequence is unique in the target genome.
  • the target sequence is double-stranded and comprises a target strand and a non-target strand.
  • the target sequence is adjacent to a protospacer adjacent motif (PAM) and the target strand of the target sequence is the strand that comprises the PAM.
  • the PAM is immediately adjacent to the target sequence and often comprise Ns, which represent any nucleotide.
  • the PAM comprises about 1 to about 10 Ns, including about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 Ns.
  • a PAM comprises 1 to 10 Ns, including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 Ns.
  • the PAM can be 5' or 3' of the target sequence on its non-target strand.
  • the PAM is 3' of the target sequence on its non-target strand for the presently disclosed guide RNAs and RGN systems.
  • the PAM is a consensus sequence of about 3-4 nucleotides, but in some embodiments, it can be 2, 3, 4, 5, 6, 7, 8, 9, or more nucleotides in length.
  • the PAM sequence recognized by the presently disclosed variant LPG10145 RGNs comprises the consensus sequence set forth as NNGG.
  • the variant LPG10145 RGNs comprise an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions differs from the corresponding amino acid residue in SEQ ID NO: 1: 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975.
  • the variant LPG10145 RGNs comprise an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions is a positively charged amino acid residue: 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975.
  • the variant LPG10145 RGNs comprise an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions is an R: 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975.
  • the variant LPG10145 RGNs comprise an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at amino acid position 778 and/or 969 differs from the corresponding amino acid residue in SEQ ID NO: 1. In some embodiments, the variant LPG10145 RGNs comprise an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at amino acid position 778 and/or 969 is a positively charged amino acid residue. In some embodiments, the variant LPG10145 RGNs comprise an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at amino acid position 778 and/or 969 is an R.
  • the variant LPG10145 RGNs comprise an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein amino acid residues at positions 778 and 856 differ from the corresponding amino acid residues in SEQ ID NO: 1. In some embodiments, the variant LPG10145 RGNs comprise an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residues at positions 778 and 856 are positively charged amino acid residues.
  • the variant LPG10145 RGNs comprise an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein amino acid residues at positions 55, 647, 778, and 969 differ from the corresponding amino acid residues in SEQ ID NO: 1. In some embodiments, the variant LPG10145 RGNs comprise an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein amino acid residues at positions 55, 647, 778, and 969 are positively charged amino acid residues.
  • a variant LPG10145 RGN having an amino acid sequence selected from: (a) the amino acid sequence set forth as SEQ ID NO: 1, wherein E at amino acid position 52 is an R; (b) the amino acid sequence set forth as SEQ ID NO: 1, wherein S at amino acid position 55 is an R; (c) the amino acid sequence set forth as SEQ ID NO: 1, wherein G at amino acid position 86 is an R; (d) the amino acid sequence set forth as SEQ ID NO: 1, wherein Y at amino acid position 472 is an R; (e) the amino acid sequence set forth as SEQ ID NO: 1, wherein K at amino acid position 533 is an R; (f) the amino acid sequence set forth as SEQ ID NO: 1, wherein K at amino acid position 541 is an R; (g) the amino acid sequence set forth as SEQ ID NO: 1, wherein Y at amino acid position 643 is substituted by an R; (h) the amino acid sequence set forth as SEQ ID NO: 1, wherein S at amino acid position 647 is an amino acid sequence set
  • a variant LPG10145 RGN having an amino acid sequence selected from: (a) the amino acid sequence set forth as SEQ ID NO: 2; (b) the amino acid sequence set forth as SEQ ID NO: 3; (c) the amino acid sequence set forth as SEQ ID NO: 4; (d) the amino acid sequence set forth as SEQ ID NO: 5; (e) the amino acid sequence set forth as SEQ ID NO: 6; (f) the amino acid sequence set forth as SEQ ID NO: 7; (g) the amino acid sequence set forth as SEQ ID NO: 8SEQ ID NO: 8; (h) the amino acid sequence set forth as SEQ ID NO: 9; (i) the amino acid sequence set forth as SEQ ID NO: 10; (j) the amino acid sequence set forth as SEQ ID NO: 11; (k) the amino acid sequence set forth as SEQ ID NO: 12; (1) the amino acid sequence set forth as SEQ ID NO: 13; (m) the amino acid sequence set forth as SEQ ID NO: 14; (n) the amino acid sequence set
  • the variant LPG10145 RGN binds to a guide sequence comprising a CRISPR repeat set forth as SEQ ID NO: 33, 244, or 245, or an active variant or fragment thereof, and a tracrRNA sequence set forth as SEQ ID NO: 34, 246, 247, or 248, or an active variant or fragment thereof.
  • the RGN systems are described further in Examples 1-6 of the present specification.
  • PAM sequence specificity for a given nuclease enzyme is affected by enzyme concentration (see, e.g., Karvelis et al. (2015) Genome Biol 16:253), which may be modified by altering the promoter used to express the RGN, or the amount of ribonucleoprotein complex delivered to the cell, organelle, or embryo.
  • the RGN Upon recognizing its corresponding PAM sequence, the RGN, if active, can cleave the target nucleotide sequence at a specific cleavage site.
  • a cleavage site is made up of the two particular nucleotides within a target nucleotide sequence at which the target strand, non-target strand, or both strands of the target nucleotide sequence is cleaved by an RGN.
  • the cleavage site can comprise the 1 st and 2 nd , 2 nd and 3 rd .
  • the cleavage site may be over 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the PAM in either the 5' or 3' direction.
  • the cleavage site is defined based on the distance of the two nucleotides from the PAM on the non-target strand of the target polynucleotide and for the target strand, the distance of the two nucleotides from the complement of the PAM.
  • compositions of the disclosure can comprise polymerases (e.g., DNA polymerases, reverse transcriptases).
  • polymerases e.g., DNA polymerases, reverse transcriptases.
  • a “polymerase” is an enzyme that catalyzes the formation of a nucleic acid polymer.
  • a polymerase can be an RNA polymerase (catalyzing an RNA polymer) or a DNA polymerase (catalyzing a DNA polymer).
  • the polymerase of the polymerase editor or system is a DNA polymerase.
  • the PE or PE system can comprise a DNA-dependent DNA polymerase (uses DNA as a template) or an RNA-dependent DNA polymerase (uses RNA as a template).
  • the DNA polymerase of the presently disclosed PEs and PE systems is an RNA-dependent DNA polymerase (i.e., reverse transcriptase).
  • RTs Reverse transcriptases
  • ssRNA single-stranded RNA
  • cDNA complementary DNA
  • RTs can also possess RNase H activity, which degrades the RNA strand of an RNA-DNA hybrid, providing a template for the synthesis of the second DNA strand.
  • a “reverse transcriptase” or “RT” is an enzyme that has polymerase activity to catalyze the formation of a nucleic acid polymer.
  • the RT synthesizes a nucleic acid polymer using a template nucleic acid molecule.
  • the polymerase activity is a DNA polymerase activity.
  • an RT catalyzes the addition of nucleotides to a nicked polynucleotide strand, using a template.
  • RTs include retroviral RTs such as HIV-1 RT, hepatitis B RT, and Murine Leukemia Virus (MLV)- RT (also known as Moloney Murine Leukemia Virus (MMLV)-RT).
  • MLV-RT serves as a model for understanding the basic mechanisms of reverse transcription.
  • Reverse transcriptases typically exhibit a "right hand" structure with three main domains: a ‘finger’ domain involved in binding the template -primer and dNTPs; the ‘palm’ domain containing the active site with highly conserved motifs responsible for catalysis; and the ‘thumb’, which maintains the enzyme's interaction with the nucleic acid substrate.
  • RTs also have distinct polymerase and RNase H domains, where the polymerase domain is responsible for nucleic acid molecule (e.g., DNA) synthesis, and the RNase H domain degrades the RNA strand in RNA-DNA hybrids to allow second-strand DNA synthesis.
  • RTs are important tools in molecular biology and biotechnology, with uses in RT-PCR, cDNA synthesis from RNA, amplification and quantification of RNA, RNA Sequencing (RNA-Seq), preparing cDNA libraries from RNA samples, and gene cloning and expression studies.
  • a polymerase (e.g., reverse transcriptase) of the disclosure includes but is not limited to the polymerases (e.g., reverse transcriptases) described herein and variants or fragments thereof, including but not limited to a reverse transcriptase comprising an amino acid sequence having at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least
  • An RT of the presently disclosed compositions and methods can lack an RNase H domain.
  • a “polymerase editor” or “PE” refers to a protein or a plurality of proteins comprising an RGN polypeptide and a polymerase editing polypeptide (e.g., DNA polymerase or reverse transcriptase) that, along with a polymerase editing guide RNA (PEgRNA) that comprises an extension arm comprising a primer binding site (PBS) and a DNA synthesis template comprising a desired edit, is capable of editing a double-stranded polynucleotide through the replacement of a target sequence using the DNA synthesis template as a template for the polymerase.
  • PEgRNA polymerase editing guide RNA
  • the RGN polypeptide and the polymerase editing polypeptide are operably linked (by fusion or insertion). In other embodiments, the RGN polypeptide and the polymerase editing polypeptide (e.g., DNA polymerase or reverse transcriptase) are not operably linked. In one particular embodiment, the RGN polypeptide and the polymerase editing polypeptide (e.g., DNA polymerase or reverse transcriptase) are two separate polypeptides.
  • the PE does not require the introduction of a doublestranded break, but rather utilizes an RGN nickase that nicks the non-target strand upstream of the sequence to be edited and upstream of the PAM, creating a 3' flap on the non-target strand.
  • the PBS of the PEgRNA is complementary to the 3' flap of the non-target strand and hybridrization of the PBS and 3' flap of the non- target strand allows for the polymerization of the replacement strand containing the edit using the DNA synthesis template and polymerase.
  • RT editors that utilize a reverse transcriptase as the polymerase are referred to herein as “RT editors” or “RTEs”.
  • the presently disclosed polymerase editors comprise a polymerase (e.g., RT) and an engineered variant LPG10145 RGN polypeptide.
  • the RT includes RTs, or active variants or fragments thereof, as described herein, including but not limited to an RT comprising an amino acid sequence having at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to the amino acid sequence set forth as SEQ ID NO: 254 or 255.
  • the engineered variant LPG10145 RGN polypeptides, or active variants or fragments thereof, include those described herein.
  • the binding and/or cleaving activity of an active variant or fragment of a variant LPG10145 RGN disclosed herein can be dependent upon recognizing a protospacer adjacent motif (PAM) adjacent and 3’ to the target sequence.
  • PAM protospacer adjacent motif
  • the PAM comprises a consensus sequence of NNGG.
  • the variant LPG10145 RGNs comprise an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions differs from the corresponding amino acid residue in SEQ ID NO: 1: 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975, and active fragments or variants thereof that retain the ability to bind to a target nucleotide sequence in an RNA- guided sequence -specific manner.
  • the variant LPG10145 RGNs comprise an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions is a positively charged amino acid residue: 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975, and active fragments or variants thereof that retain the ability to bind to a target nucleotide sequence in an RNA-guided sequence -specific manner.
  • the variant LPG10145 RGNs comprise an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions is an R: 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975, and active fragments or variants thereof that retain the ability to bind to a target nucleotide sequence in an RNA-guided sequence-specific manner.
  • the variant LPG10145 RGNs comprise an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1, wherein the amino acid residue at amino acid position 778 and/or 969 differs from the corresponding amino acid residue in SEQ ID NO: 1, and active fragments or variants thereof that retain the ability to bind to a target nucleotide sequence in an RNA-guided sequence-specific manner.
  • the variant LPG10145 RGNs comprise an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1, wherein the amino acid residue at amino acid position 778 and/or 969 is a positively charged amino acid residue, and active fragments or variants thereof that retain the ability to bind to a target nucleotide sequence in an RNA-guided sequence -specific manner.
  • the variant LPG10145 RGNs comprise an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1, wherein the amino acid residue at amino acid position 778 and/or 969 is an R, and active fragments or variants thereof that retain the ability to bind to a target nucleotide sequence in an RNA- guided sequence -specific manner.
  • the variant LPG10145 RGNs comprise an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1, wherein amino acid residues at positions 778 and 856 differ from the corresponding amino acid residues in SEQ ID NO: 1.
  • the variant LPG10145 RGNs comprise an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1, wherein amino acid residues at positions 778 and 856 are positively charged amino acid residues.
  • the variant LPG10145 RGNs comprise an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1, wherein amino acid residues at positions 55, 647, 778, and 969 differ from the corresponding amino acid residues in SEQ ID NO: 1.
  • the variant LPG10145 RGNs comprise an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1, wherein amino acid residues at positions 55, 647, 778, and 969 are positively charged amino acid residues.
  • the variant LPG10145 RGNs comprise an amino acid sequence selected from: (a) the amino acid sequence set forth as SEQ ID NO: 1, wherein E at amino acid position 52 is an R; (b) the amino acid sequence set forth as SEQ ID NO: 1, wherein S at amino acid position 55 is an R; (c) the amino acid sequence set forth as SEQ ID NO: 1, wherein G at amino acid position 86 is an R; (d) the amino acid sequence set forth as SEQ ID NO: 1, wherein Y at amino acid position 472 is an R; (e) the amino acid sequence set forth as SEQ ID NO: 1, wherein K at amino acid position 533 is an R; (f) the amino acid sequence set forth as SEQ ID NO: 1, wherein K at amino acid position 541 is an R; (g) the amino acid sequence set forth as SEQ ID NO: 1, wherein Y at amino acid position 643 is substituted by an R; (h) the amino acid sequence set forth as SEQ ID NO: 1, wherein S at amino acid position 647 is an amino acid sequence set
  • the variant LPG10145 RGNs comprise an amino acid sequence selected from: (a) the amino acid sequence set forth as SEQ ID NO: 2; (b) the amino acid sequence set forth as SEQ ID NO: 3; (c) the amino acid sequence set forth as SEQ ID NO: 4; (d) the amino acid sequence set forth as SEQ ID NO: 5; (e) the amino acid sequence set forth as SEQ ID NO: 6; (f) the amino acid sequence set forth as SEQ ID NO: 7; (g) the amino acid sequence set forth as SEQ ID NO: 8; (h) the amino acid sequence set forth as SEQ ID NO: 9; (i) the amino acid sequence set forth as SEQ ID NO: 10; (j) the amino acid sequence set forth as SEQ ID NO: 11; (k) the amino acid sequence set forth as SEQ ID NO: 12; (1) the amino acid sequence set forth as SEQ ID NO: 13; (m) the amino acid sequence set forth as SEQ ID NO: 14; (n) the amino acid sequence set forth as SEQ ID NO:
  • the presently disclosed PEs can be provided in trans, wherein the polymerase (e.g., RT) and variant LPGI0145 RGN polypeptide are separate polypeptides.
  • the polymerase (e.g., RT) and variant LPGI0145 RGN polypeptide are transcribed together and have a sequence encoding a selfcleaving peptide (e.g., 2A peptide such as P2A) in between, such that translation results in two separate polypeptides.
  • a selfcleaving peptide e.g., 2A peptide such as P2A
  • Any self-cleaving peptide known in the art can be used in such embodiments, including but not limited to, 2A peptides, which is a class of 18-22 amino acid long peptides that may function through ribosomal skipping during translation.
  • 2A peptides are T2A, P2A, E2A, and F2A.
  • the presently disclosed PEs can comprise a polymerase (e.g., RT) operably fused to a variant LPGI0145 RGN polypeptide, wherein the RT and variant LPGI0145 RGN polypeptide are fused to each other end-to-end or wherein the polymerase (e.g., RT) is inserted into the variant LPG10145 RGN polypeptide, such as those inlaid base editors described in International Appl. Publ. No. WO 2024/095245, which is herein incorporated by reference in its entirety.
  • a polymerase e.g., RT
  • the polymerase e.g., RT
  • the polymerase can be fused to the amino terminus of the variant LPG10145 RGN polypeptide or the carboxy terminus of the variant LPG10145 RGN polypeptide.
  • the presently disclosed PEs can comprise from amino terminus to carboxy terminus: the polymerase (e.g., RT) and the variant LPG10145 RGN polypeptide; or the variant LPG10145 RGN polypeptide and the polymerase (e.g., RT).
  • the polymerase e.g., RT
  • the polymerase is inserted between surface amino acid residues of the variant LPG10145 RGN polypeptide.
  • the polymerase e.g., RT
  • the RuvC domain is the RuvCIII domain.
  • a Rec or recognition lobe mediates nucleic acid binding through multiple Rec domains (e.g., Recl-3) by sensing nucleic acids, regulates the HNH conformational transition, and locks the catalytic HNH domain at the cleavage site.
  • a wedge domain is responsible for the recognition of guide RNA scaffolds.
  • a PAM-interacting domain is the domain of an RGN polypeptide that binds to a PAM site.
  • Non-limiting examples of domains within LPG10145 RGN (SEQ ID NO: 1) or a variant LPG10145 RGN (SEQ ID NOs: 2-16, 124-126, 182-196, and 271-285) include: RuvC -I from amino acid residues 1 to 42; BH from amino acid residues 43 to 79; RECI from amino acid residues 80 to 236; REC2 from amino acid residues 237 to 476; RuvC-II from amino acid residues 477 to 524; LI from amino acid residues 525 to 560; HNH from amino acid residues 561 to 676; L2 from amino acid residues 677 to 690; RuvC -III from amino acid residues 691 to 828; WED from amino acid residues 829 to 976; and PI from amino acid residues 977 to 1130.
  • the general domains of RGN polypeptides can be determined via structural comparison to RGN polypeptides with defined domains.
  • the PE comprises a variant LPG10145 RGN polypeptide comprising the amino acid sequence set forth as SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 182, 183,
  • the polymerase e.g., RT
  • the polymerase can be inserted within the variant LPG10145 RGN polypeptide, or an active variant or fragment thereof, immediately after the amino acid position selected from the group consisting of: i) amino acid position corresponding to position 347 of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 182, 183, 184,
  • the polymerase (e.g., RT) may be fused directly to the variant LPG10145 RGN polypeptide or a peptide linker can connect the polymerase (e.g., RT) and the variant LPG10145 RGN polypeptide.
  • a peptide linker can connect the polymerase (e.g., RT) and the variant LPG10145 RGN polypeptide.
  • the polymerase e.g., RT
  • there can be peptide linkers on one or both ends of the polymerase e.g., RT.
  • Any suitable peptide linker can be used to connect the polymerase (e.g., RT) and variant LPG10145 RGN polypeptide, but one suitable peptide linker comprises one or more copies of SGGS (SEQ ID NO: 241).
  • the peptide linker comprises 1 SGGS (SEQ ID NO: 241) sequence, 2 SGGS (SEQ ID NO: 241) sequences, 3 SGGS (SEQ ID NO: 241) sequences, 4 SGGS (SEQ ID NO: 241) sequences, or more, such that the linker sequence can be 4, 8, 12, or 16 amino acids long.
  • the linker between the polymerase (e.g., RT) and variant LPG10145 RGN polypeptide can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, or more amino acids in length.
  • polymerase e.g., RT
  • variant LPG10145 RGN polypeptide can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,
  • the peptide linker separating the polymerase (e.g., RT) and variant LPG10145 RGN polypeptide can also comprise an NLS, such as but not limited to those disclosed elsewhere herein, including SEQ ID NO: 36, 37, 234, or 235, wherein the NLSs can be connected by peptide linkers (such as SGGS, SEQ ID NO: 241).
  • the peptide linker separating the polymerase (e.g., RT) and variant LPG10145 RGN polypeptide comprises more than one localization sequence, such as 2, 3, or more localization sequences.
  • a peptide linker has a formula of -(SGGS) x -NLSm-(SGGS)y-NLSn-(SGGS) z -, wherein each of x, y, or z is 0, 1, 2, 3, or 4; and wherein each of m or n is 0 or 1.
  • the peptide linker has a formula of -(SGGS) x -NLSm-(SGGS)y-NLSn-(SGGS) z -, wherein each of x or y is 0, 1, 2, 3, or 4, and y is 0; and wherein one of m or n is 0, and the other is 1.
  • the peptide linker has a formula of -(SGGS) x -NLSm-(SGGS)y-NLSn-(SGGS) z -, wherein each of x, y, or z is 0, 1, 2, 3, or 4; and wherein each of m or n is 1.
  • the presently disclosed polymerases can comprise at least one nuclear localization signal (NLS) to enhance transport of the protein to the nucleus of a cell.
  • Nuclear localization signals are known in the art and generally comprise a stretch of basic amino acids (see, e.g., Lange et al., J. Biol. Chem. (2007) 282:5101-5105).
  • the polymerase e.g., RT
  • variant LPG10145 RGN polypeptide, fusion protein, or PE comprises 2, 3, 4, 5, 6 or more nuclear localization signals.
  • the nuclear localization signal(s) can be a heterologous NLS.
  • Non-limiting examples of nuclear localization signals useful for the presently disclosed polymerases are the nuclear localization signals of SV40 Large T-antigen, nucleoplasmin, and c-Myc (see, e.g., Ray et al. (2015) Bioconjug Chem 26(6): 1004-7).
  • the polymerase e.g., RT
  • variant LPG10145 RGN polypeptide, fusion protein, or PE comprises the NLS sequence set forth as SEQ ID NO: 36, 37, 234, and/or 235.
  • the polymerase (e.g., RT), variant LPG10145 RGN polypeptide, fusion protein, or PE can comprise one or more NLS sequences at its N-terminus, C- terminus, or both the N-terminus and C-terminus.
  • the polymerase (e.g., RT), variant LPG10145 RGN polypeptide, fusion protein, or PE can comprise two NLS sequences at the N-terminal region and four NLS sequences at the C-terminal region.
  • a peptide linker can connect the NLS to the polymerase (e.g., RT), variant LPG10145 RGN polypeptide, or fusion protein.
  • RTs polymerases
  • variant LPG10145 RGN polypeptides fusion proteins
  • PEs polymerases
  • plastid localization sequences mitochondrial localization sequences
  • dual-targeting signal sequences that target to both the plastid and mitochondria
  • Polymerases e.g., RTs
  • variant LPG10145 RGN polypeptides, fusion proteins, or PEs can comprise at least one cell-penetrating domain that facilitates cellular uptake of the polymerases (e.g., RTs), variant LPG10145 RGN polypeptides, fusion proteins, or PEs.
  • Cell-penetrating domains are known in the art and generally comprise stretches of positively charged amino acid residues (i.e., polycationic cell-penetrating domains), alternating polar amino acid residues and non-polar amino acid residues (i.e., amphipathic cellpenetrating domains), or hydrophobic amino acid residues (i.e., hydrophobic cell-penetrating domains) (see, e.g., Milletti F. (2012) Drug Discov Today 17:850-860).
  • a non-limiting example of a cell-penetrating domain is the trans-activating transcriptional activator (TAT) from the human immunodeficiency virus 1.
  • TAT trans-activating transcriptional activator
  • the nuclear localization signal, plastid localization signal, mitochondrial localization signal, dualtargeting localization signal, and/or cell-penetrating domain can be located at the amino-terminus (N- terminus), the carboxyl -terminus (C-terminus), and/or in an internal location of the polymerase (e.g., RT), variant LPG10145 RGN polypeptide, fusion protein, or PE.
  • the polymerase e.g., RT
  • variant LPG10145 RGN polypeptide e.g., fusion protein, or PE.
  • Polymerases e.g., RTs
  • variant LPG10145 RGN polypeptides fusion proteins
  • PEs can also comprise a purification tag, which is any molecule that can be utilized to isolate a protein or fused protein from a mixture (e.g., biological sample, culture medium).
  • purification tags include biotin, myc, maltose binding protein (MBP), glutathione-S-transferase (GST), and 3X FLAG tag.
  • a PE of the present disclosure can comprise an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to the amino acid sequence set forth as any one of SEQ ID NOs: 135, and 162-181.
  • a PE of the disclosure comprises the amino acid sequence set forth as any one of SEQ ID NOs: 135, and 162-181.
  • a PE system utilizes a polymerase editing guide RNA (“PEgRNA”).
  • the PEgRNA is a guide RNA that both specifies the target sequence and provides the template for polymerization of the replacement strand containing a desired edit by way of an extension engineered onto the RGN guide RNA or a part thereof, referred to herein as an extension arm.
  • the PEgRNA can be a single guide RNA, wherein the extension arm can be at the 5' or 3' end, or at an internal portion of the guide RNA, or multiple polynucleotides (e.g., a dual guide RNA).
  • the extension arm can be at the 5' or 3' end, or at an internal portion of the crRNA or tracrRNA molecule.
  • the template for polymerization within an extension arm is referred to herein as the DNA synthesis template.
  • the DNA synthesis template can be referred to as the reverse transcriptase template (RTT).
  • the RGN is guided to the target sequence by the PEgRNA and in those embodiments wherein the RGN is a nickase with an inactivated HNH domain and active RuvC domain, the RGN nickase nicks the non-target strand upstream of the sequence to be edited and upstream of the PAM, creating a 3' flap on the non-target strand.
  • the PEgRNA includes a primer binding site (PBS) that is complementary to the 3' flap of the non-target strand.
  • the PBS can be at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length.
  • the PEgRNA comprises a PBS that is at least 5 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) nucleotides in length.
  • the PEgRNA may comprise a PBS that is 9, 11, 12, 13, 15, or 18 nucleotides in length.
  • Hybridization of the PBS and 3' flap of the non-target strand allows polymerization of the replacement strand containing the edit using the DNA synthesis template in the extension of the PEgRNA.
  • the DNA synthesis template can be at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more nucleotides in length.
  • the PEgRNA comprises a DNA synthesis template that is at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, or at least 50 nucleotides in length.
  • the PEgRNA comprises a DNA synthesis template that is 19, 22, 23, 24, 25, 26, 29, 30, 32, 34, 37, 38, 39, 40, 42, or 46 nucleotides in length.
  • the DNA synthesis template comprises the desired edit, which can be a substitution of one or more nucleotides, a deletion of one or more nucleotides, or an addition of one or more nucleotides.
  • the extension arm of the PEgRNA can be formed from RNA or DNA.
  • the polymerase of the polymerase editor can be an RNA-dependent DNA polymerase (such as a reverse transcriptase).
  • the polymerase of the polymerase editor may be a DNA-dependent DNA polymerase.
  • a guide RNA of the disclosure comprises an extension comprising an edit template for polymerase editing.
  • An RTT of the present disclosure can comprise a nucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to the nucleotide sequence set forth as any one of SEQ ID NOs: 226- 233.
  • an RTT of the present disclosure comprises the nucleotide sequence set forth as any one of SEQ ID NOs: 226-233.
  • a PBS of the present disclosure can comprise a nucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to the nucleotide sequence set forth as any one of SEQ ID NOs: 221-223.
  • a PBS of the present disclosure comprises the nucleotide sequence set forth as any one of SEQ ID NOs: 221-223.
  • a PEgRNA of the present disclosure can comprise a nucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to the nucleotide sequence set forth as any one of SEQ ID NOs: 129-132, and 197-220.
  • a PEgRNA of the present disclosure comprises the nucleotide sequence set forth as any one of SEQ ID NOs: 129-132, and 197-220.
  • a nicking guide RNA In order to reduce the possibility that the edit introduced by a polymerase editor is removed due to mismatch repair of the edited strand, a nicking guide RNA can be used.
  • a “nicking guide RNA” is a guide RNA that targets a sequence within the unedited strand at a site nearby and opposite to the original nick and guides the RGN nickase of the PE system to this unedited strand to introduce a single -stranded nick.
  • the nicking guide RNA can be designed to match the edited sequence introduced by the PEgRNA, but not the original unedited sequence, to ensure that the nicking occurs after the editing event on the non-target strand takes place.
  • RNA-guided nucleases Encoding RNA-guided nucleases, base editing polypeptides, polymerases, fusion proteins, base editors, polymerase editors, CRISPR RNA, tracrRNA, and/or guide RNA
  • the present disclosure provides polynucleotides comprising the presently disclosed CRISPR RNAs, tracrRNAs, and/or sgRNAs and polynucleotides comprising a nucleotide sequence encoding the presently disclosed variant LPG10145 RGNs, CRISPR RNAs, tracrRNAs, sgRNAs, and/or PEgRNAs.
  • Systems of the disclosure can comprise polynucleotides comprising or encoding guide RNAs or PEgRNAs and polynucleotides comprising a nucleotide sequence encoding variant LPG10145 RGN polypeptides, fusion proteins comprising the same, polymerases (e.g., RTs), base editing polypeptides, base editors, and/or PEs.
  • polymerases e.g., RTs
  • base editing polypeptides e.g., base editors, and/or PEs.
  • polynucleotides include those comprising or encoding a CRISPR repeat comprising the nucleotide sequence set forth as SEQ ID NO: 33, 244, or 245, or an active variant or fragment thereof that when comprised within a guide RNA is capable of directing the sequence -specific binding of an associated RNA-guided nuclease to a target sequence of interest.
  • polynucleotides comprising or encoding a tracrRNA comprising the nucleotide sequence set forth as SEQ ID NO: 34, 246, 247, or 248, or an active variant or fragment thereof that when comprised within a guide RNA is capable of directing the sequence -specific binding of an associated RNA-guided nuclease to a target sequence of interest.
  • Polynucleotides are also provided that encode an RNA-guided nuclease comprising the amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions differs from the corresponding amino acid residue in SEQ ID NO: 1: 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975.
  • Polynucleotides are provided that encode an RNA-guided nuclease comprising the amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions is a positively charged amino acid residue: 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975.
  • Polynucleotides are provided that encode an RNA-guided nuclease comprising the amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions is an R: 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975.
  • Polynucleotides are also provided that encode an RNA-guided nuclease comprising the amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at amino acid position 778 and/or 969 differs from the corresponding amino acid residue in SEQ ID NO: 1.
  • Polynucleotides are provided that encode an RNA-guided nuclease comprising the amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at amino acid position 778 and/or 969 is a positively charged amino acid residue.
  • Polynucleotides are provided that encode an RNA-guided nuclease comprising the amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at amino acid position 778 and/or 969 is an R.
  • Polynucleotides are also provided that encode an RNA-guided nuclease comprising the amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein amino acid residues at positions 778 and 856 differ from the corresponding amino acid residues in SEQ ID NO: 1.
  • polynucleotides are provided that encode an RNA-guided nuclease comprising the amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residues at positions 778 and 856 are positively charged amino acid residues.
  • Polynucleotides are also provided that encode an RNA-guided nuclease comprising the amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein amino acid residues at positions 55, 647, 778, and 969 differ from the corresponding amino acid residues in SEQ ID NO: 1.
  • polynucleotides are also provided that encode an RNA-guided nuclease comprising the amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein amino acid residues at positions 55, 647, 778, and 969 are positively charged amino acid residues.
  • a polynucleotide that encodes an RNA-guided nuclease comprising the amino acid sequence selected from: (a) the amino acid sequence set forth as SEQ ID NO: 1, wherein E at amino acid position 52 is an R; (b) the amino acid sequence set forth as SEQ ID NO: 1, wherein S at amino acid position 55 is an R; (c) the amino acid sequence set forth as SEQ ID NO: 1, wherein G at amino acid position 86 is an R; (d) the amino acid sequence set forth as SEQ ID NO: 1, wherein Y at amino acid position 472 is an R; (e) the amino acid sequence set forth as SEQ ID NO: 1, wherein K at amino acid position 533 is an R; (f) the amino acid sequence set forth as SEQ ID NO: 1, wherein K at amino acid position 541 is an R; (g) the amino acid sequence set forth as SEQ ID NO: 1, wherein Y at amino acid position 643 is substituted by an R; (h) the amino acid sequence set forth as
  • a polynucleotide that encodes an RNA-guided nuclease comprising the amino acid sequence selected from: (a) the amino acid sequence set forth as SEQ ID NO: 2; (b) the amino acid sequence set forth as SEQ ID NO: 3; (c) the amino acid sequence set forth as SEQ ID NO: 4; (d) the amino acid sequence set forth as SEQ ID NO: 5; (e) the amino acid sequence set forth as SEQ ID NO: 6; (f) the amino acid sequence set forth as SEQ ID NO: 7; (g) the amino acid sequence set forth as SEQ ID NO: 8; (h) the amino acid sequence set forth as SEQ ID NO: 9; (i) the amino acid sequence set forth as SEQ ID NO: 10; (j) the amino acid sequence set forth as SEQ ID NO: 11; (k) the amino acid sequence set forth as SEQ ID NO: 12; (1) the amino acid sequence set forth as SEQ ID NO: 13; (m) the amino acid sequence set forth as SEQ ID NO
  • polynucleotide or “nucleic acid molecule” is not intended to limit the present disclosure to polynucleotides comprising DNA.
  • polynucleotides can comprise ribonucleotides (RNA) and combinations of ribonucleotides and deoxyribonucleotides.
  • RNA ribonucleotides
  • deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. These include peptide nucleic acids (PNAs), PNA-DNA chimers, locked nucleic acids (LNAs), and phosphothiorate linked sequences.
  • PNAs peptide nucleic acids
  • LNAs locked nucleic acids
  • the polynucleotides disclosed herein also encompass all forms of sequences including, but not limited to, single -stranded forms, double -stranded forms, DNA-RNA hybrids, triplex structures, stem-and-loop structures, and the like.
  • the polynucleotide encoding a presently disclosed variant LPG10145 RGN polypeptide, fusion protein comprising the same, polymerase (e.g., RT), base editing polypeptides, base editors, and/or PE is an mRNA (messenger RNA) molecule.
  • An mRNA refers to any polynucleotide which encodes a polypeptide of interest and which is capable of being translated to produce the encoded polypeptide of interest in vitro, in vivo, in situ, or ex vivo.
  • the basic components of an mRNA molecule include at least a coding region, a 5'UTR, a 3 'UTR, a 5' cap and a poly-A tail.
  • a 5' UTR situated 5' of a coding sequence and transcribed as part of an mRNA, may comprise various regulatory elements, including, e.g., 5' cap structure, G-quadruplex structure (G4), stem-loop structure, and internal ribosome entry sites (IRES), which can control translation initiation of the mRNA.
  • G4 G-quadruplex structure
  • IRS internal ribosome entry sites
  • a 3' UTR, situated 3' of a coding sequence and transcribed as part of an mRNA can be involved in numerous regulatory processes including transcript cleavage, stability and polyadenylation, translation, and mRNA localization.
  • the 3' UTR can serve as a binding site for numerous regulatory proteins and small non-coding RNAs, e.g., microRNAs.
  • a 5' UTR and/or a 3' UTR heterologous to an mRNA originates from an organism or species that is different from that of the mRNA, or if from the same organism or species as the mRNA, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.
  • inclusion of a 5' UTR and/or a 3' UTR heterologous to an mRNA encoding a variant LPG10145 RGN polypeptide, fusion protein comprising same, polymerase (e.g., RT), and/or PE of the disclosure improves polypeptide synthesis from the mRNA in a tissue (e.g., liver, or cells in vitro, such as stem cells, hepatocytes or lymphocytes).
  • Heterologous 5' UTRs and/or 3' UTRs may, for example, increase protein synthesis by increasing the time that the mRNA remains in translating polysomes (message stability) and/or the rate at which ribosomes initiate translation on the mRNA (message translation efficiency).
  • inclusion of a 5' UTR and/or a 3' UTR heterologous to an mRNA encoding variant LPG10145 RGN polypeptide, fusion protein comprising the same, polymerase (e.g., RT), base editing polypeptides, base editors, and/or PE of the disclosure can lead to prolonged and/or increased polypeptide synthesis, enabling improved editing of a target polynucleotide by the PE or PE system.
  • the enhanced polypeptide synthesis from an mRNA occurs in a tissue-specific manner.
  • Heterologous UTR sequences are described, for example, in US 2023/0050143 and US 2017/0252461.
  • an mRNA encoding a variant LPG10145 RGN, fusion protein comprising the same, polymerase (e.g., RT), base editing polypeptides, base editors, and/or PE useful in the presently disclosed methods and compositions can include one or more structural and/or chemical modifications or alterations which impart useful properties to the polynucleotide.
  • a useful property of an mRNA includes the lack of a substantial induction of the innate immune response of a cell into which the mRNA is introduced.
  • a “structural” feature or modification is one in which two or more linked nucleotides are inserted, deleted, duplicated, inverted or randomized in an mRNA without significant chemical modification to the nucleotides themselves.
  • Chemical modifications to mRNA can involve inclusion of 5 -methylcytosine, Nl-methyl-pseudouridine, pseudouridine, 2-thiouridine, 4-thiouridine, 5-methoxyuridine, 2'Fluoroguanosine, 2'Fluorouridine, 5- bromouridine, 5-(2-carbomethoxyvinyl) uridine, 5-[3(l-E-propenylamino)] uridine, a-thiocytidine, N6- methyladenosine, 5 -methylcytidine, N4-acetylcytidine, 5 -formylcytidine, or combinations thereof, in an mRNA.
  • nucleic acid molecules encoding variant LPG10145 RGNs, fusion proteins comprising the same, polymerases (e.g., RTs), base editing polypeptides, base editors, and/or PEs can be codon optimized for expression in an organism of interest.
  • a "codon-optimized” coding sequence is a polynucleotide coding sequence having its frequency of codon usage designed to mimic the frequency of preferred codon usage or transcription conditions of a particular host cell. Expression in the particular host cell or organism is enhanced as a result of the alteration of one or more codons at the nucleic acid level such that the translated amino acid sequence is not changed.
  • Nucleic acid molecules can be codon optimized, either wholly or in part.
  • Codon tables and other references providing preference information for a wide range of organisms are available in the art (see, e.g., Campbell and Gowri (1990) Plant Physiol. 92: 1-11 for a discussion of plantpreferred codon usage). Methods are available in the art for synthesizing plant-preferred genes or mammalian (for example human) codon-optimized coding sequences. See, for example, U.S. Patent Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference.
  • Polynucleotides encoding the variant LPG10145 RGNs, fusion proteins comprising the same, polymerases (e.g., RTs), base editing polypeptides, base editors, PEs, crRNAs, tracrRNAs, sgRNAs, and/or PEgRNAs provided herein can be provided in expression cassettes for in vitro expression or expression in a cell, organelle, embryo, or organism of interest.
  • the cassette will include 5' and 3' regulatory sequences operably linked to a polynucleotide encoding a variant LPG10145 RGN, fusion protein comprising the same, polymerase (e.g., RT), base editing polypeptides, base editors, PE, crRNA, tracrRNAs, sgRNAs, and/or PEgRNAs provided herein that allows for expression of the polynucleotide.
  • the cassette may additionally contain at least one additional gene or genetic element to be cotransformed into the organism. Where additional genes or elements are included, the components are operably linked.
  • the term “operably linked” is intended to mean a functional linkage between two or more elements.
  • an operable linkage between a promoter and a coding region of interest is a functional link that allows for expression of the coding region of interest.
  • Operably linked elements may be contiguous or non-contiguous.
  • polypeptides that are “operably fused” or “operably linked” means that the structure and/or biological activity of each individual peptide is also present in the fusion.
  • the additional gene(s) or element(s) can be provided on multiple expression cassettes.
  • the nucleotide sequence encoding a presently disclosed variant LPG10145 RGN, fusion protein comprising the same, polymerase (e.g., RT), base editing polypeptide, base editor, or PE can be present on one expression cassette, whereas the nucleotide sequence encoding a crRNA, tracrRNA, or complete guide RNA (or PEgRNA) can be on a separate expression cassette.
  • Such an expression cassette is provided with a plurality of restriction sites and/or recombination sites for insertion of the polynucleotides to be under the transcriptional regulation of the regulatory regions.
  • the expression cassette may additionally contain a selectable marker gene.
  • the expression cassette will include in the 5'-3' direction of transcription, a transcriptional (and, in some embodiments, translational) initiation region (z.e., a promoter), a variant LPG10145 RGN-, a fusion protein-, a polymerase- (e.g., RT-), base editing polypeptide-, base editor-, PE-, crRNA-, tracrRNA-, sgRNA-, and/or PEgRNA- encoding polynucleotide of the invention, and a transcriptional (and in some embodiments, translational) termination region (i. e. , termination region) functional in the organism of interest.
  • the promoters of the invention are capable of directing or driving expression of a coding sequence in a host cell.
  • the regulatory regions may be endogenous or heterologous to the host cell or to each other.
  • heterologous in reference to a regulatory region that is heterologous to another regulatory region or to the host cell, is a regulatory region that is not found with another regulatory region or in the host cell in nature.
  • the heterologous regulatory region can originate from a foreign species or from the same species.
  • the heterologous regulatory region can be in its native form or is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.
  • a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence.
  • a nucleic acid molecule that is heterologous to another nucleic acid molecule is a nucleic acid molecule that is not found with another nucleic acid molecule in nature.
  • the heterologous nucleic acid molecule can originate from a foreign species or from the same species.
  • the heterologous nucleic acid molecule can be in its native form or is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.
  • an mRNA encoding a variant LPG10145 RGN polypeptide can comprise a heterologous 5’ UTR, wherein the 5’ UTR is not present as part of the mRNA encoding the variant LPG10145 RGN polypeptide in nature.
  • Convenient termination regions include ones from simian virus (SV40), human growth hormone (hGH), bovine growth hormone (BGH), and rabbit beta-globin (rbGlob). See also Proudfoot (1991) Cell 64:671-674; Munroe et al. (1990) Gene 91: 151-158; Schek et al. (1992) Molecular and Cellular Biology 12( 12): 5386-5393; Gil and Proudfoot (1987) Cell 49(3)399-406; Goodwin and Rottman (1992) The Journal of Biological Chemistry 267(23): 16330-16334; and Lanoix and Acheson (1988) EMBO J. 7(8): 2515-2522. Additional termination regions are available from the Ti-plasmid of A.
  • SV40 simian virus
  • hGH human growth hormone
  • BGH bovine growth hormone
  • rbGlob rabbit beta-globin
  • iumefaciens such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262: 141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5: 141-149; Mogen et a/. (1990) Plant Cell 2: 1261-1272; Munroe et al. (1990) Gene 91: 151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acids Res. 15:9627-9639.
  • Additional regulatory signals include, but are not limited to, transcriptional initiation start sites, operators, activators, enhancers, other regulatory elements, ribosomal binding sites, an initiation codon, termination signals, and the like. See, for example, U.S. Pat. Nos. 5,039,523 and 4,853,331; EPO 0480762A2; Sambrook et al. (1992) Molecular Cloning: A Laboratory Manual, ed. Maniatis et al. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), hereinafter "Sambrook 11"; Davis et al., eds. (1980) Advanced Bacterial Genetics (Cold Spring Harbor Laboratory Press), Cold Spring Harbor, N.Y., and the references cited therein.
  • the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame.
  • adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like.
  • in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions may be involved.
  • a number of promoters can be used in the practice of the invention.
  • the promoters can be selected based on the desired outcome.
  • the nucleic acids can be combined with constitutive, inducible, growth stage-specific, cell type-specific, tissue-preferred, tissue-specific, or other promoters for expression in the organism of interest.
  • Exemplary constitutive promoters for expression in cells of the present disclosure include: an SV40 early promoter; a mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter; a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE); a rous sarcoma virus (RSV) promoter; a human ubiquitin C promoter (UBC); a human U6 small nuclear promoter (U6); an enhanced U6 promoter; a human Hl promoter from RNA polymerase III (Hl); a human elongation factor la promoter (EF1A); a human betaactin promoter (ACTB); a human or mouse phosphoglycerate kinase 1 promoter (PGK); a chicken P-Actin promoter coupled with CMV early enhancer (CAGG); a yeast transcription elongation factor promoter (
  • constitutive promoters also include CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2: 163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et a/. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); and MAS (Velten t a/. (1984) EMBO J. 3:2723-2730).
  • CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812)
  • rice actin McElroy et al. (1990) Plant Cell 2: 163-171
  • ubiquitin Christensen et al. (1989) Plant Mol. Biol
  • inducible promoters examples include the Adhl promoter which is inducible by hypoxia or cold stress, the Hsp70 promoter which is inducible by heat stress, the PPDK promoter and the pepcarboxylase promoter which are both inducible by light. Also useful are promoters which are chemically inducible, such as the In2-2 promoter which is safener induced (U.S. Pat. No.
  • tissue-specific or tissue-preferred promoters can be utilized to target expression of an expression construct within a particular tissue.
  • the tissue-specific or tissue-preferred promoters are active in mammalian tissue.
  • tissue-specific or tissue-preferred promoters include promoters that initiate transcription preferentially in certain tissues, such as white blood cells (e.g., CD4 T cell), heart, kidney, liver, CNS, eye, pancreas, skeletal muscle, and testis.
  • the tissue-specific or tissue-preferred promoters are active in plant tissue. Examples of promoters under developmental control in plants include promoters that initiate transcription preferentially in certain tissues, such as leaves, roots, fruit, seeds, or flowers.
  • tissue specific promoter is a promoter that initiates transcription only in certain tissues. Unlike constitutive expression of genes, tissue-specific expression is the result of several interacting levels of gene regulation. As such, promoters from homologous or closely related plant species can be preferable to use to achieve efficient and reliable expression of transgenes in particular tissues.
  • the expression comprises a tissue-preferred promoter.
  • tissue preferred is a promoter that initiates transcription preferentially, but not necessarily entirely or solely in certain tissues.
  • the nucleic acid molecules encoding a variant LPGI0145 RGN, fusion protein comprising the same, polymerase (e.g., RT), base editing polypeptide, base editor, PE, crRNA, tracrRNA, sgRNA, and/or PEgRNA comprise a cell type-specific promoter.
  • a "cell type specific" promoter is a promoter that primarily drives expression in certain cell types in one or more organs. Some examples of cells in which cell type specific promoters may be primarily active include, for example, a primary cell, a neuronal cell, a glial cell, an adipocyte, a cardiomyocyte, a smooth muscle cell, a photoreceptor cell, and a retinal ganglia cell.
  • cell type specific promoters functional in plants may be primarily active include, for example, BETL cells, vascular cells in roots, leaves, stalk cells, and stem cells.
  • the nucleic acid molecules can also include cell type preferred promoters.
  • a "cell type preferred" promoter is a promoter that primarily drives expression mostly, but not necessarily entirely or solely in certain cell types in one or more organs.
  • Some examples of cells in which cell type preferred promoters may be preferentially active include, for example, a primary cell, a neuron, an adipocyte, a cardiomyocyte, a smooth muscle cell, and a photoreceptor cell.
  • plant cells in which cell type preferred promoters functional in plants may be preferentially active include, for example, BETL cells, vascular cells in roots, leaves, stalk cells, and stem cells.
  • the nucleic acid sequences encoding the variant LPGI0145 RGNs, fusion proteins comprising the same, polymerases (e.g., RTs), base editing polypeptides, base editors, PEs, crRNAs, tracrRNAs, sgRNAs, and/or PEgRNAs can be operably linked to a promoter sequence that is recognized by a phage RNA polymerase for example, for in vitro mRNA synthesis.
  • the in vv/ro-transcribcd RNA can be purified for use in the methods described herein.
  • the promoter sequence can be a T7, T3, or SP6 promoter sequence or a variation of a T7, T3, or SP6 promoter sequence.
  • the expressed protein and/or RNAs can be purified for use in the methods of genome modification described herein.
  • the polynucleotide encoding the variant LPGI0145 RGN, fusion protein comprising the same, polymerase (e.g., RT), base editing polypeptide, base editor, PE, crRNA, tracrRNA, sgRNA, and/or PEgRNA also can be linked to a polyadenylation signal (e.g., SV40 polyA signal and other signals functional in plants) and/or at least one transcriptional termination sequence.
  • the sequence encoding the RGN also can be linked to sequence(s) encoding at least one nuclear localization signal, at least one cell-penetrating domain, and/or at least one signal peptide capable of trafficking proteins to particular subcellular locations, as described elsewhere herein.
  • the polynucleotide encoding the variant LPG10145 RGN, fusion protein comprising the same, polymerase (e.g., RT), base editing polypeptide, base editor, PE, crRNA, tracrRNA, sgRNA, and/or PEgRNA can be present in a vector or multiple vectors.
  • a “vector” refers to a polynucleotide composition for transferring, delivering, or introducing a nucleic acid into a host cell. Suitable vectors include plasmid vectors, phagemids, cosmids, artificial/mini-chromosomes, transposons, and viral vectors (e.g., lentiviral vectors, adeno-associated viral vectors, baculoviral vector).
  • the vector can comprise additional expression control sequences (e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcriptional termination sequences), selectable marker sequences (e.g., antibiotic resistance genes), origins of replication, and the like. Additional information can be found in “Current Protocols in Molecular Biology” Ausubel et al., John Wiley & Sons, New York, 2003 or "Molecular Cloning: A Laboratory Manual” Sambrook & Russell, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 3rd edition, 2001.
  • additional expression control sequences e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcriptional termination sequences
  • selectable marker sequences e.g., antibiotic resistance genes
  • the vector can also comprise a selectable marker gene for the selection of transformed cells.
  • Selectable marker genes are utilized for the selection of transformed cells or tissues.
  • Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D).
  • Marker genes can include genes that allow selection for growth on a particular nutrient or substance, such as dihydrofolate reductase (DHFR; Simonsen and Levinson (1983) Proc.
  • DHFR dihydrofolate reductase
  • the expression cassette or vector comprising the polynucleotide encoding the variant LPG10145 RGN polypeptide, fusion protein comprising the same, polymerase (e.g., RT), base editing polypeptide, base editor, and/or PE can further comprise a polynucleotide encoding a crRNA and/or a tracrRNA, or the crRNA and tracrRNA combined to create a sgRNA or combined to be a part of a PEgRNA.
  • the polynucleotide sequence(s) encoding the crRNA, tracrRNA, gRNA, and/or PEgRNA can be operably linked to at least one transcriptional control sequence for expression of the crRNA, tracrRNA, gRNA, and/or PEgRNA in the organism or host cell of interest.
  • the polynucleotide encoding the crRNA, tracrRNA, gRNA, and/or PEgRNA can be operably linked to a promoter sequence that is recognized by RNA polymerase III (Pol III).
  • Suitable Pol III promoters include, but are not limited to, mammalian U6, U3, Hl, and 7SL RNA promoters and rice U6 and U3 promoters, such as the human U6 promoter set forth as SEQ ID NO: 41, as well as the promoters disclosed in International Publication No. WO 2022/261394, which is herein incorporated by reference in its entirety, including those set forth herein as SEQ ID NOs: 114-123.
  • expression constructs comprising nucleotide sequences encoding the variant LPG10145 RGNs, fusion proteins comprising the same, polymerases (e.g., RTs), base editing polypeptides, base editors, and/or PEs, crRNAs, tracrRNAs, sgRNAs, and/or PEgRNAs can be used to transform organisms of interest.
  • Methods for transformation involve introducing a nucleotide construct into an organism of interest. By "introducing” is intended to introduce the nucleotide construct to the host cell in such a manner that the construct gains access to the interior of the host cell.
  • the methods of the invention do not require a particular method for introducing a nucleotide construct to a host organism, only that the nucleotide construct gains access to the interior of at least one cell of the host organism.
  • the host cell can be a eukaryotic or prokaryotic cell.
  • the eukaryotic host cell is a plant cell, a mammalian cell, an avian cell, or an insect cell.
  • the eukaryotic cell that comprises or expresses a presently disclosed variant LPG10145 RGN, fusion protein comprising the same, polymerase (e.g., RT), base editing polypeptide, base editor, or PE, or that has been modified by a presently disclosed variant LPG10145 RGN, fusion protein comprising the same, polymerase (e.g., RT), base editing polypeptide, base editor, or PE, is a human cell.
  • the eukaryotic cell that comprises or expresses a presently disclosed variant LPG10145 RGN, fusion protein comprising the same, polymerase (e.g., RT), base editing polypeptide, base editor, or PE, or that has been modified by a presently disclosed variant LPG10145 RGN, fusion protein comprising the same, polymerase (e.g., RT), base editing polypeptide, base editor, or PE, is a primary cell.
  • the term "primary cell” refers to a cell isolated directly from a multicellular organism. Primary cells typically have undergone very few population doublings and are therefore more representative of the main functional component of the tissue from which they are derived in comparison to continuous (tumor or artificially immortalized) cell lines.
  • primary cells are cells that have been isolated and then used immediately. In other cases, primary cells cannot divide indefinitely and thus cannot be cultured for long periods of time in vitro.
  • a primary cell is a primary T cell.
  • the eukaryotic cell that comprises or expresses a presently disclosed variant LPG10145 RGN, fusion protein comprising the same, polymerase (e.g., RT), base editing polypeptide, base editor, or PE, or that has been modified by a presently disclosed variant LPG10145 RGN, fusion protein comprising the same, polymerase (e.g., RT), base editing polypeptide, base editor, or PE is a cell of hematopoietic origin, such as an immune cell (i.e., a cell of the innate or adaptive immune system) including but not limited to a B cell, a T cell, a natural killer (NK) cell, a pluripotent stem cell, an induced pluripotent stem cell, a
  • an immune cell i.e.
  • the eukaryotic cell that comprises or expresses a presently disclosed variant LPG10145 RGN, fusion protein comprising the same, polymerase (e.g., RT), base editing polypeptide, base editor, or PE, or that has been modified by a presently disclosed variant LPG10145 RGN, fusion protein comprising the same, polymerase (e.g., RT), base editing polypeptide, base editor, or PE, is an ocular cell, muscle cell (e.g., skeletal muscle cell), epithelial cell (e.g., lung epithelial cell), diseased cell (e.g., tumor cell).
  • Methods for introducing nucleotide constructs into plants and other host cells are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus- mediated methods.
  • the methods result in a transformed organism, such as a plant, including whole plants, as well as plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, propagules, embryos and progeny of the same.
  • Plant cells can be differentiated or undifferentiated (e.g. callus, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells, pollen).
  • Transgenic organisms or “transformed organisms” or “stably transformed” organisms or cells or tissues refers to organisms that have incorporated or integrated a polynucleotide encoding a variant LPG10145 RGN, fusion protein comprising the same, polymerase (e.g., RT), base editing polypeptide, base editor, PE, crRNA, tracrRNA, gRNA, and/or PEgRNA of the invention. It is recognized that other exogenous or endogenous nucleic acid sequences or DNA fragments may also be incorporated into the host cell. Agrobacterium-and biolistic-mediated transformation remain the two predominantly employed approaches for transformation of plant cells.
  • transformation of a host cell may be performed by infection, transfection, microinjection, electroporation, microprojection, biolistics or particle bombardment, electroporation, silica/carbon fibers, ultrasound mediated, PEG mediated, calcium phosphate coprecipitation, polycation DMSO technique, DEAE dextran procedure, and viral mediated, liposome mediated and the like.
  • Viral-mediated introduction of a polynucleotide encoding a variant LPG10145 RGN, fusion protein comprising the same, polymerase (e.g., RT), base editing polypeptide, base editor, PE, crRNA, tracrRNA, gRNA, and/or PEgRNA includes retroviral, lentiviral, adenoviral, and adeno-associated viral mediated introduction and expression, as well as the use of Caulimoviruses, Geminiviruses, and RNA plant viruses.
  • Transformation protocols as well as protocols for introducing polypeptides or polynucleotide sequences into plants may vary depending on the type of host cell (e.g, monocot or dicot plant cell) targeted for transformation.
  • Methods for transformation are known in the art and include those set forth in US Patent Nos: 8,575,425; 7,692,068; 8,802,934; 7,541,517; each of which is herein incorporated by reference. See, also, Rakoczy-Trojanowska, M. (2002) Cell Mol Biol Lett. 7:849-858; Jones et al. (2005) Plant Methods 1:5; Rivera et al. (2012) Physics of Life Reviews 9:308-345; Bartlett et al.
  • Transformation may result in stable or transient incorporation of the nucleic acid into the cell.
  • Stable transformation is intended to mean that the nucleotide construct introduced into a host cell integrates into the genome of the host cell and is capable of being inherited by the progeny thereof.
  • Transient transformation is intended to mean that a polynucleotide is introduced into the host cell and does not integrate into the genome of the host cell.
  • plastid transformation can be accomplished by transactivation of a silent plastid-borne transgene by tissue-preferred expression of a nuclear-encoded and plastid-directed RNA polymerase.
  • tissue-preferred expression of a nuclear-encoded and plastid-directed RNA polymerase Such a system has been reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91:7301- 7305.
  • the cells that have been transformed may be grown into a transgenic organism, such as a plant, in accordance with conventional ways. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having constitutive expression of the desired phenotypic characteristic identified.
  • Two or more generations may be grown to ensure that the polynucleotide encoding a variant LPG10145 RGN, fusion protein comprising the same, polymerase (e.g., RT), base editing polypeptide, base editor, PE, crRNA, tracrRNA, gRNA, and/or PEgRNA is stably maintained and inherited and then seeds harvested to ensure the presence of the polynucleotide encoding a variant LPG10145 RGN, fusion protein comprising the same, polymerase (e.g., RT), base editing polypeptide, base editor, PE, crRNA, tracrRNA, gRNA, and/or PEgRNAs.
  • polymerase e.g., RT
  • base editing polypeptide base editor
  • PE crRNA
  • tracrRNA gRNA
  • gRNA gRNA
  • the present invention provides a transformed plant or plant part having a nucleotide construct of the invention, for example, an expression cassette of the invention, stably incorporated into their genome.
  • seed having an expression cassette of the disclosure stably incorporated into their genome can be referred to as "transgenic seed” .
  • cells that have been transformed may be introduced into an organism. These cells could have originated from the organism, wherein the cells are transformed in an ex vivo approach. These cells can be autologous (originated and returned to the same subject), allogeneic (the donor and recipient subjects are of the same species).
  • sequences provided herein may be used for transformation of any plant species, including, but not limited to, monocots and dicots.
  • plants of interest include, but are not limited to, com (maize), sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed rape, Brassica sp., alfalfa, rye, millet, safflower, peanuts, sweet potato, cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond, oats, vegetables, ornamentals, and conifers.
  • Vegetables include, but are not limited to, tomatoes, lettuce, green beans, lima beans, peas, and members of the genus Curcumis such as cucumber, cantaloupe, and musk melon. Ornamentals include, but are not limited to, azalea, hydrangea, hibiscus, roses, tulips, daffodils, petunias, carnation, poinsettia, and chrysanthemum.
  • plants of the present invention are crop plants (for example, maize, sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, oilseed rape, etc.).
  • the term plant includes plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, and the like. Grain is intended to mean the mature seed produced by commercial growers for purposes other than growing or reproducing the species. Progeny, variants, and mutants of the regenerated plants are also included within the scope of the invention, provided that these parts comprise the introduced polynucleotides. Further provided is a processed plant product or byproduct that retains the sequences disclosed herein, including for example, soymeal.
  • polymerases e.g., RTs
  • base editing polypeptides e.g., base editors
  • PEs, crRNAs, tracrRNAs, gRNAs, and/or PEgRNAs or comprising the crRNAs, tracrRNAs, gRNAs, and/or PEgRNAs can also be used to transform any prokaryotic species, including but not limited to, archaea and bacteria (e.g., Bacillus sp., Klebsiella sp.
  • Streptomyces sp. Rhizobium sp., Escherichia sp., Pseudomonas sp., Salmonella sp., Shigella sp., Vibrio sp., Yersinia sp., Mycoplasma sp., Agrobacterium, Lactobacillus sp.).
  • polymerases e.g., RTs
  • base editing polypeptides e.g., base editors
  • PEs, crRNAs, tracrRNAs, gRNAs, and/or PEgRNAs or comprising the crRNAs, tracrRNAs, gRNAs, and/or PEgRNAs can be used to transform any eukaryotic species, including but not limited to animals (e.g., mammals, insects, fish, birds, and reptiles), fungi, amoeba, algae, and yeast.
  • Non- viral vector delivery systems include DNA plasmids, RNA (e.g. a transcript of a vector described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome.
  • Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell.
  • Methods of non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid: nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA.
  • Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam TM and LipofectinTM).
  • Cationic and neutral lipids that are suitable for efficient receptorrecognition lipofection of polynucleotides include those of Feigner, WO 91/17424; WO 91/16024. Delivery can be to cells (e.g. in vitro or ex vivo administration) or target tissues (e.g. in vivo administration).
  • lipidmucleic acid complexes including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291- 297 (1995); Behr et al., Bioconjugate Chem.
  • RNA or DNA viral based systems for the delivery of nucleic acids takes advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus.
  • Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro, and the modified cells may optionally be administered to patients (ex vivo).
  • Conventional viral based systems could include retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues.
  • retroviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system would therefore depend on the target tissue. Retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting UTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression.
  • Widely used retroviral vectors include those based upon murine leukemia virus (MuUV), gibbon ape leukemia virus (GaUV), Simian Immuno deficiency virus (SIV), human immuno deficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Viral. 66:2731-2739 (1992); Johann et al., J. Viral. 66: 1635-1640 (1992); Sommnerfelt et al., Viral. 176:58-59 (1990); Wilson et al., J. Viral. 63:2374-2378 (1989); Miller et al., 1. Viral. 65:2220-2224 (1991); WO 1994/026877).
  • MiUV murine leukemia virus
  • GaUV gibbon ape leukemia virus
  • SIV Simian Immuno deficiency virus
  • HAV human immuno deficiency virus
  • Adenoviral based systems may be used.
  • Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system.
  • Adeno-associated virus (“AAV”) vectors may also be used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93/24641; Katin, Human Gene Therapy 5:793-801 (1994);
  • AAV adeno-associated virus
  • AAV adeno-associated virus
  • AAV adeno-associated virus
  • AAV adeno-associated virus
  • Multiple serotypes of this virus are known to be suitable for gene delivery; all known serotypes can infect cells from various tissue types. At least 11, sequentially numbered, have been described.
  • Non-limiting exemplary serotypes useful in the compositions and methods disclosed herein include any of the 11 serotypes (e.g., AAV2, AAV5, AAV6, AAV8, AAV9), or variant serotypes, e.g., AAV-DJ.
  • AAV is advantageous over other viral vectors for in vivo delivery of genes (e.g., encoding gene editing components) due to their low toxicity and low probability of causing insertional mutagenesis because it typically does not integrate into the host genome.
  • AAV has a packaging limit of about 4.5 to 4.75 Kb.
  • Packaging cells are typically used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and q/J2 cells or PA317 cells, which package retrovirus.
  • Viral vectors used in gene therapy are usually generated by producing a cell line that packages a nucleic acid vector into a viral particle.
  • the vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the polynucleotide(s) to be expressed.
  • the missing viral functions are typically supplied in trans by the packaging cell line.
  • AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome.
  • Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences.
  • the cell line may also be infected with adenovirus as a helper.
  • the helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid.
  • the helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV. Additional methods for the delivery of nucleic acids to cells are known to those skilled in the art. See, for example, US20030087817, incorporated herein by reference.
  • a host cell is transiently or non-transiently transfected with one or more vectors described herein.
  • a cell is transfected as it naturally occurs in a subject.
  • a cell that is transfected is taken from a subject.
  • a cell that is transfected is a eukaryotic cell.
  • the eukaryotic cell is an animal cell (e.g., mammals, humans, insects, fish, birds, and reptiles).
  • a cell that is transfected is a human cell.
  • a cell that is transfected is a cell of hematopoietic origin, such as an immune cell (i.e., a cell of the innate or adaptive immune system) including but not limited to a B cell, a T cell, a natural killer (NK) cell, a pluripotent stem cell, an induced pluripotent stem cell, a chimeric antigen receptor T (CAR-T) cell, a monocyte, a macrophage, and a dendritic cell.
  • an immune cell i.e., a cell of the innate or adaptive immune system
  • a B cell i.e., a cell of the innate or adaptive immune system
  • NK natural killer
  • pluripotent stem cell i.e., an induced pluripotent stem cell
  • CAR-T chimeric antigen receptor T
  • the cell is derived from cells taken from a subject, such as a cell line.
  • the cell or cell line is prokaryotic.
  • the cell or cell line is eukaryotic.
  • the cell or cell line may be mammalian, such as for example human, monkey, mouse, cow, swine, goat, hamster, rat, cat, or dog.
  • the cell or cell line is derived from insect, avian, plant, or fungal species. A wide variety of cell lines for tissue culture are known in the art.
  • cell lines include, but are not limited to, C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLaS3, Huhl, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panel, PC-3, TF1, CTLL-2, CIR, Rat6, CVI, RPTE, A1O, T24, 182, A375, ARH-77, Calul, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI- 231, HB56, TIB55, lurkat, 145.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4.
  • a cell transfected with one or more polynucleotides or vectors described herein is used to establish a new cell line comprising one or more vector-derived sequences.
  • a cell transiently transfected with the components of an RGN, base editing, or PE system as described herein (such as by transient transfection of one or more vectors, or transfection with RNA), and modified through the activity of an RGN, base editing, or PE system, is used to establish a new cell line comprising cells containing the modification but lacking any other exogenous sequence.
  • cells transiently or non-transiently transfected with one or more vectors described herein, or cell lines derived from such cells are used in assessing one or more test compounds.
  • one or more vectors described herein are used to produce a non-human transgenic animal or transgenic plant.
  • the transgenic animal is a mammal, such as a mouse, rat, hamster, rabbit, cow, or pig.
  • the transgenic animal is a bird, such as a chicken or a duck.
  • the transgenic animal is an insect, such as a mosquito or a tick.
  • the present disclosure provides active engineered variants of a naturally-occurring (i. e. , wild-type) LPG10145 RNA-guided nuclease.
  • the wild-type LPG10145 RGN comprises the amino acid sequence set forth as SEQ ID NO: 1.
  • a variant LPG10145 RGN of the disclosure comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions differs from the corresponding amino acid residue in SEQ ID NO: 1 at one or more corresponding amino acid residues.
  • variant LPG10145 RGN that “comprises an amino acid sequence having at least x% (e.g., 85%) sequence identity to SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions differs”, it is intended to mean that the variant LPG10145 RGN comprises an amino acid sequence that has at least x% (e.g., 85%) sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, and the amino acid residues at the recited positions differ from the corresponding amino acid residues in SEQ ID NO: 1.
  • variant LPG10145 RGN that “comprises an amino acid sequence set forth as SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions differs”, it is intended to mean that the variant LPG10145 RGN comprises an amino acid sequence that is identical to the amino acid sequence set forth in SEQ ID NO: 1, except that amino acid residues at the recited positions differ from the corresponding amino acid residues in SEQ ID NO: 1.
  • a variant LPG10145 RGN of the disclosure comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions differs from the corresponding amino acid residue in SEQ ID NO: 1: 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975.
  • a variant LPG10145 RGN of the disclosure comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions is a positively charged amino acid residue: 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975.
  • a variant LPG10145 RGN of the disclosure comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions is an R: 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975.
  • a variant LPG10145 RGN of the disclosure comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at amino acid position 778 and/or 969 differs from the corresponding amino acid residue in SEQ ID NO: 1.
  • a variant LPG10145 RGN of the disclosure comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at amino acid position 778 and/or 969 is a positively charged amino acid residue.
  • a variant LPG10145 RGN of the disclosure comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at amino acid position 778 and/or 969 is an R.
  • a variant LPG10145 RGN of the disclosure comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein amino acid residues at positions 778 and 856 differ from the corresponding amino acid residues in SEQ ID NO: 1. In some embodiments, a variant LPG10145 RGN of the disclosure comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residues at positions 778 and 856 are positively charged amino acid residues.
  • a variant LPG10145 RGN of the disclosure comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein amino acid residues at positions 55, 647, 778, and 969 differ from the corresponding amino acid residues in SEQ ID NO: 1. In some embodiments, a variant LPG10145 RGN of the disclosure comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein amino acid residues at positions 55, 647, 778, and 969 are positively charged amino acid residues.
  • a variant LPG10145 RGN of the disclosure comprises an amino acid sequence selected from: (a) the amino acid sequence set forth as SEQ ID NO: 1, wherein E at amino acid position 52 is an R; (b) the amino acid sequence set forth as SEQ ID NO: 1, wherein S at amino acid position 55 is an R; (c) the amino acid sequence set forth as SEQ ID NO: 1, wherein G at amino acid position 86 is an R; (d) the amino acid sequence set forth as SEQ ID NO: 1, wherein Y at amino acid position 472 is an R; (e) the amino acid sequence set forth as SEQ ID NO: 1, wherein K at amino acid position 533 is an R; (f) the amino acid sequence set forth as SEQ ID NO: 1, wherein K at amino acid position 541 is an R; (g) the amino acid sequence set forth as SEQ ID NO: 1, wherein Y at amino acid position 643 is substituted by an R; (h) the amino acid sequence set forth as SEQ ID NO: 1, wherein S at amino acid position 6
  • a variant LPG10145 RGN of the disclosure comprises an amino acid sequence selected from: (a) the amino acid sequence set forth as SEQ ID NO: 2; (b) the amino acid sequence set forth as SEQ ID NO: 3; (c) the amino acid sequence set forth as SEQ ID NO: 4; (d) the amino acid sequence set forth as SEQ ID NO: 5; (e) the amino acid sequence set forth as SEQ ID NO: 6; (f) the amino acid sequence set forth as SEQ ID NO: 7; (g) the amino acid sequence set forth as SEQ ID NO: 8; (h) the amino acid sequence set forth as SEQ ID NO: 9; (i) the amino acid sequence set forth as SEQ ID NO: 10; (j) the amino acid sequence set forth as SEQ ID NO: 11; (k) the amino acid sequence set forth as SEQ ID NO: 12; (1) the amino acid sequence set forth as SEQ ID NO: 13; (m) the amino acid sequence set forth as SEQ ID NO: 14; (n) the amino acid sequence set forth as SEQ ID NO:
  • the present disclosure provides active variants and fragments of naturally- occurring CRISPR repeats, such as the sequence set forth as SEQ ID NO: 33, 244, or 245, and active variants and fragments of naturally-occurring tracrRNAs, such as the sequence set forth as SEQ ID NO: 34, 246, 247, or 248, and polynucleotides encoding the same.
  • a variant or fragment While the activity of a variant or fragment may be altered compared to the polynucleotide or polypeptide of interest, the variant and fragment should retain the functionality of the polynucleotide or polypeptide of interest. For example, a variant or fragment may have increased activity, decreased activity, different spectrum of activity or any other alteration in activity when compared to the polynucleotide or polypeptide of interest.
  • Variant LPG10145 RGN polypeptides such as those disclosed herein, will retain sequence-specific, RNA-guided DNA-binding activity.
  • fragments and variants of variant LPG10145 RGN polypeptides, such as those disclosed herein, will retain nuclease activity (single-stranded or double-stranded).
  • the binding and/or cleaving activity of an active variant or fragment of a variant LPG10145 RGN disclosed herein can be dependent upon recognizing a protospacer adjacent motif (PAM) adjacent and 3’ to the target sequence.
  • PAM protospacer adjacent motif
  • the PAM comprises a consensus sequence of NNGG.
  • the present disclosure provides an active variant or fragment of a variant LPG10145 RGN that has increased nuclease activity as compared to the RGN of SEQ ID NO: 1.
  • an active variant or fragment of a variant LPG10145 RGN disclosed herein comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NOs: 2-16, 182-196, and 271-285 has increased nuclease activity as compared to the RGN of SEQ ID NO: 1.
  • an active variant or fragment of a variant LPG10145 RGN disclosed herein comprising the amino acid sequence of any one of SEQ ID NOs: 2-16, 182-196, and 271-285 has increased nuclease activity as compared to the RGN of SEQ ID NO: 1.
  • an active variant or fragment of a variant LPG10145 RGN disclosed herein has nuclease activity that is from about 80% to about 500%, from about 80% to about 200%, or from about 90% to about 150%, or from about 95% to about 120%, or is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least
  • a reference LPG10145 RGN has the amino acid sequence set forth as SEQ ID NO: 1.
  • a reference LPG10145 RGN is a variant of SEQ ID NO: 1 that lacks the corresponding mutations of the active variant or fragment thereof that has nuclease activity, e.g., as described above.
  • a reference LPG10145 RGN is a non-identical variant LPG10145 RGN.
  • a reference LPG10145 RGN is a non-identical variant LPG10145 RGN.
  • Nuclease activity can be measured by assays known to one of skill in the art, including but not limited to, Tracking of Indels by DEcomposition (TIDE) analysis, flow cytometry, in vitro or in vivo cleavage assays wherein cleavage is confirmed using PCR, sequencing, or gel electrophoresis, with or without the attachment of an appropriate label (e.g., radioisotope, fluorescent substance) to the target sequence to facilitate detection of degradation products.
  • an appropriate label e.g., radioisotope, fluorescent substance
  • NTEXPAR nicking triggered exponential amplification reaction
  • NTEXPAR nicking triggered exponential amplification reaction
  • the efficiency of cleaving a target sequence is assessed by measuring the percentage of a target sequence or cells comprising the target sequence that comprise altered expression of the target sequence or of a polypeptide encoded by the target sequence.
  • the expression is measured by quantitative PCR, microarray, RNA-seq, flow cytometry, immunoblot, enzyme-linked immunosorbent assay (ELISA), protein immunoprecipitation, immunostaining, high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC/MS), mass spectrometry, or a combination thereof.
  • the target sequence encodes a cell surface expressed protein
  • the efficiency of cleaving the target sequence is assessed by measuring the percentage of cells comprising a reduction of the cell surface expressed protein as measured by flow cytometry.
  • an active variant or fragment of a variant LPG10145 RGN disclosed herein when operably fused to a deaminase, has increased base editing activity as compared to the RGN of SEQ ID NO: 1.
  • an active variant or fragment of a variant LPG10145 RGN disclosed herein comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NOs: 2-16, 182-196, and 271-285, when operably fused to a deaminase, has increased base editing activity as compared to the RGN of SEQ ID NO: 1.
  • an active variant or fragment of a variant LPG10145 RGN disclosed herein comprising the amino acid sequence of any one of SEQ ID NOs: 2-16, 182-196, and 271-285, when operably fused to a deaminase, has increased base editing activity as compared to the RGN of SEQ ID NO: 1.
  • an active variant or fragment of a variant LPG10145 RGN disclosed herein when operably fused to a deaminase, has base editing activity that is from about 80% to about 500%, from about 80% to about 200%, or from about 90% to about 150%, or from about 95% to about 120%, or is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 11
  • a reference LPG10145 RGN has the amino acid sequence set forth as SEQ ID NO: 1.
  • a reference LPG10145 RGN is a variant of SEQ ID NO: 1 that lacks the corresponding mutations of the active variant or fragment thereof that has base editing activity, e.g., as described above.
  • a reference LPG10145 RGN is a non-identical variant LPG10145 RGN.
  • Base editing activity can be measured by assays known to one of skill in the art, including but not limited to, transfection of mammalian cells with a base editor (e.g., comprising a variant LPG10145 RGN and a deaminase) and a guide RNA and detecting base editing by PCR amplification of the target sequence and next generation sequencing (NGS), as described in Example 4 of the present disclosure.
  • Base editing assays are also described in International Publication No. WO 2022/056254, which is herein incorporated by reference in its entirety.
  • the efficiency of base editing a target sequence is assessed by measuring the percentage of a target sequence or cells comprising the target sequence that comprise altered expression of the target sequence or of a polypeptide encoded by the target sequence.
  • the expression is measured by quantitative PCR, microarray, RNA-seq, flow cytometry, immunoblot, enzyme- linked immunosorbent assay (ELISA), protein immunoprecipitation, immunostaining, high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC/MS), mass spectrometry, or a combination thereof.
  • an active variant or fragment of a variant LPG10145 RGN disclosed herein when operably fused to a polymerase (e.g., RT), has increased editing activity as compared to SEQ ID NO: 1.
  • an active variant or fragment of a variant LPG10145 RGN disclosed herein comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NOs: 2-16, 182-196, and 271-285, when operably fused to a polymerase (e.g., RT), has increased editing activity as compared to the RGN of SEQ ID NO: 1.
  • an active variant or fragment of a variant LPG10145 RGN disclosed herein comprising the amino acid sequence of any one of SEQ ID NOs: 2-16, 182-196, and 271-285, when operably fused to a polymerase (e.g., RT), has increased editing activity as compared to the RGN of SEQ ID NO: 1.
  • a polymerase e.g., RT
  • an active variant or fragment of a variant LPG10145 RGN disclosed herein when operably fused to a polymerase (e.g., RT), has editing activity that is from about 80% to about 500%, from about 80% to about 200%, or from about 90% to about 150%, or from about 95% to about 120%, or is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least
  • a reference LPG10145 RGN has the amino acid sequence set forth as SEQ ID NO: 1.
  • a reference LPG10145 RGN is a variant of SEQ ID NO: 1 that lacks the corresponding mutations of the active variant or fragment thereof that has editing activity, e.g., as described above.
  • a reference LPG10145 RGN is a non-identical variant LPG10145 RGN.
  • Editing activity can be measured by assays known to one of skill in the art, including but not limited to, transfection of mammalian cells with a polymerase editor (e.g., comprising a variant LPG10145 RGN and a reverse transcriptase) and a PEgRNA and detecting editing by PCR amplification of the target sequence and next generation sequencing (NGS), as described in Examples 5 and 6 of the present disclosure.
  • the editing rate (RT Edit %) can be determined by calculating the percent of read counts assigned to alleles containing the desired edit and, optionally, no additional edits.
  • the efficiency of editing a target sequence is assessed by measuring the percentage of a target sequence or cells comprising the target sequence that comprise altered expression of the target sequence or of a polypeptide encoded by the target sequence.
  • the expression is measured by quantitative PCR, microarray, RNA-seq, flow cytometry, immunoblot, enzyme-linked immunosorbent assay (ELISA), protein immunoprecipitation, immunostaining, high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC/MS), mass spectrometry, or a combination thereof.
  • Active variants and fragments of CRISPR repeats will retain the ability, when part of a guide RNA (comprising a tracrRNA), to bind to and guide an RNA-guided nuclease or base editor or PE comprising the same (complexed with the guide RNA) to a target nucleotide sequence in a sequence-specific manner.
  • a guide RNA comprising a tracrRNA
  • Active variants and fragments of tracrRNAs will retain the ability, when part of a guide RNA (comprising a CRISPR RNA), to guide an RNA-guided nuclease or base editor or PE comprising the same (complexed with the guide RNA) to a target nucleotide sequence in a sequencespecific manner.
  • a guide RNA comprising a CRISPR RNA
  • Active variants and fragments of PEgRNAs disclosed herein will retain the ability to bind and guide a PE to a target nucleotide sequence in a sequence-specific manner.
  • Active variants and fragments of base editors disclosed herein will retain the ability to, when associated with a guide RNA, chemically modify (e.g., deaminate) a nucleobase, resulting in conversion from one nucleobase to another.
  • Active variants and fragments of PEs disclosed herein will retain the ability to, when associated with a PEgRNA, edit a double-stranded polynucleotide through the replacement of a target sequence using the template sequence of the PEgRNA.
  • fragment refers to a portion of a polynucleotide or polypeptide sequence of the invention.
  • “Fragments”, “active fragments”, or “biologically active portions” include polynucleotides comprising a sufficient number of contiguous nucleotides to retain the biological activity (z. e. , binding to and directing an RGN in a sequence-specific manner to a target nucleotide sequence when comprised within a guide RNA).
  • “Fragments” or “biologically active portions” include polypeptides comprising a sufficient number of contiguous amino acid residues to retain the biological activity (i.e.
  • fragments of the RGN proteins include those that are shorter than the full-length sequences due to the use of an alternate downstream start site.
  • Such biologically active portions can be prepared by recombinant techniques and evaluated for activity.
  • a biologically active portion of an RGN protein can be a polypeptide that comprises, for example, 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, or more contiguous amino acid residues of an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions differs from the corresponding amino acid residue in SEQ ID NO: 1: 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975.
  • a biologically active portion of an RGN protein can be a polypeptide that comprises, for example, 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, or more contiguous amino acid residues of an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions is a positively charged amino acid residue: 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975.
  • a biologically active portion of an RGN protein can be a polypeptide that comprises, for example, 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, or more contiguous amino acid residues of an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions is an R: 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975.
  • a biologically active portion of an RGN protein can be a polypeptide that comprises, for example, 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, or more contiguous amino acid residues of an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at amino acid position 778 and/or 969 differs from the corresponding amino acid residue in SEQ ID NO: 1.
  • a biologically active portion of an RGN protein can be a polypeptide that comprises, for example, 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, or more contiguous amino acid residues of an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at amino acid position 778 and/or 969 is a positively charged amino acid residue.
  • a biologically active portion of an RGN protein can be a polypeptide that comprises, for example, 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, or more contiguous amino acid residues of an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at amino acid position 778 and/or 969 is an R.
  • a biologically active portion of an RGN protein can be a polypeptide that comprises, for example, 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, or more contiguous amino acid residues of an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein amino acid residues at positions 778 and 856 differ from the corresponding amino acid residues in SEQ ID NO: 1.
  • a biologically active portion of an RGN protein can be a polypeptide that comprises, for example, 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, or more contiguous amino acid residues of an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein amino acid residues at positions 778 and 856 are positively charged amino acid residues.
  • a biologically active portion of an RGN protein can be a polypeptide that comprises, for example, 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, or more contiguous amino acid residues of an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein amino acid residues at positions 55, 647, 778, and 969 differ from the corresponding amino acid residues in SEQ ID NO: 1.
  • a biologically active portion of an RGN protein can be a polypeptide that comprises, for example, 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, or more contiguous amino acid residues of an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein amino acid residues at positions 55, 647, 778, and 969 are positively charged amino acid residues.
  • a biologically active portion of an RGN protein can be a polypeptide that comprises, for example, 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, or more contiguous amino acid residues of an amino acid sequence selected from: (a) the amino acid sequence set forth as SEQ ID NO: 1, wherein E at amino acid position 52 is an R; (b) the amino acid sequence set forth as SEQ ID NO: 1, wherein S at amino acid position 55 is an R; (c) the amino acid sequence set forth as SEQ ID NO: 1, wherein G at amino acid position 86 is an R; (d) the amino acid sequence set forth as SEQ ID NO: 1, wherein Y at amino acid position 472 is an R; (e) the amino acid sequence set forth as SEQ ID NO: 1, wherein K at amino acid position 533 is an R; (f) the amino acid sequence set forth as
  • a biologically active portion of an RGN protein can be a polypeptide that comprises, for example, 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, or more contiguous amino acid residues of an amino acid sequence selected from: (a) the amino acid sequence set forth as SEQ ID NO: 2; (b) the amino acid sequence set forth as SEQ ID NO: 3; (c) the amino acid sequence set forth as SEQ ID NO: 4; (d) the amino acid sequence set forth as SEQ ID NO: 5; (e) the amino acid sequence set forth as SEQ ID NO: 6; (f) the amino acid sequence set forth as SEQ ID NO: 7; (g) the amino acid sequence set forth as SEQ ID NO: 8; (h) the amino acid sequence set forth as SEQ ID NO: 9; (i) the amino acid sequence set forth as SEQ ID NO: 10; (a) the amino
  • a biologically active fragment of a CRISPR repeat can comprise at least 8 contiguous amino acids of SEQ ID NO: 33, 244, or 245.
  • a biologically active portion of a CRISPR repeat can be a polynucleotide that comprises, for example, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 contiguous nucleotides of SEQ ID NO: 33, 244, or 245.
  • a biologically active portion of a tracrRNA can be a polynucleotide that comprises, for example, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more contiguous nucleotides of SEQ ID NO: 34, 246, 247, or 248.
  • a biologically active fragment of a guide RNA backbone (comprising the CRISPR repeat and tracrRNA, and optionally a nucleotide linker) can be a polynucleotide that comprises, for example, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more contiguous nucleotides of any one of SEQ ID NOs: 249-252.
  • a biologically active fragment of a PEgRNA can be a polynucleotide that comprises, for example, 100, 105, 110, 115, 120, 125, 130, or more contiguous nucleotides of any one of SEQ ID NOs: 129-132, and 197-220.
  • a biologically active portion of a polymerase can be a polypeptide that comprises, for example, 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or more contiguous amino acid residues of SEQ ID NO: 254 or 255.
  • a biologically active portion of a base editing polypeptide can be a polypeptide that comprises, for example, 10, 25, 50, 75, 100, 125, or more contiguous amino acid residues of any one of SEQ ID NOs: 42-113, and 257.
  • a biologically active portion of a base editor can be a polypeptide that comprises, for example, 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, or more contiguous amino acid residues of any one of SEQ ID NOs: 258-270.
  • a biologically active portion of a PE can be a polypeptide that comprises, for example, 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, or more contiguous amino acid residues of any one of SEQ ID NOs: 135, and 162-181.
  • variants or “active variant” is intended to mean substantially similar sequences.
  • a variant comprises a deletion and/or addition of one or more nucleotides at one or more internal sites within the native polynucleotide and/or a substitution of one or more nucleotides at one or more sites in the native polynucleotide.
  • a "native” or “wild type” polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively.
  • conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the native amino acid sequence of the gene of interest.
  • Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques as outlined below.
  • Variant polynucleotides also include synthetically derived polynucleotides, such as those generated, for example, by using site-directed mutagenesis but which still encode the polypeptide or the polynucleotide of interest.
  • variants of a particular polynucleotide disclosed herein will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular polynucleotide as determined by sequence alignment programs and parameters described elsewhere herein.
  • Variants of a particular polynucleotide disclosed herein can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide. Percent sequence identity between any two polypeptides can be calculated using sequence alignment programs and parameters described elsewhere herein.
  • the percent sequence identity between the two encoded polypeptides is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.
  • the presently disclosed polynucleotides encode an RNA-guided nuclease polypeptide comprising an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater identity to the amino acid sequence set forth as SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions differs from the corresponding amino acid residue in SEQ ID NO: 1: 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975.
  • the presently disclosed polynucleotides encode an RNA-guided nuclease polypeptide comprising an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater identity to the amino acid sequence set forth as SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions is a positively charged amino acid residue: 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975.
  • the presently disclosed polynucleotides encode an RNA-guided nuclease polypeptide comprising an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater identity to the amino acid sequence set forth as SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions is an R: 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975.
  • the presently disclosed polynucleotides encode an RNA-guided nuclease polypeptide comprising an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater identity to the amino acid sequence set forth as SEQ ID NO: 1, wherein the amino acid residue at amino acid position 778 and/or 969 differs from the corresponding amino acid residue in SEQ ID NO: 1.
  • the presently disclosed polynucleotides encode an RNA-guided nuclease polypeptide comprising an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater identity to the amino acid sequence set forth as SEQ ID NO: 1, wherein the amino acid residue at amino acid position 778 and/or 969 is a positively charged amino acid residue.
  • the presently disclosed polynucleotides encode an RNA-guided nuclease polypeptide comprising an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater identity to the amino acid sequence set forth as SEQ ID NO: 1, wherein the amino acid residue at amino acid position 778 and/or 969 is an R.
  • the presently disclosed polynucleotides encode an RNA-guided nuclease polypeptide comprising an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater identity to the amino acid sequence set forth as SEQ ID NO: 1, wherein amino acid residues at positions 778 and 856 differs from the corresponding amino acid residues in SEQ ID NO: 1.
  • the presently disclosed polynucleotides encode an RNA-guided nuclease polypeptide comprising an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater identity to the amino acid sequence set forth as SEQ ID NO: 1, wherein amino acid residues at positions 778 and 856 are positively charged amino acid residues.
  • the presently disclosed polynucleotides encode an RNA-guided nuclease polypeptide comprising an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater identity to the amino acid sequence set forth as SEQ ID NO: 1, wherein amino acid residues at positions 55, 647, 778, and 969 differs from the corresponding amino acid residues in SEQ ID NO: 1.
  • the presently disclosed polynucleotides encode an RNA-guided nuclease polypeptide comprising an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater identity to the amino acid sequence set forth as SEQ ID NO: 1, wherein amino acid residues at positions 55, 647, 778, and 969 are positively charged amino acid residues.
  • the presently disclosed polynucleotides encode an RNA-guided nuclease polypeptide comprising an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater identity to the amino acid sequence selected from: (a) the amino acid sequence set forth as SEQ ID NO: 1, wherein E at amino acid position 52 is an R; (b) the amino acid sequence set forth as SEQ ID NO: 1, wherein S at amino acid position 55 is an R; (c) the amino acid sequence set forth as SEQ ID NO: 1, wherein G at amino acid position 86 is an R; (d) the amino acid sequence set forth as SEQ ID NO: 1, wherein Y at amino acid position 472 is an R; (e) the amino acid sequence set forth as
  • the presently disclosed polynucleotides encode an RNA-guided nuclease polypeptide comprising an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater identity to the amino acid sequence selected from: (a) the amino acid sequence set forth as SEQ ID NO: 2, wherein Q at amino acid position 822 is an R, G at amino acid position 856 is an R, S at amino acid position 647 is an R, S at amino acid position 55 is an R, and E at amino acid position 778 is an R; (b) the amino acid sequence set forth as SEQ ID NO: 3, wherein Q at amino acid position 822 is an R, S at amino acid position 647 is an R, S at amino acid position 55 is an R, and E
  • a biologically active variant of a polymerase can comprise an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to the amino acid sequence set forth as SEQ ID NO: 254 or 255.
  • a biologically active variant of a base editing polypeptide can comprise an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to the amino acid sequence set forth as any one of SEQ ID NOs: 42-113, and 257.
  • a biologically active variant of a base editor can comprise an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to the amino acid sequence set forth as any one of SEQ ID NOs: 258-270.
  • a biologically active variant of a PE can comprise an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to the amino acid sequence set forth as any one of SEQ ID NOs: 135, and 162-181.
  • a biologically active variant of an RGN polypeptide, a fusion protein comprising an RGN polypeptide, a polymerase (e.g., RT), a base editing polypeptide, a base editor, or a PE of the invention may differ by as few as about 1-15 amino acid residues, as few as about 1-10, such as about 6-10, as few as 5, as few as 4, as few as 3, as few as 2, or as few as 1 amino acid residue.
  • the polypeptides can comprise an N-terminal or a C-terminal truncation, which can comprise at least a deletion of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100 amino acids or more from either the N or C terminus of the polypeptide.
  • the presently disclosed polynucleotides comprise or encode a CRISPR repeat comprising a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to the nucleotide sequence set forth as SEQ ID NO: 33, 244, or 245.
  • the presently disclosed polynucleotides can comprise or encode a CRISPR repeat comprising a nucleotide sequence set forth as SEQ ID NO: 33, 244, or 245, or that differs from SEQ ID NO: 33, 244, or 245 by 1 to 5 nucleotides.
  • the CRISPR repeat comprises a nucleotide sequence that differs from SEQ ID NO: 33, 244, or 245 by 5 nucleotides.
  • the CRISPR repeat comprises a nucleotide sequence that differs from SEQ ID NO: 33, 244, or 245 by 4 nucleotides.
  • the CRISPR repeat comprises a nucleotide sequence that differs from SEQ ID NO: 33, 244, or 245 by 3 nucleotides. In some embodiments, the CRISPR repeat comprises a nucleotide sequence that differs from SEQ ID NO: 33, 244, or 245 by 2 nucleotides. In some embodiments, the CRISPR repeat comprises a nucleotide sequence that differs from SEQ ID NO: 33, 244, or 245 by 1 nucleotide.
  • the presently disclosed polynucleotides can comprise or encode a tracrRNA comprising a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to the nucleotide sequence set forth as SEQ ID NO: 34, 246, 247, or 248.
  • the presently disclosed polynucleotides can comprise or encode a guide RNA backbone comprising a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to the nucleotide sequence set forth as any one of SEQ ID NOs: 249-252.
  • the presently disclosed polynucleotides can comprise or encode a PEgRNA comprising a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to the nucleotide sequence set forth as any one of SEQ ID NOs: 129-132, and 197-220.
  • Biologically active variants of a CRISPR repeat, tracrRNA, guide RNA backbone, or PEgRNA of the invention may differ by as few as about 1-15 nucleotides, as few as about 1-10, such as about 6-10, as few as 5, as few as 4, as few as 3, as few as 2, or as few as 1 nucleotide.
  • the polynucleotides can comprise a 5' or 3' truncation, which can comprise at least a deletion of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 95, 100, 105, 110 nucleotides or more from either the 5' or 3' end of the polynucleotide.
  • Variants of guide RNAs disclosed herein include guide RNAs that have modified nucleotides, sugars, phosphate backbone, and/or nucleobases.
  • Variant guide RNAs can include modifications including: 2'-O-methyl (2'-O-Me) modification; 2'-fluoro (2'-F) modification; 2'F-4'Ca-OMe modification; 2',4'-di-Ca- OMe modification; 2'-O-methyl 3'-phosphorothioate (MS) modification; 2'-O-methyl 3'thiophosphonoacetate (MSP; 2'-O-methyl 3'thioPACE) modification; 2'-O-methyl 3'phosphonoacetate (MP) modification; phosphorothioate (PS) modification; bridged nucleic acid (BNA) modification (e.g., 2', 4' BNA, locked nucleic acid (LNA), N-methyl substituted bridged nucleic acid BNA NC [N-Me], 2'-
  • RGN polypeptides fusion proteins comprising the same, polymerases (e.g., RTs), base editing polypeptides, base editors, PEs, CRISPR repeats, tracrRNAs, guide RNAs, and PEgRNAs provided herein creating variant proteins and polynucleotides. Changes designed by man may be introduced through the application of site-directed mutagenesis techniques. Alternatively, native, as yet-unknown or as yet unidentified polynucleotides and/or polypeptides structurally and/or functionally-related to the sequences disclosed herein may also be identified that fall within the scope of the present invention.
  • Conservative amino acid substitutions may be made in nonconserved regions that do not alter the function of the RGN proteins, fusion proteins comprising the same, polymerases (e.g., RTs), base editing polypeptides, base editors, or PEs. Alternatively, modifications may be made that improve the activity of the RGN proteins, fusion proteins comprising the same, polymerases (e.g., RTs), base editing polypeptides, base editors, or PEs.
  • Variant polynucleotides and proteins also encompass sequences and proteins derived from a mutagenic and recombinogenic procedure such as DNA shuffling.
  • a mutagenic and recombinogenic procedure such as DNA shuffling.
  • one or more variant LPG10145 RGN protein, fusion protein comprising the same, polymerase (e.g., RT), base editing polypeptide, base editor, or PE disclosed herein is manipulated to create a new RGN protein, fusion protein comprising the same, polymerase (e.g., RT), base editing polypeptide, base editor, or PE possessing the desired properties.
  • libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo.
  • sequence motifs encoding a domain of interest may be shuffled between the RGN, fusion protein comprising the same, polymerase (e.g., RT), base editing polypeptide, base editor, or PE sequences provided herein and other known RGN, fusion protein comprising the same, polymerase (e.g., RT), base editing polypeptide, base editor, or PE genes to obtain a new gene coding for a protein with an improved property of interest, such as an increased K m in the case of an enzyme.
  • a "shuffled" nucleic acid is a nucleic acid produced by a shuffling procedure such as any shuffling procedure set forth herein.
  • Shuffled nucleic acids are produced by recombining (physically or virtually) two or more nucleic acids (or character strings), for example in an artificial, and optionally recursive, fashion.
  • one or more screening steps are used in shuffling processes to identify nucleic acids of interest; this screening step can be performed before or after any recombination step.
  • the overall process of recombination and selection are optionally repeated recursively.
  • shuffling can refer to an overall process of recombination and selection, or, alternately, can simply refer to the recombinational portions of the overall process.
  • sequence identity in the context of two polynucleotides or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. It is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity”. Means for making this adjustment are well known to those of skill in the art.
  • a conservative substitution is given a score between zero and 1.
  • the scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, California).
  • percentage of sequence identity means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (z. e. , gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.
  • sequence identity/similarity values provided herein refer to the value obtained using GAP Version 10 using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix; or any equivalent program thereof.
  • equivalent program is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.
  • Two sequences are "optimally aligned” when they are aligned for similarity scoring using a defined amino acid substitution matrix (e.g., BLOSUM62), gap existence penalty and gap extension penalty so as to arrive at the highest score possible for that pair of sequences.
  • Amino acid substitution matrices and their use in quantifying the similarity between two sequences are well-known in the art and described, e.g., in Dayhoff et al. (1978) "A model of evolutionary change in proteins.” In “Atlas of Protein Sequence and Structure,” Vol. 5, Suppl. 3 (ed. M. O. Dayhoff), pp. 345-352. Natl. Biomed. Res. Found., Washington, D.C. and Henikoff et al.
  • the BLOSUM62 matrix is often used as a default scoring substitution matrix in sequence alignment protocols.
  • the gap existence penalty is imposed for the introduction of a single amino acid gap in one of the aligned sequences, and the gap extension penalty is imposed for each additional empty amino acid position inserted into an already opened gap.
  • the alignment is defined by the amino acid positions of each sequence at which the alignment begins and ends, and optionally by the insertion of a gap or multiple gaps in one or both sequences, so as to arrive at the highest possible score.
  • BLAST 2.0 a computer-implemented alignment algorithm
  • BLAST 2.0 a computer-implemented alignment algorithm
  • Optimal alignments including multiple alignments, can be prepared using, e.g., PSI-BLAST, available through www.ncbi.nlm.nih.gov and described by Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.
  • an amino acid residue “corresponds to” the position in the reference sequence with which the residue is paired in the alignment.
  • the "position” is denoted by a number that sequentially identifies each amino acid in the reference sequence based on its position relative to the N-terminus. Owing to deletions, insertion, truncations, fusions, etc., that must be taken into account when determining an optimal alignment, in general the amino acid residue number in a test sequence as determined by simply counting from the N-terminal will not necessarily be the same as the number of its corresponding position in the reference sequence.
  • polymerases e.g., RTs
  • base editing polypeptides e.g., base editors
  • PEs e.g., ribonucleoproteins comprising the RGN polypeptides or fusion proteins of the present disclosure.
  • Engineered variant LPGI0145 RGN polypeptides include those having the amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions differs from the corresponding amino acid residue in SEQ ID NO: 1: 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975.
  • antibodies of the present disclosure bind to RGN polypeptides or ribonucleoproteins comprising the RGN polypeptides having the amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions is a positively charged amino acid residue: 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975.
  • antibodies of the present disclosure bind to RGN polypeptides or ribonucleoproteins comprising the RGN polypeptides having the amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions is an R: 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975.
  • Antibodies of the disclosure bind to the RGN polypeptides or ribonucleoproteins comprising the RGN polypeptides of the present disclosure, including those having the amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at amino acid position 778 and/or 969 differs from the corresponding amino acid residue in SEQ ID NO: 1.
  • antibodies of the present disclosure bind to RGN polypeptides or ribonucleoproteins comprising the RGN polypeptides having the amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at amino acid position 778 and/or 969 is a positively charged amino acid residue.
  • antibodies of the present disclosure bind to RGN polypeptides or ribonucleoproteins comprising the RGN polypeptides having the amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at amino acid position 778 and/or 969 is an R.
  • antibodies of the present disclosure bind to RGN polypeptides or ribonucleoproteins comprising the RGN polypeptides having the amino acid sequence selected from: the amino acid sequence set forth as SEQ ID NO: 1, wherein E at amino acid position 52 is an R; (b) the amino acid sequence set forth as SEQ ID NO: 1, wherein S at amino acid position 55 is an R; (c) the amino acid sequence set forth as SEQ ID NO: 1, wherein G at amino acid position 86 is an R; (d) the amino acid sequence set forth as SEQ ID NO: 1, wherein Y at amino acid position 472 is an R; (e) the amino acid sequence set forth as SEQ ID NO: 1, wherein K at amino acid position 533 is an R; (f) the amino acid sequence set forth as SEQ ID NO: 1, wherein K at amino acid position 541 is an R; (g) the amino acid sequence set forth as SEQ ID NO: 1, wherein Y at amino acid position 643 is substituted by an R; (h) the amino acid sequence selected from: the
  • antibodies of the present disclosure bind to RGN polypeptides or ribonucleoproteins comprising the RGN polypeptides having the amino acid sequence selected from: (a) the amino acid sequence set forth as SEQ ID NO: 2; (b) the amino acid sequence set forth as SEQ ID NO: 3; (c) the amino acid sequence set forth as SEQ ID NO: 4; (d) the amino acid sequence set forth as SEQ ID NO: 5; (e) the amino acid sequence set forth as SEQ ID NO: 6; (f) the amino acid sequence set forth as SEQ ID NO: 7; (g) the amino acid sequence set forth as SEQ ID NO: 8; (h) the amino acid sequence set forth as SEQ ID NO: 9; (i) the amino acid sequence set forth as SEQ ID NO: 10; (j) the amino acid sequence set forth as SEQ ID NO: 11; (k) the amino acid sequence set forth as SEQ ID NO: 12; (1) the amino acid sequence set forth as SEQ ID NO: 13; (m) the amino acid sequence set
  • kits comprising antibodies that specifically bind to the polypeptides or ribonucleoproteins described herein, including, for example, polypeptides comprising the amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions differs from the corresponding amino acid residue in SEQ ID NO: 1: 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975.
  • the polypeptides comprise the amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions is a positively charged amino acid residue: 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975.
  • the polypeptides comprise the amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions is an R: 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975.
  • the polypeptides can comprise the amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at amino acid position 778 and/or 969 differs from the corresponding amino acid residue in SEQ ID NO: 1. In some embodiments, the polypeptides comprise the amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at amino acid position 778 and/or 969 is a positively charged amino acid residue. In some embodiments, the polypeptides comprise the amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at amino acid position 778 and/or 969 is an R.
  • kits can comprise antibodies that specifically bind to the polypeptides comprising the amino acid sequence selected from: the amino acid sequence set forth as SEQ ID NO: 1, wherein E at amino acid position 52 is an R; (b) the amino acid sequence set forth as SEQ ID NO: 1, wherein S at amino acid position 55 is an R; (c) the amino acid sequence set forth as SEQ ID NO: 1, wherein G at amino acid position 86 is an R; (d) the amino acid sequence set forth as SEQ ID NO: 1, wherein Y at amino acid position 472 is an R; (e) the amino acid sequence set forth as SEQ ID NO: 1, wherein K at amino acid position 533 is an R; (f) the amino acid sequence set forth as SEQ ID NO: 1, wherein K at amino acid position 541 is an R; (g) the amino acid sequence set forth as SEQ ID NO: 1, wherein Y at amino acid position 643 is substituted by an R; (h) the amino acid sequence set forth as SEQ ID NO: 1, wherein S at amino acid position 647 is an R
  • the present disclosure provides a system for binding a target sequence of interest, wherein the system comprises at least one guide RNA or a nucleotide sequence encoding the same, and at least one variant LPGI0145 RNA-guided nuclease or a nucleotide sequence encoding the same.
  • the guide RNA hybridizes to the target sequence of interest and also forms a complex with the variant LPGI0145 RGN polypeptide, thereby directing the variant LPGI0145 RGN polypeptide to bind to the target sequence.
  • the present disclosure also provides a base editing system for binding and editing a target sequence of interest, wherein the base editing system comprises an engineered variant LPGI0145 RGN polypeptide (or a polynucleotide encoding the same), a base editing polypeptide (e.g., deaminase) (or a polynucleotide encoding the same), along with one or more guide RNA (or one or more polynucleotide sequences encoding the same).
  • the base editing system comprises an engineered variant LPGI0145 RGN polypeptide (or a polynucleotide encoding the same), a base editing polypeptide (e.g., deaminase) (or a polynucleotide encoding the same), along with one or more guide RNA (or one or more polynucleotide sequences encoding the same).
  • the base editing system may comprise a base editor wherein the base editor comprises a single fusion protein (or a polynucleotide encoding the same) comprising the variant LPGI0145 RGN polypeptide and the base editing polypeptide (e.g., deaminase), or a base editor wherein the variant LPGI0145 RGN polypeptide and the base editing polypeptide (e.g., deaminase) are two separate polypeptides.
  • a base editing system can comprise a plurality of engineered variant LPGI0145 RGN polypeptides (or polynucleotides encoding the same), a plurality of base editing polypeptide (e.g., deaminases) (or polynucleotides encoding the same), and/or a plurality of guide RNAs (or polynucleotides encoding the same).
  • LPGI0145 RGN polypeptides or polynucleotides encoding the same
  • base editing polypeptide e.g., deaminases
  • guide RNAs or polynucleotides encoding the same
  • a base editor of the disclosure can comprise an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to any one of SEQ ID NOs: 258-270.
  • a base editor disclosed herein comprises the amino acid sequence set forth as any one of SEQ ID NOs: 258-270.
  • the present disclosure also provides a PE system for binding and editing a target sequence of interest, wherein the PE system comprises an engineered variant LPG10145 RGN polypeptide (or a polynucleotide encoding the same), a polymerase (e.g., RT) (or a polynucleotide encoding the same), along with one or more PEgRNAs (or one or more polynucleotide sequences encoding the same).
  • a polymerase e.g., RT
  • PEgRNAs or one or more polynucleotide sequences encoding the same.
  • the PE system may comprise a PE wherein the PE comprises a single fusion protein (or a polynucleotide encoding the same) comprising the variant LPG10145 RGN polypeptide and the polymerase (e.g., RT), or a PE wherein the variant LPG10145 RGN polypeptide and the polymerase (e.g., RT) are two separate polypeptides.
  • the PE comprises a single fusion protein (or a polynucleotide encoding the same) comprising the variant LPG10145 RGN polypeptide and the polymerase (e.g., RT), or a PE wherein the variant LPG10145 RGN polypeptide and the polymerase (e.g., RT) are two separate polypeptides.
  • a PE of the disclosure can comprise an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to any one of SEQ ID NOs: 135, and 162- 181.
  • a PE disclosed herein comprises the amino acid sequence set forth as any one of SEQ ID NOs: 135, and 162-181.
  • a dual PE (DPE) system for dual polymerase editing comprises: a) a first PE system comprising a first PE (or one or more polynucleotides encoding the same; or a combination of protein(s) and encoding polynucleotide(s)) and a first PEgRNA (or a polynucleotide encoding the same); and b) a second PE system comprising a second PE (or one or more polynucleotides encoding the same; or a combination of protein(s) and encoding polynucleotide(s)) and a second PEgRNA (or a polynucleotide encoding the same).
  • a first PE system comprising a first PE (or one or more polynucleotides encoding the same; or a combination of protein(s) and encoding polynucleotide(s)) and a first PEgRNA (or a polynucleotide encoding
  • the first and second PE can be the same or different and can be any one of the PEs described herein.
  • the DPE system can also comprise a PE (or one or more polynucleotides encoding the same; or a combination of protein(s) and encoding polynucleotide(s)) and a first PEgRNA (or a polynucleotide encoding the same) and a second PEgRNA (or a polynucleotide encoding the same).
  • the first and second PEgRNAs can bind to opposite strands of a target polynucleotide such that the intended repair event is installed on both strands of the target DNA.
  • the DNA synthesis template of the first PEgRNA and the DNA synthesis template of the second PEgRNA each encode a singlestranded DNA sequence that is complementary (full or partial) to each other so that the region of the DNA between the two nicked sites is replaced.
  • the DPE system comprises PEgRNAs wherein the DNA synthesis templates are designed such that the replacement sequence (comprising the complementary single-stranded DNA sequences encoded by the first and second DNA synthesis templates) comprises a first recombinase site, as well as a donor DNA comprising a second recombinase site and the corresponding site-specific recombinase that recognizes the first and second recombinase site.
  • the recombination of the replacement sequence and donor DNA results in an insertion of exogenous DNA.
  • recombinase refers to a site-specific enzyme that catalyzes the recombination of DNA between recombinase sites that results in the excision, integration, inversion, or exchange of DNA fragments between the recombinase sites.
  • Non-limiting examples of recombinases are serine recombinases (e.g., Hin, Gin, Tn3, -six, CinH, ParA, y5, Bxbl, (pC31, TP901, TGI, cpBTl, R4, cpRVl, cpFCl, MR11, Al 18, U153, and gp29) and tyrosine recombinases (e.g., Cre, FLP, R, Lambda, HK101, HK022, and pSAM2).
  • serine recombinases e.g., Hin, Gin, Tn3, -six, CinH, ParA, y5, Bxbl, (pC31, TP901, TGI, cpBTl, R4, cpRVl, cpFCl, MR11, Al 18, U153, and gp29
  • tyrosine recombinases e.g., Cre, FL
  • recombinase site refers to a target nucleotide sequence recognized by a recombinase that undergoes strand exchange with another nucleotide sequence having a similar recombinase site.
  • Non-limiting examples of recombinase sites are the attB/attP sites recognized by the HK022 and ⁇ j)C31 recombinases.
  • the variant LPG10145 RGNs comprise an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions differs from the corresponding amino acid residue in SEQ ID NO: 1: 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975.
  • the variant LPG10145 RGNs comprise an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions is a positively charged amino acid residue: 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975.
  • the variant LPG10145 RGNs comprise an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions is an R: 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975.
  • the variant LPG10145 RGNs comprise an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at amino acid position 778 and/or 969 differs from the corresponding amino acid residue in SEQ ID NO: 1. In some embodiments, the variant LPG10145 RGNs comprise an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at amino acid position 778 and/or 969 is a positively charged amino acid residue. In some embodiments, the variant LPG10145 RGNs comprise an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at amino acid position 778 and/or 969 is an R.
  • the variant LPG10145 RGNs comprise an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein amino acid residues at positions 778 and 856 differ from the corresponding amino acid residues in SEQ ID NO: 1. In some embodiments, the variant LPG10145 RGNs comprise an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residues at positions 778 and 856 are positively charged amino acid residues.
  • the variant LPG10145 RGNs comprise an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein amino acid residues at positions 55, 647, 778, and 969 differ from the corresponding amino acid residues in SEQ ID NO: 1. In some embodiments, the variant LPG10145 RGNs comprise an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein amino acid residues at positions 55, 647, 778, and 969 are positively charged amino acid residues.
  • the variant LPG10145 RGN comprises the amino acid sequence selected from: (a) the amino acid sequence set forth as SEQ ID NO: 1, wherein E at amino acid position 52 is an R; (b) the amino acid sequence set forth as SEQ ID NO: 1, wherein S at amino acid position 55 is an R; (c) the amino acid sequence set forth as SEQ ID NO: 1, wherein G at amino acid position 86 is an R; (d) the amino acid sequence set forth as SEQ ID NO: 1, wherein Y at amino acid position 472 is an R; (e) the amino acid sequence set forth as SEQ ID NO: 1, wherein K at amino acid position 533 is an R; (f) the amino acid sequence set forth as SEQ ID NO: 1, wherein K at amino acid position 541 is an R; (g) the amino acid sequence set forth as SEQ ID NO: 1, wherein Y at amino acid position 643 is substituted by an R; (h) the amino acid sequence set forth as SEQ ID NO: 1, wherein S at amino acid position 647 is
  • the variant LPG10145 RGN comprises the amino acid sequence selected from: (a) the amino acid sequence set forth as SEQ ID NO: 2; (b) the amino acid sequence set forth as SEQ ID NO: 3; (c) the amino acid sequence set forth as SEQ ID NO: 4; (d) the amino acid sequence set forth as SEQ ID NO: 5; (e) the amino acid sequence set forth as SEQ ID NO: 6; (f) the amino acid sequence set forth as SEQ ID NO: 7; (g) the amino acid sequence set forth as SEQ ID NO: 8; (h) the amino acid sequence set forth as SEQ ID NO: 9; (i) the amino acid sequence set forth as SEQ ID NO: 10; (j) the amino acid sequence set forth as SEQ ID NO: 11; (k) the amino acid sequence set forth as SEQ ID NO: 12; (1) the amino acid sequence set forth as SEQ ID NO: 13; (m) the amino acid sequence set forth as SEQ ID NO: 14; (n) the amino acid sequence set forth as SEQ ID NO: 5; (e
  • the guide RNA comprises a CRISPR repeat sequence comprising the nucleotide sequence set forth as SEQ ID NO: 33, 245, or 245, or an active variant or fragment thereof.
  • the guide RNA comprises a tracrRNA comprising the nucleotide sequence set forth as SEQ ID NO: 34, 246, 247, or 248, or an active variant or fragment thereof.
  • the guide RNA of the system can be a single guide RNA or a dual-guide RNA.
  • the system comprises an RNA- guided nuclease that is heterologous to the guide RNA, wherein the RGN and guide RNA are not found complexed to one another (i.e., bound to one another) in nature.
  • the system for binding a target sequence of interest can be a ribonucleoprotein complex, which is at least one molecule of an RNA bound to at least one protein.
  • the ribonucleoprotein (RNP) complexes provided herein can comprise at least one guide RNA as the RNA component and an RNA-guided nuclease as the protein component.
  • An RNP complex provided herein can comprise at least one guide RNA as the RNA component and a base editor as the protein component.
  • An RNP complex provided herein can comprise at least one PEgRNA as the RNA component and a PE as the protein component.
  • Such ribonucleoprotein complexes can be purified from a cell or organism that naturally expresses the protein component (e.g., an RGN polypeptide) and has been engineered to express a particular guide RNA that is specific for a target sequence of interest.
  • the protein component e.g., an RGN polypeptide
  • the ribonucleoprotein complex can be purified from a cell or organism that has been transformed with polynucleotides that encode a protein component (e.g., an RGN polypeptide, a base editor, or a PE) and an RNA component (e.g., a guide RNA or a PEgRNA) (or a polynucleotide that comprises the RNA component) and cultured under conditions to allow for the expression of the protein and RNA components (e.g., RGN polypeptide and guide RNA, base editor and guide RNA, or PE and PEgRNA).
  • a protein component e.g., an RGN polypeptide, a base editor, or a PE
  • RNA component e.g., a guide RNA or a PEgRNA
  • an RGN polypeptide e.g., a polymerase (e.g., RT), a base editing polypeptide, a fusion protein comprising an RGN, a base editor, or a PE, or an RNP complex comprising these polypeptides.
  • a polymerase e.g., RT
  • a base editing polypeptide e.g., a base editing polypeptide
  • a fusion protein comprising an RGN, a base editor, or a PE
  • RNP complex comprising these polypeptides.
  • Such methods comprise culturing a cell comprising a nucleotide sequence encoding a protein component (e.g., an RGN polypeptide, a polymerase (e.g., RT), a base editing polypeptide, a fusion protein comprising an RGN, a base editor, or PE), and in some embodiments a nucleotide sequence encoding an RNA component (e.g., a guide RNA or PEgRNA), under conditions in which the protein component (e.g., RGN polypeptide, a polymerase (e.g., RT), a base editing polypeptide, a fusion protein comprising an RGN, a base editor, or PE) (and in some embodiments, the RNA component (e.g., guide RNA or PEgRNA) is expressed.
  • a protein component e.g., an RGN polypeptide, a polymerase (e.g., RT), a base editing polypeptide, a fusion protein comprising an
  • the RGN polypeptide, a polymerase (e.g., RT), a base editing polypeptide, a fusion protein comprising an RGN, a base editor, a PE, or RNP complex can then be purified from a lysate of the cultured cells.
  • the nucleotide sequence encoding an RGN polypeptide, a polymerase (e.g., RT), a base editing polypeptide, a fusion protein comprising an RGN, a base editor, or PE includes a mRNA (messenger RNA).
  • methods for assembling an RNP complex comprise combining one or more of the presently disclosed guide RNAs and one or more of the presently disclosed RGN polypeptides, base editors, or PEs under conditions suitable for formation of the RNP complex.
  • methods for assembling an RNP complex comprise combining one or more of the presently disclosed RNA components (e.g., guide RNAs or PEgRNAs) and one or more of the presently disclosed protein components (e.g., RGN polypeptide, base editor, PE) under conditions suitable for formation of the RNP complex.
  • Methods for purifying an RGN polypeptide, a polymerase (e.g., RT), a base editing polypeptide, a fusion protein comprising an RGN, a base editor, a PE, or RNP complex from a lysate of a biological sample are known in the art (e.g., size exclusion and/or affinity chromatography, 2D-PAGE, HPLC, reversed-phase chromatography, immunoprecipitation).
  • the RGN polypeptide, polymerase (e.g., RT), base editing polypeptide, fusion protein comprising an RGN, base editor, PE, or RNP complex is recombinantly produced and comprises a purification tag to aid in its purification, including but not limited to, glutathione-S-transferase (GST), chitin binding protein (CBP), maltose binding protein, thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG (e.g., 3X FLAG tag), HA, nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, SI, T7, V5, VSV-G, 6xHis, lOxHis, biotin carboxyl carrier protein (BCCP), and calmodulin.
  • GST glutathione-S-transferase
  • the tagged RGN polypeptide, polymerase (e.g., RT), base editing polypeptide, fusion protein comprising an RGN, base editor, PE, or RGN ribonucleoprotein complex is purified using immobilized metal affinity chromatography. It will be appreciated that other similar methods known in the art may be used, including other forms of chromatography or for example immunoprecipitation, either alone or in combination.
  • an "isolated” or “purified” polypeptide, or biologically active portion thereof, is substantially or essentially free from components that normally accompany or interact with the polypeptide as found in its naturally occurring environment.
  • an isolated or purified polypeptide is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • a protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein.
  • optimally culture medium represents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or non-protein-of-interest chemicals.
  • an “isolated” polynucleotide or nucleic acid molecule is removed from its naturally occurring environment.
  • An isolated polynucleotide is substantially free of chemical precursors or other chemicals when chemically synthesized or has been removed from a genomic locus via the breaking of phosphodiester bonds.
  • An isolated polynucleotide can be part of a vector, a composition of matter or can be contained within a cell so long as the cell is not the original environment of the polynucleotide.
  • In vitro assembly of an RNPcomplex can be performed using any method known in the art in which a protein component (e.g., an RGN polypeptide, base editor, or PE) is contacted with an RNA component (e.g., guide RNA or PEgRNA) under conditions to allow for binding of the protein component (e.g., RGN polypeptide, base editor, or PE) to the RNA component (e.g., guide RNA or PEgRNA).
  • RNA component e.g., guide RNA or PEgRNA
  • contact contacting
  • contacted refer to making one entity in touch with one or more other entities with or without any intermediate means.
  • “contact”, “contacting”, or “contacted” include but is not limited to placing the components of a desired reaction together under conditions suitable for carrying out the desired reaction, or placing the entities in the same container or the same solution, or transfection, transformation, viral delivery, or LNP delivery of the one entity and the one or more other entities.
  • the RGN polypeptide, base editor, or PE can be purified from a biological sample, cell lysate, or culture medium, produced via in vitro translation, or chemically synthesized.
  • the guide RNA or PEgRNA can be purified from a biological sample, cell lysate, or culture medium, transcribed in vitro, or chemically synthesized.
  • the protein component e.g., RGN polypeptide, base editor, or PE
  • RNA component e.g., guide RNA or PEgRNA
  • solution e.g., buffered saline solution
  • the kit comprises a vector system and instructions for using the kit.
  • the vector system comprises (a) a first regulatory element operably linked to a DNA sequence encoding the crRNA sequence and one or more insertion sites for inserting a guide sequence upstream of the encoded crRNA sequence, wherein when expressed, the guide sequence directs sequencespecific binding of an RGN complex to a target sequence in a eukaryotic cell, wherein the RGN complex comprises an RGN enzyme complexed with the guide RNA polynucleotide; and/or (b) a second regulatory element operably linked to an enzyme coding sequence encoding said RGN enzyme comprising a nuclear localization sequence.
  • kits can include engineered variant LPG10145 RGN polypeptides, polymerases (e.g., RTs), base editing polypeptides, fusion proteins comprising variant LPG10145 RGN polypeptides, base editors, PEs, guide RNAs, PEgRNAs, or polynucleotides encoding the same; cells; and complete RGN, base editing, or PE systems.
  • Elements may be provided individually or in combinations, and may be provided in any suitable container, such as a vial, a bottle, or a tube.
  • kits includes instructions in one or more languages.
  • a kit comprises one or more reagents for use in a process utilizing one or more of the elements described herein.
  • Reagents may be provided in any suitable container.
  • a kit may provide one or more reaction or storage buffers.
  • Reagents may be provided in a form that is usable in a particular assay, or in a form that requires addition of one or more other components before use (e.g. in concentrate or lyophilized form).
  • a buffer can be any buffer, including but not limited to a sodium carbonate buffer, a sodium bicarbonate buffer, a borate buffer, a Tris buffer, a MOPS buffer, a HEPES buffer, and combinations thereof.
  • the buffer is alkaline.
  • the buffer has a pH from about 7 to about 10.
  • the kit comprises one or more oligonucleotides corresponding to a guide sequence for insertion into a vector so as to operably link the guide sequence and a regulatory element.
  • the kit comprises a homologous recombination template polynucleotide.
  • the disclosure provides methods for using one or more elements of an RGN, base editing, or PE system. The RGN, base editing, or PE system of the invention provides an effective means for modifying a target polynucleotide.
  • the RGN, base editing, or PE system of the invention has a wide variety of utility including modifying (e.g., deleting, inserting, translocating, inactivating, activating, base editing) a target polynucleotide in a multiplicity of cell types.
  • modifying e.g., deleting, inserting, translocating, inactivating, activating, base editing
  • the RGN, base editing, or PE system of the invention has a broad spectrum of applications in, e.g., gene therapy, drug screening, disease diagnosis, and prognosis.
  • An exemplary RGN system, or RGN complex comprises an RGN enzyme complexed with a guide RNA hybridized to a target sequence within the target polynucleotide.
  • An exemplary base editing system, or RNP complex comprising a base editor comprises a base editor complexed with a guide RNA hybridized to a target sequence within the target polynucleotide.
  • An exemplary PE system, or RNP complex comprising a PE comprises a PE complexed with a PEgRNA hybridized to a target sequence within the target polynucleotide.
  • the present disclosure provides methods for binding, cleaving, and/or modifying a target nucleotide sequence of interest.
  • the methods include delivering a system comprising at least one guide RNA or a polynucleotide encoding the same, and at least one variant LPG10145 RGN polypeptide or a polynucleotide encoding the same to the target sequence or a cell, organelle, or embryo comprising the target sequence.
  • the methods include delivering a system comprising at least one guide RNA or a polynucleotide encoding the same, and at least one base editor comprising a variant LPG10145 RGN polypeptide (or one or more polynucleotides encoding the same, or a combination of protein(s) and encoding polynucleotide(s)) to the target sequence or a cell, organelle, or embryo comprising the target sequence.
  • the methods include delivering a system comprising at least one PEgRNA or a polynucleotide encoding the same, and at least one PE comprising a variant LPG10145 RGN polypeptide (or one or more polynucleotides encoding the same, or a combination of protein(s) and encoding polynucleotide(s)) to the target sequence or a cell, organelle, or embryo comprising the target sequence.
  • the methods for modifying a target polynucleotide (e.g., target DNA) of interest comprising a target sequence can be performed ex vivo or in vitro.
  • the methods for modifying a target polynucleotide (e.g., target DNA) of interest are not methods for treatment of the human or animal body by therapy or are not processes for editing the germ line genetic identity of a human being.
  • the RGN comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions differs from the corresponding amino acid residue in SEQ ID NO: 1: 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975.
  • the RGN comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions is a positively charged amino acid residue: 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975.
  • the RGN comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions is an R: 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975.
  • the RGN comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at amino acid position 778 and/or 969 differs from the corresponding amino acid residue in SEQ ID NO: 1. In some of these embodiments, the RGN comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at amino acid position 778 and/or 969 is a positively charged amino acid residue. In some of these embodiments, the RGN comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at amino acid position 778 and/or 969 is an R.
  • the RGN comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein amino acid residues at positions 778 and 856 differ from the corresponding amino acid residues in SEQ ID NO: 1. In some of these embodiments, the RGN comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein amino acid residues at positions 778 and 856 are positively charged amino acid residues.
  • the RGN comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein amino acid residues at positions 55, 647, 778, and 969 differ from the corresponding amino acid residues in SEQ ID NO: 1. In some of these embodiments, the RGN comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein amino acid residues at positions 55, 647, 778, and 969 are positively charged amino acid residues.
  • the RGN comprises the amino acid sequence selected from: (a) the amino acid sequence set forth as SEQ ID NO: 1, wherein E at amino acid position 52 is an R; (b) the amino acid sequence set forth as SEQ ID NO: 1, wherein S at amino acid position 55 is an R; (c) the amino acid sequence set forth as SEQ ID NO: 1, wherein G at amino acid position 86 is an R; (d) the amino acid sequence set forth as SEQ ID NO: 1, wherein Y at amino acid position 472 is an R; (e) the amino acid sequence set forth as SEQ ID NO: 1, wherein K at amino acid position 533 is an R; (f) the amino acid sequence set forth as SEQ ID NO: 1, wherein K at amino acid position 541 is an R; (g) the amino acid sequence set forth as SEQ ID NO: 1, wherein Y at amino acid position 643 is substituted by an R; (h) the amino acid sequence set forth as SEQ ID NO: 1, wherein S at amino acid position 647 is an R; (i) the amino acid sequence set
  • the RGN comprises the amino acid sequence selected from: (a) the amino acid sequence set forth as SEQ ID NO: 2; (b) the amino acid sequence set forth as SEQ ID NO: 3; (c) the amino acid sequence set forth as SEQ ID NO: 4; (d) the amino acid sequence set forth as SEQ ID NO: 5; (e) the amino acid sequence set forth as SEQ ID NO: 6; (f) the amino acid sequence set forth as SEQ ID NO: 7; (g) the amino acid sequence set forth as SEQ ID NO: 8; (h) the amino acid sequence set forth as SEQ ID NO: 9; (i) the amino acid sequence set forth as SEQ ID NO: 10; (j) the amino acid sequence set forth as SEQ ID NO: 11; (k) the amino acid sequence set forth as SEQ ID NO: 12; (1) the amino acid sequence set forth as SEQ ID NO: 13; (m) the amino acid sequence set forth as SEQ ID NO: 14; (n) the amino acid sequence set forth as SEQ ID NO: 15; and
  • the guide RNA comprises a CRISPR repeat comprising the nucleotide sequences set forth as SEQ ID NO: 33, 244, or 245, or an active variant or fragment thereof.
  • the guide RNA comprises a tracrRNA comprising the nucleotide sequence set forth as SEQ ID NO: 34, 246, 247, or 248, or an active variant or fragment thereof.
  • the guide RNA of the system can be a single guide RNA or a dual-guide RNA.
  • the RGN of the system may be a nuclease dead RGN, have nickase activity, or may be a fusion polypeptide.
  • the fusion polypeptide comprises a base-editing polypeptide, for example a cytosine deaminase or an adenine deaminase.
  • the RGN fusion protein is a PE.
  • the PE can comprise, for example, a polymerase (e.g., reverse transcriptase).
  • the RGN fusion protein comprises a polypeptide that recruits members of a functional nucleic acid repair complex, such as a member of the nucleotide excision repair (NER) or transcription coupled-nucleotide excision repair (TC-NER) pathway (Wei et al., 2015, PNAS USA 112(27):E3495-504 ; Troelstra et al., 1992, Cell 71:939-953; Mamef et al., 2017, J Mol Biol 429(9): 1277-1288), as described in U.S. Provisional Application No. 62/966,203, which was filed on January 27, 2020, and is incorporated by reference in its entirety.
  • NER nucleotide excision repair
  • TC-NER transcription coupled-nucleotide excision repair
  • the RGN fusion protein comprises CSB (van den Boom et al., 2004, J Cell Biol 166(l):27-36; van Gool et al., 1997, EMBO J 16(19):5955-65; an example of which is set forth as SEQ ID NO: 39), which is a member of the TC-NER (nucleotide excision repair) pathway and functions in the recruitment of other members.
  • the RGN fusion protein comprises an active domain of CSB, such as the acidic domain of CSB which comprises amino acid residues 356-394 of SEQ ID NO: 39 (Teng et al., 2018, Nat Commun 9( 1):4115).
  • the RGN, base editor, PE, and/or guide RNA is heterologous to the cell, organelle, or embryo to which the RGN, base editor, PE, and/or guide RNA (or polynucleotide(s) encoding at least one of the RGN and guide RNA) are introduced.
  • the method comprises contacting a target polynucleotide with an RNP complex.
  • the contacting can be ex vivo, in vitro, or in vivo.
  • the RNP complex may comprise an RGN that is nuclease dead or has nickase activity.
  • the RGN of the RNP complex is a fusion polypeptide comprising a base-editing polypeptide. In some embodiments, the RGN of the RNP complex is a fusion polypeptide comprising a polymerase (e.g., RT). In certain embodiments, the method comprises introducing into a cell, organelle, or embryo comprising a target polynucleotide an RNP complex.
  • the RNP complex can be one that has been purified from a biological sample, recombinantly produced and subsequently purified, or in w/ro-asscmblcd as described herein.
  • the method can further comprise the in vitro assembly of the complex prior to contact with the target polynucleotide, cell, organelle, or embryo.
  • a purified or in vitro assembled RNP complex can be introduced into a cell, organelle, or embryo using any method known in the art, including, but not limited to electroporation.
  • an RGN polypeptide, base editor, PE, and/or polynucleotide encoding or comprising the guide RNA can be introduced into a cell, organelle, or embryo using any method known in the art (e.g., electroporation).
  • delivery of a polynucleotide encoding a guide RNA, PEgRNA, RGN polypeptide, base editor, and/or PE to a cell or embryo is not a method for treatment of the human or animal body by therapy or is not a process for modifying the germ line genetic identity of a human being.
  • a method comprising contacting a target polynucleotide with an RNP complex is not a method for treatment of the human or animal body by therapy or is not a process for modifying the germ line genetic identity of a human being.
  • the embryo is a non-human embryo.
  • the guide RNA directs the RGN, base editor, or PE to bind to the target sequence in a sequence-specific manner.
  • the RGN polypeptide cleaves the target sequence of interest upon binding.
  • the target sequence can subsequently be modified via endogenous repair mechanisms, such as non-homologous end joining, or homology-directed repair with a provided donor polynucleotide.
  • the base editor comprises an RGN nickase
  • a single strand of a double-stranded DNA target sequence is nicked and the base editing polypeptide (e.g., deaminase) of the base editor edits a nucleobase in the target sequence.
  • the PE comprises an RGN nickase
  • a single strand of a double -stranded DNA target sequence is nicked and the DNA synthesis template within the extension arm of the associated PEgRNA is used as a template to introduce a desired edit into the target sequence.
  • Methods to measure binding of an RGN polypeptide to a target sequence include chromatin immunoprecipitation assays, gel mobility shift assays, DNA pull-down assays, reporter assays, microplate capture and detection assays.
  • methods to measure cleavage or modification of a target sequence include in vitro or in vivo cleavage assays wherein cleavage is confirmed using PCR, sequencing, or gel electrophoresis, with or without the attachment of an appropriate label (e.g., radioisotope, fluorescent substance) to the target sequence to facilitate detection of degradation products.
  • an appropriate label e.g., radioisotope, fluorescent substance
  • NTEXPAR nicking triggered exponential amplification reaction
  • the methods involve the use of a single type of RGN, base editor, or PE complexed with more than one guide RNA.
  • the more than one guide RNA can target different regions of a single gene or can target multiple genes.
  • the methods comprise dual polymerase editing wherein a single PE (or a polynucleotide encoding the same) and two PEgRNAs (or polynucleotides encoding the same) that associate with the PE are contacted with a target DNA molecule for the programmable replacement or excision of DNA sequences.
  • a first PE or a polynucleotide encoding the same and its associated first PEgRNA (or a polynucleotide encoding the same) and a second PE (or a polynucleotide encoding the same) and its associated second PEgRNA (or a polynucleotide encoding the same) are contacted with a target DNA molecule, wherein the first and second PEs are different from each other.
  • the two PEgRNAs bind to opposite strands of a target DNA so that the intended repair event is installed on both strands of the target DNA.
  • the DNA synthesis template of the first PEgRNA and the DNA synthesis template of the second PEgRNA each encode a single-stranded DNA sequence that is complementary (full or partial) to each other so that the region of the DNA between the two nicked sites is replaced.
  • the DPE DNA synthesis templates are designed such that the replacement sequence comprises a first recombinase site, and a donor DNA comprising a second recombinase site is introduced, as well as the corresponding site-specific recombinase that recognizes the first and second recombinase site, which results in site-specific integration of exogenous DNA into the target DNA molecule.
  • a double -stranded break introduced by an RGN polypeptide can be repaired by a non-homologous end-joining (NHEJ) repair process. Due to the error-prone nature of NHEJ, repair of the double-stranded break can result in a modification to the target sequence.
  • NHEJ non-homologous end-joining
  • a “modification” in reference to a nucleic acid molecule refers to a change in the nucleotide sequence of the nucleic acid molecule, which can be a deletion, insertion, or substitution of one or more nucleotides, or a combination thereof. Modification of the target sequence can result in the expression of an altered protein product or inactivation of a coding sequence.
  • the donor sequence in the donor polynucleotide can be integrated into or exchanged with the target nucleotide sequence during the course of repair of the introduced double-stranded break, resulting in the introduction of the exogenous donor sequence.
  • a donor polynucleotide thus comprises a donor sequence that is desired to be introduced into a target sequence of interest.
  • the donor sequence alters the original target nucleotide sequence such that the newly integrated donor sequence will not be recognized and cleaved by the RGN.
  • homology arms have a length of at least 30 base pairs, at least 35 base pairs, at least 40 base pairs, at least 45 base pairs, at least 50 base pairs, at least 55 base pairs, at least 60 base pairs, at least 65 base pairs, at least 70 base pairs, at least 75 base pairs, at least 80 base pairs, at least 85 base pairs, at least 90 base pairs, at least 95 base pairs, at least 100 base pairs, and up to 2000 base pairs or more, and have at least
  • the donor polynucleotide can comprise a donor sequence flanked by compatible overhangs, allowing for direct ligation of the donor sequence to the cleaved target nucleotide sequence comprising overhangs by a non-homologous repair process during repair of the double-stranded break.
  • the method can comprise introducing two RGN nickases that target identical or overlapping target sequences and cleave different strands of the polynucleotide.
  • an RGN nickase that only cleaves the positive (+) strand of a double-stranded polynucleotide can be introduced along with a second RGN nickase that only cleaves the negative (-) strand of a double-stranded polynucleotide.
  • a method for binding a target nucleotide sequence and detecting the target sequence, wherein the method comprises introducing into a cell, organelle, or embryo at least one guide RNA or a polynucleotide encoding the same, and at least one RGN polypeptide or a polynucleotide encoding the same, expressing the guide RNA and/or RGN polypeptide (if coding sequences are introduced), wherein the RGN polypeptide is a nuclease-dead RGN and further comprises a detectable label, and the method further comprises detecting the detectable label.
  • the detectable label may be fused to the RGN as a fusion protein (e.g., fluorescent protein) or may be a small molecule conjugated to or incorporated within the RGN polypeptide that can be detected visually or by other means.
  • the methods comprise introducing into a cell, organelle, or embryo at least one guide RNA or a polynucleotide encoding the same, and at least one RGN polypeptide or a polynucleotide encoding the same, expressing the guide RNA and/or RGN polypeptide (if coding sequences are introduced), wherein the RGN polypeptide is a nuclease-dead RGN.
  • the nuclease-dead RGN is a fusion protein comprising an expression modulator domain (z.e., epigenetic modification domain, transcriptional activation domain or a transcriptional repressor domain) as described herein.
  • an expression modulator domain z.e., epigenetic modification domain, transcriptional activation domain or a transcriptional repressor domain
  • the present disclosure also provides methods for binding and/or modifying a target nucleotide sequence of interest.
  • the methods include delivering a system comprising at least one guide RNA or a polynucleotide encoding the same, and at least one fusion polypeptide comprising an RGN of the invention and a base-editing polypeptide, for example a cytosine deaminase or an adenine deaminase, or a polynucleotide encoding the fusion polypeptide, to the target sequence or a cell, organelle, or embryo comprising the target sequence.
  • the methods include delivering a system comprising at least one guide RNA or a polynucleotide encoding the same, and at least one fusion polypeptide comprising an RGN of the invention and a polymerase editing polypeptide, for example a DNA polymerase or a reverse transcriptase, or a polynucleotide encoding the fusion polypeptide, to the target sequence or a cell, organelle, or embryo comprising the target sequence.
  • a polymerase editing polypeptide for example a DNA polymerase or a reverse transcriptase, or a polynucleotide encoding the fusion polypeptide
  • the binding of the fusion polypeptide to a target sequence results in the modification of nucleotide(s) adjacent to the target sequence.
  • the nucleobase adjacent to the target sequence that is modified by the deaminase may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 base pairs from the 5’ or 3’ end of the target sequence.
  • the binding of the PE or PE system results in the modification of nucleotide(s) within or adjacent to the target sequence through the use of a DNA synthesis template of varying lengths such that the editing window using a polymerase editor can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 base pairs from the 5’ or 3’ end of the target sequence.
  • one or more components of the mismatch repair system can be inactivated using, for example, a dominant negative version thereof.
  • a component of the mismatch repair system that can be inactivated or have its activity reduced is MLH1.
  • a dominant negative MLH1 or a polynucleotide encoding the same can be introduced into the cell, along with, preceding or subsequent to the introduction of the PE system.
  • a non-limiting example of a dominant-negative MLH1 is the sequence set forth as SEQ ID NO: 256.
  • the DNA construct encoding the PE comprises the dominant-negative MLH1 -encoding sequence on its N-terminus or its C-terminus and the dominant-negative MLH1 -encoding sequence can connect to the PE-encoding sequence through a peptide linker and/or a NLS.
  • methods comprise the use of a single RGN polypeptide in combination with multiple, distinct guide RNAs, which can target multiple, distinct sequences within a single gene and/or multiple genes. Also encompassed herein are methods wherein multiple, distinct guide RNAs are introduced in combination with multiple, distinct RGN polypeptides. These guide RNAs and guide RNA/RGN polypeptide systems can target multiple, distinct sequences within a single gene and/or multiple genes.
  • the method comprises delivering an RGN system, a base editing system, a PE system, or an RNP complex described herein to a target sequence or to a cell comprising the target sequence.
  • the RGN system, base editing system, PE system, or the RNP complex comprises a variant LPG10145 RGN described herein such that cleavage or modification of the target nucleic acid molecule occurs at greater efficiency as compared to cleavage or modification of the target nucleic acid molecule by a method comprising delivering to the same target nucleic acid molecule a reference RGN system, base editing system, PE system, or RNP complex.
  • the reference RGN system, base editing system, PE system, or RNP complex does not comprise a variant LPG10145 RGN described herein.
  • the reference RGN system, base editing system, PE system, or RNP complex comprises an LPG10145 RGN having the amino acid sequence set forth as SEQ ID NO: 1.
  • the reference RGN system, base editing system, PE system, or RNP complex comprises a variant LPG10145 RGN that is not identical to the variant LPG10145 RGN of the RGN system, base editing system, PE system, or RNP complex being tested for efficiency of cleaing and/or modifying a nucleic acid molecule.
  • the efficiency of cleaving and/or modifying a target sequence is increased by 2 -fold to 100-fold, or 2-fold to 80-fold, or 2-fold to 5-fold. In some embodiments, the efficiency of cleaving and/or modifying a target sequence is increased by 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, or more. In some embodiments, the efficiency of cleaving and/or modifying a target sequence is increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or more.
  • gene editing efficiency is measured by next generation sequencing, Tracking of Indels by DEcomposition (TIDE) analysis, flow cytometry, or a combination thereof.
  • the efficiency of cleaving and/or modifying the target sequence is assessed by measuring the percentage of a target sequence or cells comprising the target sequence that comprise altered expression of the target sequence or of a polypeptide encoded by the target sequence.
  • the expression is measured by quantitative PCR, microarray, RNA-seq, flow cytometry, immunoblot, enzyme-linked immunosorbent assay (ELISA), protein immunoprecipitation, immunostaining, high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC/MS), mass spectrometry, or a combination thereof.
  • the target sequence encodes a cell surface expressed protein, and the efficiency of cleaving and/or modifying the target sequence is assessed by measuring the percentage of cells comprising a reduction of the cell surface expressed protein as measured by flow cytometry.
  • the disclosure provides for methods of modifying a target polynucleotide comprising a target sequence or modifying the expression of a target polynucleotide in a eukaryotic cell, which may be in vivo, ex vivo, or in vitro.
  • the method comprises sampling a cell or population of cells from a human or non-human animal or plant (including microalgae) and modifying the cell or cells. Culturing may occur at any stage ex vivo. The cell or cells may even be re-introduced into the non-human animal or plant (including micro-algae).
  • plant breeders combine most useful genes for desirable qualities, such as yield, quality, uniformity, hardiness, and resistance against pests. These desirable qualities also include growth, day length preferences, temperature requirements, initiation date of floral or reproductive development, fatty acid content, insect resistance, disease resistance, nematode resistance, fungal resistance, herbicide resistance, tolerance to various environmental factors including drought, heat, wet, cold, wind, and adverse soil conditions including high salinity
  • the sources of these useful genes include native or foreign varieties, heirloom varieties, wild plant relatives, and induced mutations, e.g., treating plant material with mutagenic agents.
  • plant breeders are provided with a new tool to induce mutations. Accordingly, one skilled in the art can analyze the genome for sources of useful genes, and in varieties having desired characteristics or traits employ the present invention to induce the rise of useful genes, with more precision than previous mutagenic agents and hence accelerate and improve plant breeding programs.
  • the target polynucleotide of an RGN, base editing, or PE system can be any polynucleotide endogenous or exogenous to the eukaryotic cell.
  • the target polynucleotide can be a polynucleotide residing in the nucleus of the eukaryotic cell.
  • the target polynucleotide can be a sequence coding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or a junk DNA).
  • the target sequence should be associated with a PAM (protospacer adjacent motif); that is, a short sequence recognized by the RGN, base editing, or PE system.
  • PAMs are typically 2-7 base pair sequences adjacent to the protospacer (that is, the target sequence).
  • the target polynucleotide of an RGN, base editing, or PE system may include a number of disease- associated genes and polynucleotides as well as signaling biochemical pathway-associated genes and polynucleotides.
  • target polynucleotides include a sequence associated with a signaling biochemical pathway, e.g., a signaling biochemical pathway-associated gene or polynucleotide.
  • target polynucleotides include a disease associated gene or polynucleotide.
  • a “disease-associated" gene or polynucleotide refers to any gene or polynucleotide which is yielding transcription or translation products at an abnormal level or in an abnormal form in cells derived from a disease-affected tissues compared with tissues or cells of a non-disease control. It may be a gene that becomes expressed at an abnormally high level; it may be a gene that becomes expressed at an abnormally low level, where the altered expression correlates with the occurrence and/or progression of the disease.
  • a disease-associated gene also refers to a gene possessing mutation(s) or genetic variation that is directly responsible or is in linkage disequilibrium with a gene(s) that is responsible for the etiology of a disease (e.g., a causal mutation).
  • the causal mutation may be an expanded repeat (present at higher numbers than those in normal individuals) that leads to instability of transcribed mRNA, altered splicing, or instability of a translated product or a translated product with reduced activity.
  • the transcribed or translated products may be known or unknown, and further may be at a normal or abnormal level.
  • the disease may be an animal disease.
  • the disease may be an avian disease.
  • the disease may be a mammalian disease.
  • the disease may be a human disease.
  • RGN systems are particularly useful for their relative ease in targeting to genomic sequences of interest, there still remains an issue of what the RGN can do to address a causal mutation.
  • One approach is to produce a fusion protein between an RGN (preferably an inactive or nickase variant of the RGN) and a base-editing enzyme or the active domain of a base editing enzyme, such as a cytosine deaminase or an adenine deaminase base editor (U.S. Patent No. 9,840,699, herein incorporated by reference).
  • the methods comprise contacting a DNA molecule with (a) a fusion protein comprising a variant LPG10145 RGN of the invention or a nickase variant thereof and a base-editing polypeptide such as a deaminase; and (b) a gRNA targeting the fusion protein of (a) to a target nucleotide sequence of the DNA molecule; wherein the DNA molecule is contacted with the fusion protein and the gRNA in an amount effective and under conditions suitable for the deamination of a nucleobase.
  • the target DNA sequence comprises a sequence associated with a disease or disorder, and wherein the deamination of the nucleobase results in a sequence that is not associated with a disease or disorder.
  • the target DNA sequence resides in an allele of a crop plant, wherein the particular allele of the trait of interest results in a plant of lesser agronomic value. The deamination of the nucleobase results in an allele that improves the trait and increases the agronomic value of the plant.
  • a fusion protein can be generated comprising an RGN (preferably an inactive or nickase variant of the RGN) and a polymerase editing polypeptide, such as a DNA polymerase or reverse transcriptase as described herein.
  • the methods comprise contacting a DNA molecule with (a) a fusion protein comprising a variant LPG10145 RGN of the disclosure or a nickase variant thereof and a polymerase editing polypeptide such as a reverse transcriptase; and (b) a PEgRNA targeting the fusion protein of (a) to a target nucleotide sequence of the DNA molecule; wherein the DNA molecule is contacted with the fusion protein and the PEgRNA in an amount effective and under conditions suitable for the editing of the target nucleotide sequence.
  • the target DNA sequence comprises a sequence associated with a disease or disorder, and wherein the editing of the target DNA sequence results in a sequence that is not associated with a disease or disorder.
  • the target DNA sequence resides in an allele of a crop plant, wherein the particular allele of the trait of interest results in a plant of lesser agronomic value. The editing of the target DNA sequence results in an allele that improves the trait and increases the agronomic value of the plant.
  • the target DNA sequence comprises a T->C or A->G point mutation associated with a disease or disorder, and wherein the deamination of the mutant C or G base or editing of the target DNA sequence comprising the point mutation results in a sequence that is not associated with a disease or disorder. In some embodiments, the deamination or editing corrects a point mutation in the sequence associated with the disease or disorder.
  • the sequence associated with the disease or disorder encodes a protein
  • the deamination or editing introduces a stop codon into the sequence associated with the disease or disorder, resulting in a truncation of the encoded protein.
  • the contacting is performed in vivo in a subject susceptible to having, having, or diagnosed with the disease or disorder.
  • the disease or disorder is a disease associated with a point mutation, or a single-base mutation, in the genome.
  • the disease is a genetic disease, a cancer, a metabolic disease, or a lysosomal storage disease.
  • compositions of the disclosure can comprise: the presently disclosed variant LPG10145 RGN polypeptides and active variants and fragments thereof (or base editors or PEs comprising the same), as well as polynucleotides encoding the same; the presently disclosed gRNAs, or active variants and fragments thereof, or polynucleotides encoding the same; the presently disclosed RGN systems comprising variant LPG10145 RGN polypeptides and/or gRNAs; base editing systems comprising variant LPG10145 RGN polypeptides and/or gRNAs described herein; PE systems comprising variant LPG10145 RGN polypeptides and/or PEgRNAs described herein (including DPE systems); or cells comprising any of the variant LPG10145 RGN polypeptides (or base editors or PEs comprising the same) or variant LPG10145 RGN-encoding, polymerase-encoding, base editing polypeptide -encoding polynucleotides, gRNA or gRNA- encoding
  • a pharmaceutical composition is a composition that is employed to prevent, reduce in intensity, cure or otherwise treat a target condition or disease that comprises an active ingredient (i.e., RGN polypeptides, RGN-encoding polynucleotides, base editors, base editor-encoding polynucleotides, PEs, PE-encoding polynucleotides, gRNA, gRNA-encoding polynucleotides, RNP complexes, RGN systems, base editing systems, PE systems, or cells comprising any one of these) and a pharmaceutically acceptable carrier.
  • an active ingredient i.e., RGN polypeptides, RGN-encoding polynucleotides, base editors, base editor-encoding polynucleotides, PEs, PE-encoding polynucleotides, gRNA, gRNA-encoding polynucleotides, RNP complexes, RGN systems, base editing systems, PE systems, or cells comprising any one of these
  • a “pharmaceutically acceptable carrier” refers to a material that does not cause significant irritation to an organism and does not abrogate the activity and properties of the active ingredient (i.e., RGN polypeptides, RGN-encoding polynucleotides, base editors, base editor-encoding polynucleotides, PEs, PE-encoding polynucleotides, gRNA, gRNA-encoding polynucleotides, RNP complexes, RGN systems, base editing systems, PE systems, or cells comprising any one of these).
  • Carriers must be of sufficiently high purity and of sufficiently low toxicity to render them suitable for administration to a subject being treated.
  • the carrier can be inert, or it can possess pharmaceutical benefits.
  • a pharmaceutically acceptable carrier comprises one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration to a human or other vertebrate animal.
  • the pharmaceutically acceptable carrier is not naturally-occurring.
  • the pharmaceutically acceptable carrier and the active ingredient are not found together in nature.
  • compositions used in the presently disclosed methods can be formulated with suitable carriers, excipients, and other agents that provide suitable transfer, delivery, tolerance, and the like.
  • suitable carriers excipients, and other agents that provide suitable transfer, delivery, tolerance, and the like.
  • suitable carriers excipients, and other agents that provide suitable transfer, delivery, tolerance, and the like.
  • Suitable formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTIN vesicles), lipid nanoparticles, DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax.
  • Pharmaceutical compositions for oral or parenteral use may be prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.
  • compositions comprising lipid-based formulations including an active ingredient (i.e., RGN polypeptides, RGN-encoding polynucleotides, base editors, base editor-encoding polynucleotides, PEs, PE-encoding polynucleotides, gRNA, gRNA-encoding polynucleotides, RNP complexes, RGN systems, base editing systems, PE systems, or cells comprising any one of these).
  • the lipid-based formulations can include liposomes.
  • the lipid-based formulations can include lipid nanoparticles (LNPs).
  • an active ingredient is encapsulated in the lipid particle and/or disposed on the surface of the lipid particle.
  • an active ingredient is covalently attached to the lipid particle.
  • an active ingredient is non-covalently associated with the lipid particle.
  • a covalent attachment includes the sharing of electrons in a chemical bond.
  • Non-covalent interactions include dispersed electromagnetic interactions such as hydrogen bonds, ionic bonds, van der Waals interactions, and hydrophobic bonds.
  • an active ingredient is encapsulated in the lipid particle.
  • encapsulate means to enclose, surround or encase. As it relates to the formulation of the compounds of the disclosure, encapsulation may be substantial, complete or partial.
  • substantially encapsulated or substantially encapsulation means that greater than 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or greater of the pharmaceutical composition or active ingredient of the disclosure may be enclosed, surrounded, or encased within a delivery agent (e.g., liposome or LNP).
  • a delivery agent e.g., liposome or LNP
  • partial encapsulated means that less than 50%, 40%, 30%, 20%, 10%, or less of the pharmaceutical composition or active ingredient of the disclosure may be enclosed, surrounded, or encased within the delivery agent. Encapsulation may be determined by measuring the escape or the activity of the pharmaceutical composition or active ingredient of the disclosure using fluorescence and/or electron microscopy. For example, at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or greater of the pharmaceutical composition or active ingredient of the disclosure is encapsulated in a delivery agent (e.g., liposome or LNP).
  • a delivery agent e.g., liposome or LNP
  • Liposomes are spherical vesicle structures composed of a uni- or multilamellar lipid bilayer surrounding internal aqueous compartments and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes have gained considerable attention as drug delivery carriers because they are biocompatible, nontoxic, can deliver both hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes, and transport their load across biological membranes and the blood brain barrier (BBB) (see, e.g., Spuch and Navarro (2011) Journal of drug delivery 2011).
  • BBB blood brain barrier
  • Liposomes can be made from several different types of lipids (e.g. ionizable lipids, structural lipids, helper lipids, and pegylated lipids); however, phospholipids are most commonly used to generate liposomes as drug carriers. Although liposome formation is spontaneous when a lipid fdm is mixed with an aqueous solution, it can also be expedited by applying force in the form of shaking by using a homogenizer, sonicator, or an extrusion apparatus (see, e.g., Spuch and Navarro (2011) Journal of drug delivery 2011).
  • lipids e.g. ionizable lipids, structural lipids, helper lipids, and pegylated lipids
  • phospholipids are most commonly used to generate liposomes as drug carriers.
  • liposome formation is spontaneous when a lipid fdm is mixed with an aqueous solution, it can also be expedited by applying force in the form of shaking by using
  • a conventional liposome formulation is mainly comprised of natural phospholipids and phospholipids such as l,2-distearoryl-5 «-glycero-3 -phosphatidyl choline (DSPC), sphingomyelin, egg phosphatidylcholines, and monosialoganglioside.
  • DSPC distalo-3 -phosphatidyl choline
  • DOPE diolcoyl-s'w-glyccro-3- phosphoethanolamine
  • Additives may be added to liposomes in order to modify their structure and properties.
  • cholesterol and/or sphingomyelin may be added to a liposomal mixture to help stabilize the liposomal structure and to prevent leakage of the liposomal inner cargo.
  • addition of cholesterol to a conventional liposome formulation reduces rapid release of the encapsulated active ingredient (i.e., RGN polypeptides, RGN-encoding polynucleotides, base editors, base editor-encoding polynucleotides, PEs, PE-encoding polynucleotides, gRNA, gRNA-encoding polynucleotides, RNP complexes, RGN systems, base editing systems, PE systems, or cells comprising any one of these) into the plasma.
  • liposomes are prepared from hydrogenated egg phosphatidylcholine or egg phosphatidylcholine, cholesterol, and dicetyl phosphate.
  • mean liposome vesicle size is adjusted to about 50 or 100 nm.
  • Trojan Horse liposomes also known as Molecular Trojan Horses or PEGylated immunoliposomes
  • BBB blood brain barrier
  • compositions comprising Trojan Horse liposomes may be used to deliver an active ingredient (i.e., RGN polypeptides, RGN-encoding polynucleotides, base editors, base editor-encoding polynucleotides, PEs, PE-encoding polynucleotides, gRNA, gRNA-encoding polynucleotides, RNP complexes, RGN systems, base editing systems, PE systems, or cells comprising any one of these) to the brain via an intravascular injection.
  • an active ingredient i.e., RGN polypeptides, RGN-encoding polynucleotides, base editors, base editor-encoding polynucleotides, PEs, PE-encoding polynucleotides, gRNA, gRNA-encoding polynucleotides, RNP complexes, RGN systems, base editing systems, PE systems, or cells comprising any one of these
  • liposomes include stable nucleic-acid-lipid particles (SNALP) (see, e.g., Morrissey et al. (2005) Nature Biotechnology 23(8): 1002-1007; Zimmerman et al. (2006) Nature 441: 111- 114).
  • SNALPs include a mixture of cationic and fusogenic lipids and coated with polyethylene glycol (PEG) that allow cellular uptake and endosomal release of an active ingredient cargo.
  • PEG polyethylene glycol
  • a SNALP is a class of LNP and includes an ionizable lipid that is cationic at low pH (e.g., DLinDMA, COATSOME® SS-OC), a neutral helper lipid (e.g.
  • a SNALP formulation includes the following lipids: 3- JV-(-methoxy poly(ethylene glycol)2000) carbamoyl- 1,2-dimyrestyloxy-propylamine (PEG-cDMA); 1,2- dilinolcyloxy- '. '-dimcthyl-3-aminopropanc (DLinDMA); l,2-distearoyl-5 «-glycero-3-phosphocholine (DSPC); and cholesterol.
  • a SNALP includes synthetic cholesterol, dipalmitoylphosphatidylcholine (DOPC), PEG-cDMA, and DLinDMA (see, e.g., Geisbert et al. (2010) Lancet 375: 1896-1905).
  • a SNALP includes synthetic cholesterol, DSPC, PEG- cDMA, and DLinDMA (see, e.g., Judge et al. (2009) J. Clin. Invest. 119:661-673).
  • a SNALP formulation includes COATSOME® SS-OC, DSPC, Brij S100, and cholesterol.
  • a SNALP formulation includes an ionizable lipid, DSPC, cholesterol, and a PEG lipid.
  • a SNALP formulation includes the ionizable lipids and/or PEG lipids disclosed in WO2022173531 or WO2022173531 each of which is herein incorporated by reference in its entirety.
  • SNALP liposomes are about 80-100 nm in size. SNALPs have been used as effective delivery molecules to highly vascularized HepG2 -derived liver tumors (see, e.g., Li et al. (2012) Gene Therapy 19:775- 780).
  • the ionizable lipid serves to condense lipid with an active ingredient (e.g., a polynucleotide) during particle formation.
  • an active ingredient e.g., a polynucleotide
  • the ionizable lipid may mediate the fusion of a SNALP with the endosomal membrane, enabling release of the active ingredient into the cytoplasm.
  • the PEG-lipid may stabilize the particle and reduce aggregation during formulation, and subsequently may provide a neutral hydrophilic exterior that improves pharmacokinetic properties.
  • SNALP liposomes are prepared by formulating DLinDMA and PEG-cDMA with DSPC, cholesterol and an active ingredient using a 25: 1 lipid: active ingredient ratio and a 48:40: 10:2 molar ratio of cholesterol: DLinDMA: DSPC: PEG-cDMA.
  • a pharmaceutical composition of the disclosure includes LNPs.
  • lipids may be formulated with an active ingredient of the present disclosure to form LNPs.
  • An LNP comprises a plurality of lipid molecules physically associated with each other by intermolecular forces.
  • LNPs include liposomes.
  • LNPs differ from liposomes in not having a continuous lipid bilayer.
  • LNPs comprise solid particles having a mixture of solid and liquid lipids.
  • LNPs include dendrimer lipid nanoparticles (DLNPs), SNALPs, and lipid-like nanoparticles (LLNPs).
  • nanoparticle refers to any particle having a diameter of less than 1000 nanometers (nm). In some embodiments, nanoparticles have a diameter of 500 nm or less. In some embodiments, nanoparticles have a diameter ranging between 25 nm and 200 nm, or 100 nm or less. In some embodiments, nanoparticles have a diameter ranging between 35 nm and 60 nm. In some embodiments, an LNP includes a lipid particle between about 1 and about 100 nm in size.
  • LNPs include four components: ionizable cationic lipids, fusogenic zwitterionic phospholipids, cholesterol, and PEGylated (PEG) lipids.
  • the ionizable cationic lipid component complexes a negatively charged polynucleotide and enhances endosomal escape).
  • the phospholipid component functions in modifying lipid bilayer structure.
  • the cholesterol component helps to stabilize an LNP.
  • the PEG lipid component decreases LNP aggregation and non-specific uptake.
  • the LNP includes an ionizable lipid that is cationic at low pH (e.g., DLinDMA, COATSOME® SS-OC), a neutral helper lipid (e.g. DSPC), cholesterol, and a diffusible polyethylene glycol (PEG)-lipid (e.g. Brij S100).
  • an ionizable lipid that is cationic at low pH e.g., DLinDMA, COATSOME® SS-OC
  • a neutral helper lipid e.g. DSPC
  • cholesterol e.g., a diffusible polyethylene glycol (PEG)-lipid
  • PEG polyethylene glycol
  • the LNP includes the following lipids: 3-A'-(-mcthoxy poly(ethylene glycol)2000) carbamoyl- 1,2-dimyrestyloxy-propylamine (PEG-cDMA); 1 ,2-dil inoley loxy- 'A'-dimcthy 1 -3 -aminopropane (DLinDMA) ; 1 ,2-distearoyl-5 «-glycero-3 -phosphocholine (DSPC); and cholesterol.
  • the LNP includes synthetic cholesterol, dipalmitoylphosphatidylcholine (DOPC), PEG-cDMA, and DLinDMA (see, e.g., Geisbert et al.
  • the LNP includes synthetic cholesterol, DSPC, PEG-cDMA, and DLinDMA (see, e.g., Judge et al. (2009) J. Clin. Invest. 119:661-673).
  • Ionizable cationic lipids useful in LNPs include: COATSOME® SS-OC, l,2-dilineoyl-3- dimethylammonium-propane (DLinDAP); DLinDMA; l.2-dilinolcyloxy-kcto-A.A-dimcthyl-3-aminopropanc (DlinK-DMA); l,2-dilinoleyl-4-(2-dimethylaminoethyl)-[ 1,3] -dioxolane (DlinKC2-DMA); 5A2-SC8 (Zhou et al. (2016) Proc. Natl Acad. Sci.
  • DSDMA 1,2- diolcyloxy- 'A' -dimethyl-3 -aminopropane
  • DODMA 1,2- diolcyloxy- 'A' -dimethyl-3 -aminopropane
  • DLenDMA 1.2-dilinolcnyloxy- 'A' -dimethyl-3 -aminopropane
  • Dlin-MC3-DMA dilinoleylmethyl-4-dimethylaminobutyrate
  • Cationic lipids are further described in International Publication Nos. WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638, W02010080724, W0201021865, WO2022173531, WO2022150485 and W02008103276, US Patent Nos. 7,893,302 and 7,404,969 and US Patent Publication No. US20100036115, each of which is herein incorporated by reference in its entirety.
  • Zwitterionic phospholipids useful for LNPs include DSPC, DOPE, and DOPC.
  • PEG lipids useful for LNPs include: l,2-dimyristoyl-rac-glycero-3 -methoxypolyethylene glycol (PEG-DMG); (3-o-[2 - (methoxypolyethyleneglycol 2000) succinoyl]-l,2-dimyristoyl-sn-glycol (PEG-S-DMG); R-3-[(co-methoxy- poly(ethylene glycol)2000) carbamoyl] -l,2-dimyristyloxlpropyl-3 -amine (PEG-C-DOMG); and C16 PEG- ceramide.
  • an LNP includes 50: 10: 38.5: 1.5 molar ratio of DlinKC2-DMA or C12- 200: DSPC: cholesterol: PEG-DMG (see, e.g., Basha et al. (2011) Molecular Therapy 19(12):2186-2200).
  • an LNP includes 26.5: 20: 52: 1.5 ionizable lipid: DOPE: cholesterol: PEG lipid (see, e.g., Han et al. (2022) Sci Adv 8(3): eabj6901; Kim et al. (2021) Sci Adv 7(9): eabf4398).
  • PEG lipids are further described in WO2012099755, WO2022173531 and WO2022150485 each of which is herein incorporated by reference in its entirety.
  • the ratio of PEG in the LNP formulations may be increased or decreased and/or the carbon chain length of the PEG lipid may be modified from C14 to Cl 8 to alter the pharmacokinetics and/or biodistribution of the LNP formulations.
  • a LNP formulation includes COATSOME® SS-OC, DSPC, Brij S100, and cholesterol.
  • a LNP formulation includes an ionizable lipid, DSPC, cholesterol, and a PEG lipid.
  • a LNP formulation includes the ionizable lipids and/or PEG lipids disclosed in WO2022173531 or WO2022150485 each of which is herein incorporated by reference in its entirety.
  • a LNP formulation includes the ionizable lipids and/or PEG lipids disclosed in WO2022173531 or WO2022150485 (each of which is herein incorporated by reference in its entirety), DSPC, and cholesterol.
  • a LNP formulation includes any one of CAT1-CAT35 and any one of CHM-001 to CHM-016 disclosed in WO2022173531 or WO2022150485 each of which is herein incorporated by reference in its entirety.
  • a LNP formulation includes any one of CAT1-CAT35 and any one of CHM-001 to CHM-016 disclosed in WO2022173531 or WO2022150485 (each of which is herein incorporated by reference in its entirety), DSPC, and cholesterol.
  • the LNP of the disclosure comprises 44-60 mol % of the cationic lipid, 19-25 mol % of the helper lipid, 25-33 mol % of the structural lipid, and 0.2-0.8 mol % of the PEG-lipid, inclusive of the endpoints. In some embodiments, the LNP of the disclosure comprises 44-54 mol % of the cationic lipid, 19-25 mol % of the helper lipid, 24-32 mol % of the structural lipid, and 1.2-1.8 mol % of the PEG- lipid, inclusive of the endpoints.
  • the LNP of the disclosure comprises 44-54 mol % of the cationic lipid, 8-14 mol % of the helper lipid, 35-43 mol % of the structural lipid, and 1.2-1.8 mol % of the PEG-lipid, inclusive of the endpoints. In some embodiments, the LNP of the disclosure comprises 45-55 mol % of the cationic lipid, 5-9 mol % of the helper lipid, 36-44 mol % of the structural lipid, and 2.5-3.5 mol % of the PEG-lipid, inclusive of the endpoints.
  • the LNP of the disclosure comprises 49 mol % of the cationic lipid, 22 mol % of the helper lipid, 28.5 % of the structural lipid, and .05 mol % of the PEG-lipid. In some embodiments, the LNP of the disclosure comprises 49 mol % of SS-OC, 22 mol % DSPC, 28.5 mol % cholesterol, and .05 mol % Brij S100.
  • the LNP of the disclosure comprises 50 mol % of the cationic lipid, 7 mol % of the helper lipid, 40 % of the structural lipid, and 3 mol % of the PEG-lipid. In some embodiments, the LNP of the disclosure comprises 50 mol % of the ionizable lipid, 7 mol % DSPC, 40 mol % cholesterol, and .05 mol % the PEG-lipid.
  • the LNP of the disclosure comprises 50 mol % any one of CAT1-CAT35 disclosed in WO2022173531 or WO2022150485 (each of which is herein incorporated by reference in its entirety), 7 mol % DSPC, 40 mol % cholesterol, and .05 mol % any one of CHM-001 - CHM-016 disclosed in WO2022173531 or WO2022150485 (each of which is herein incorporated by reference in its entirety).
  • the LNP of the disclosure comprises 50 mol % CAT7 disclosed in WO2022173531 or WO2022150485 (each of which is herein incorporated by reference in its entirety), 7 mol % DSPC, 40 mol % cholesterol, and .05 mol % CHM-006 disclosed in WO2022173531 or WO2022150485 (each of which is herein incorporated by reference in its entirety).
  • LNPs are about 80-100 run in size. In some embodiments, the LNPs are about 80 run, about 81 nm, about 82 nm, about 83 nm, about 84 nm, about 85 nm, about 86 nm, about 87 nm, about 88 nm, about 89 nm, about 90 nm, about 91 nm, about 92 nm, about 93 nm, about 94 nm, about 95 nm, about 96 nm, about 97 nm, about 98 nm, about 99 nm, or about 100 nm in size.
  • the individual LNPs in a population of LNPs may vary in size by about +5 nm.
  • the charge of an LNP is taken into consideration.
  • Cationic lipids may combine with negatively charged lipids to induce non-bilayer structures that facilitate intracellular delivery. Because charged LNPs are rapidly cleared from circulation following intravenous injection, ionizable cationic lipids with pKa values below 7 were developed (see, e.g., Basha et al. (2011) Molecular Therapy 19( 12):2186-2200). Negatively charged polymers such as polynucleotides may be loaded into LNPs at low pH values (e.g., pH 4) where the ionizable lipids display a positive charge. However, at physiological pH values, the LNPs exhibit a low surface charge compatible with longer circulation times.
  • Lipids are commercially available (e.g., from Tekmira Pharmaceuticals, Vancouver, Canada; Avanti Polar Lipids, Inc., Alabaster, AL) or may be synthesized (e.g., Kim et al. (2021) Sci Adv 7(9): eabf4398). Synthesis of cationic lipids are also described in International Publication Nos. W02012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638, W02010080724 and W0201021865. Cholesterol is commercially available (e.g., from Sigma-Aldrich, St Louis, MO).
  • encapsulation may be performed by dissolving lipid mixtures comprising cationic lipid (e.g., Dlin-DMA): phospholipid (e.g., DSPC, DOPE): cholesterol: PEG-lipid (e.g., at 40: 10: 40: 10 molar ratio) in ethanol.
  • An active ingredient e.g., a polynucleotide comprising or encoding a guide RNA or RGN of the disclosure
  • an acidic buffer e.g., citrate, acetate
  • the lipid solution and active ingredient solution may be mixed using a microfluidics system (Chen et al. (2012) J. Amer. Chem.
  • Removal of ethanol and neutralization of formulation buffer may be performed by dialysis for, e.g., 16 hours or overnight, against phosphate-buffered saline (PBS) using dialysis cassettes (e.g., 3500 molecular weight cutoff cassettes from Life Technologies).
  • Dynamic light scattering may be used to assess LNP size, polydispersity index (PDI), and zeta potential.
  • Encapsulation efficiency of an active ingredient such as RNA may be determined by assays such as Quant-itTM Ribogreen Assay (Thermo Fisher).
  • the polynucleotide may be extracted from the eluted nanoparticles and quantified at 260 nm.
  • LNP pKa may be assessed using a 2-(p-toluidino)-6- napthalene sulfonic acid (TNS) assay (Zhang et al. (2011) Langmuir 27(5): 1907-1914).
  • a final lipid: active ingredient weight ratio includes 12: 1, 11: 1, 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, and 5: 1.
  • a pharmaceutical composition comprises a ribonucleoprotein (RNP) complex encapsulated in an LNP
  • inclusion of an additional permanent cationic lipid e.g., l,2-dioleoyl-3- trimethylammonium-propane (DOTAP)
  • DOTAP l,2-dioleoyl-3- trimethylammonium-propane
  • the permanent cationic lipid is included at 10 to 20 mole % of total lipids in LNPs.
  • the LNP formulations described herein may additionally comprise a permeability enhancer molecule. Non-limiting permeability enhancer molecules are described in US2005/0222064.
  • the LNP compositions are biodegradable, in that they do not accumulate to cytotoxic levels in vivo at a therapeutically effective dose.
  • LNP formulations may be improved by replacing the cationic lipid with a biodegradable cationic lipid which is known as a rapidly eliminated lipid nanoparticle (reLNP).
  • reLNP rapidly eliminated lipid nanoparticle
  • the rapid metabolism of the rapidly eliminated lipids can improve the tolerability and therapeutic index of LNPs by an order of magnitude from a 1 mg/kg dose to a 10 mg/kg dose in rat.
  • Inclusion of an enzymatically degraded ester linkage can improve the degradation and metabolism profile of the cationic component, while still maintaining the activity of the reLNP formulation.
  • the ester linkage can be internally located within the lipid chain or it may be terminally located at the terminal end of the lipid chain. The internal ester linkage may replace any carbon in the lipid chain.
  • the LNP compositions do not cause an innate immune response that leads to substantial adverse effects at a therapeutic dose level. In some embodiments, the LNP compositions provided herein do not cause toxicity at a therapeutic dose level.
  • the active ingredient i.e., RGN polypeptides, RGN-encoding polynucleotides, base editors, base editor-encoding polynucleotides, PEs, PE-encoding polynucleotides, gRNA, gRNA-encoding polynucleotides, RNP complexes, RGN systems, base editing systems, PE systems, or cells comprising any one of these
  • a solid lipid nanoparticle may be spherical with an average diameter between 10 to 1000 nm. SLN possess a solid lipid core matrix that can solubilize lipophilic molecules and may be stabilized with surfactants and/or emulsifiers.
  • the lipid nanoparticle may be a self-assembly lipidpolymer nanoparticle (see, e.g., Zhang et al. (2008) ACS Nano 2(8): 1696-1702).
  • a lipid-based formulation including an active ingredient i.e., RGN polypeptides, RGN-encoding polynucleotides, base editors, base editor-encoding polynucleotides, PEs, PE- encoding polynucleotides, gRNA, gRNA-encoding polynucleotides, RNP complexes, RGN systems, base editing systems, PE systems, or cells comprising any one of these
  • an active ingredient i.e., RGN polypeptides, RGN-encoding polynucleotides, base editors, base editor-encoding polynucleotides, PEs, PE- encoding polynucleotides, gRNA, gRNA-encoding polynucleotides, RNP complexes, RGN systems, base editing systems, PE systems, or cells comprising any one of these
  • controlled release refers to a pharmaceutical composition or compound release profile that conforms to a particular pattern of release to effect a therapeutic outcome.
  • a lipid-based formulation including an active ingredient includes at least one controlled release coating.
  • an active ingredient i.e., RGN polypeptides, RGN-encoding polynucleotides, base editors, base editor-encoding polynucleotides, PEs, PE- encoding polynucleotides, gRNA, gRNA-encoding polynucleotides, RNP complexes, RGN systems, base editing systems, PE systems, or cells comprising any one of these
  • an active ingredient i.e., RGN polypeptides, RGN-encoding polynucleotides, base editors, base editor-encoding polynucleotides, PEs, PE- encoding polynucleotides, gRNA, gRNA-encoding polynucleotides, RNP complexes, RGN systems, base editing systems, PE systems, or cells comprising any one of these
  • at least one controlled release coating includes at least one controlled release coating.
  • Controlled release coatings include: OPADRY® (Colorcon Inc., Harleysville, PA); polyvinylpyrrolidone/vinyl acetate copolymer; polyvinylpyrrolidone; hydroxypropyl methylcellulose; hydroxypropyl cellulose; hydroxyethyl cellulose; EUDRAGIT RL® (Evonik, Essen, Germany); EUDRAGIT RS® (Evonik, Essen, Germany); and cellulose derivatives such as ethylcellulose aqueous dispersions (AQUACOAT® and SURELEASE®, Colorcon Inc., Harleysville, PA).
  • the controlled release and/or targeted delivery formulation may comprise at least one degradable polyester which may contain polycationic side chains.
  • Degradable polyesters include poly(serine ester), poly(L- lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), and combinations thereof.
  • the degradable polyesters may include a PEG conjugation to form a PEGylated polymer.
  • LNP formulations may be prepared such that they passively or actively are directed to different cell types in vivo, including hepatocytes, immune cells, tumor cells, endothelial cells, antigen presenting cells, and leukocytes (Akinc et al. (2010) Mol Ther. 18: 1357-1364; Song et al. (2005) Nat Biotechnol. 23:709-717; Judge et al. (2009) J Clin Invest. 119:661-673; Kaufmann et al. (2010) Microvasc Res 80:286-293; Santel et al. (2006) Gene Ther 13: 1222-1234; Santel et al. (2006) Gene Ther 13: 1360-1370; Gutbier et al. (2010) Pulm Pharmacol.
  • One example of passive targeting of formulations to liver cells includes the Dlin- DMA, Dlin-KC2-DMA and MC3 -based lipid nanoparticle formulations which have been shown to bind to apolipoprotein E and promote binding and uptake of these formulations into hepatocytes in vivo (Akinc et al. (2010) Mol Ther. 18: 1357-1364).
  • LNP formulations can also be selectively targeted through expression of different ligands on their surface such as folate, transferrin, N-acetylgalactosamine (GalNAc), and antibody targeted approaches (Kolhatkar et al. (2011) Curr Drug Discov Technol. 8: 197-206; Musacchio and Torchilin (2011) Front Biosci. 16: 1388-1412; Yu et al. (2010) Mol Membr Biol. 27:286-298; Patil et al. (2008) Crit Rev Ther Drug Carrier Syst. 25: 1- 61; Benoit et al. (2011) Biomacromolecules. 12:2708-2714; Zhao et al. (2008) Expert Opin Drug Deliv.
  • an active ingredient i.e., RGN polypeptides, RGN-encoding polynucleotides, base editors, base editor-encoding polynucleotides, PEs, PE-encoding polynucleotides, gRNA, gRNA-encoding polynucleotides, RNP complexes, RGN systems, base editing systems, PE systems, or cells comprising any one of these
  • an active ingredient i.e., RGN polypeptides, RGN-encoding polynucleotides, base editors, base editor-encoding polynucleotides, PEs, PE-encoding polynucleotides, gRNA, gRNA-encoding polynucleotides, RNP complexes, RGN systems, base editing systems, PE systems, or cells comprising any one of these
  • an active ingredient i.e., RGN polypeptides, RGN-encoding polynucleotides, base editors, base editor-encoding polynucleotides
  • the polymer, hydrogel or surgical sealant includes: poly(lactic-co- glycolic acid (PLGA); ethylene vinyl acetate (EV Ac); poloxamer; GELSITE® (Nanotherapeutics, Inc. Alachua, FL); HYLENEX® (Halozyme Therapeutics, San Diego CA); surgical sealants such as fibrinogen polymers (Ethicon Inc., Cornelia, GA) and TISSELL® (Baxter International, Inc Deerfield, IL); PEG-based sealants; and COSEAL® (Baxter International, Inc Deerfield, IL).
  • PLGA poly(lactic-co- glycolic acid
  • EV Ac ethylene vinyl acetate
  • poloxamer GELSITE® (Nanotherapeutics, Inc. Alachua, FL)
  • HYLENEX® HyLENEX® (Halozyme Therapeutics, San Diego CA)
  • surgical sealants such as fibrinogen polymers (Ethicon Inc., Cornelia, GA) and
  • LNPs and LNP formulations are further described in, e.g., U.S. Pat. Nos. 7,982,027; 7,799,565; 8,058,069; 8,283,333; 7,901,708; 7,745,651; 7,803,397; 8,101,741; 8,188,263; 7,915,399; 8,236,943 and 7,838,658; European Pat. Nos. 1766035; 1519714; 1781593; and 1664316.
  • cells comprising or modified with the presently disclosed RGN polypeptides, base editors, PEs, gRNAs, RNP complexes, RGN systems, base editing systems, PE systems, or polynucleotides encoding the same are administered to a subject
  • the cells are administered as a suspension with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier to be used in a cell composition will not include buffers, compounds, cryopreservation agents, preservatives, or other agents in amounts that substantially interfere with the viability of the cells to be delivered to the subject.
  • a formulation comprising cells can include e.g., osmotic buffers that permit cell membrane integrity to be maintained, and optionally, nutrients to maintain cell viability or enhance engraftment upon administration.
  • osmotic buffers that permit cell membrane integrity to be maintained, and optionally, nutrients to maintain cell viability or enhance engraftment upon administration.
  • Such formulations and suspensions are known to those of skill in the art and/or can be adapted for use with the cells described herein using routine experimentation.
  • a cell composition can also be emulsified or presented as a liposome composition, provided that the emulsification procedure does not adversely affect cell viability.
  • the cells and any other active ingredient can be mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient, and in amounts suitable for use in the therapeutic methods described herein.
  • Additional agents included in a cell composition can include pharmaceutically acceptable salts of the components therein.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids, such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases, such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.
  • Physiologically tolerable and pharmaceutically acceptable carriers are well known in the art.
  • Exemplary liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline.
  • aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes.
  • Liquid compositions can also contain liquid phases in addition to and to the exclusion of water.
  • Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions.
  • the amount of an active compound used in the cell compositions that is effective in the treatment of a particular disorder or condition can depend on the nature of the disorder or condition, and can be determined by standard clinical techniques.
  • RGN polypeptides can be formulated with pharmaceutically acceptable excipients such as carriers, solvents, stabilizers, adjuvants, diluents, etc., depending upon the particular mode of administration and dosage form.
  • these pharmaceutical compositions are formulated to achieve a physiologically compatible pH, and range from a pH of about 3 to a pH of about 11, about pH 3 to about pH 7, depending on the formulation and route of administration.
  • the pH can be adjusted to a range from about pH 5.0 to about pH 8.
  • compositions can comprise a therapeutically effective amount of at least one compound as described herein, together with one or more pharmaceutically acceptable excipients.
  • compositions comprise a combination of the compounds described herein, or include a second active ingredient useful in the treatment or prevention of bacterial growth (for example and without limitation, anti-bacterial or anti-microbial agents), or include a combination of reagents of the present disclosure.
  • Suitable excipients include, for example, carrier molecules that include large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles.
  • Other exemplary excipients can include antioxidants (for example and without limitation, ascorbic acid), chelating agents (for example and without limitation, EDTA), carbohydrates (for example and without limitation, dextrin, hydroxyalkylcellulose, and hydroxyalkylmethylcellulose), stearic acid, liquids (for example and without limitation, oils, water, saline, glycerol and ethanol), wetting or emulsifying agents, pH buffering substances, and the like.
  • the formulations are provided in unit-dose or multi-dose containers, for example sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring the addition of the sterile liquid carrier, for example, saline, water-for-inj ection, a semi-liquid foam, or gel, immediately prior to use.
  • sterile liquid carrier for example, saline, water-for-inj ection, a semi-liquid foam, or gel
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • the active ingredient is dissolved in a buffered liquid solution that is frozen in a unit-dose or multi -dose container and later thawed for injection or kept/stabilized under refrigeration until use.
  • the therapeutic agent(s) may be contained in controlled release systems.
  • delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
  • the use of a long-term sustained release implant may be particularly suitable for treatment of chronic conditions. Long-term sustained release implants are well-known to those of ordinary skill in the art.
  • Methods of treating a disease in a subject in need thereof comprise administering to a subject in need thereof an effective amount of: a presently disclosed RGN polypeptide or active variant or fragment thereof or a polynucleotide encoding the same; a base editor, or active variant or fragment thereof, described herein, or a polynucleotide encoding the same; a PE, or active variant or fragment thereof, described herein, or a polynucleotide encoding the same; a presently disclosed gRNA or a polynucleotide encoding the same; a presently disclosed RGN, base editing, or PE system; or a cell modified by or comprising any one of these compositions.
  • the treatment comprises in vivo gene editing by administering a presently disclosed RGN polypeptide, base editor, PE, gRNA, or system (e.g., RGN, base editing, or PE), or polynucleotide(s) encoding the same.
  • the treatment comprises ex vivo gene editing wherein cells are genetically modified ex vivo with a presently disclosed RGN polypeptide, base editor, PE, gRNA, or system(e.g., RGN, base editing, or PE), or polynucleotide (s) encoding the same and then the modified cells are administered to a subject.
  • the genetically modified cells originate from the subject that is then administered the modified cells, and the transplanted cells are referred to herein as autologous.
  • the genetically modified cells originate from a different subject (i.e., donor) within the same species as the subject that is administered the modified cells (i.e., recipient), and the transplanted cells are referred to herein as allogeneic.
  • the cells can be expanded in culture prior to administration to a subject in need thereof.
  • the disease to be treated with the presently disclosed compositions is one that can be treated with immunotherapy, such as with a chimeric antigen receptor (CAR) T cell.
  • immunotherapy such as with a chimeric antigen receptor (CAR) T cell.
  • CAR chimeric antigen receptor
  • the disease to be treated with the presently disclosed compositions is associated with a genetic defect, e.g., a lysosomal storage disorder or a metabolic disease, such as, for example, type I diabetes.
  • a genetic defect e.g., a lysosomal storage disorder or a metabolic disease, such as, for example, type I diabetes.
  • compositions of the disclosure are used to edit a genomic sequence causal for the disease or disorder or symptoms of such disease or disorder. For example, the deamination of a target nucleobase or editing of a target polynucleotide results in the correction of a genetic defect such that loss of function of a gene product is restored.
  • the disease to be treated with the presently disclosed compositions is associated with a mutation or causal mutation.
  • a “causal mutation” or “mutation associated with a disease” refers to a particular nucleotide, nucleotides, or nucleotide sequence in the genome that contributes to the severity or presence of a disease or disorder in a subject. The correction of the causal mutation leads to the improvement of at least one symptom resulting from a disease or disorder.
  • the causal mutation is adjacent to a PAM site recognized by a variant LPG10145 RGN disclosed herein.
  • the causal mutation can be corrected with a presently disclosed variant LPG10145 RGN or a fusion polypeptide comprising a presently disclosed variant LPG10145 RGN and a base-editing polypeptide (i.e., a base editor) or a fusion polypeptide comprising a presently disclosed variant LPG10145 RGN and a polymerase (i.e., a PE).
  • diseases associated with a mutation or causal mutation include cystic fibrosis, Hurler syndrome, Friedreich’s Ataxia, Huntington’s Disease, and sickle cell disease.
  • Non-limiting examples of disease-associated genes and mutations are available from McKusick- Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, Md.), available on the World Wide Web.
  • the methods provided herein are used to introduce a deactivating point mutation into a gene or allele that encodes a gene product that is associated with a disease or disorder.
  • methods are provided herein that employ a RGN, base editing, or PE system to introduce a deactivating point mutation into an oncogene (e.g., in the treatment of a proliferative disease).
  • a deactivating mutation may, in some embodiments, generate a premature stop codon in a coding sequence, which results in the expression of a truncated gene product, e.g., a truncated protein lacking the function of the full-length protein.
  • the purpose of the methods provided herein is to restore the function of a dysfunctional gene via genome editing.
  • the RGN, base editing, or PE system can be validated for gene editing -based human therapeutics in vitro, e.g., by correcting a disease associated mutation in human cell culture. It will be understood by the skilled artisan that a base editor comprising an RGN and a base editing polypeptide, or a PE comprising an RGN and a polymerase, can be used to correct a single point mutation.
  • a method of treating a disease in a subject in need thereof comprises creating an induced pluripotent stem cell (iPSC) or isolating a mesenchymal stem cell from the subject, contacting the iPSC or mesenchymal stem cell with any one of the RGN polypeptides, base editors, PE, RGN systems, base editing systems, PE systems, compositions comprising the same, or pharmaceutical compositions disclosed herein in order to genetically modify a target polynucleotide within the cell, differentiating the modified iPSC or the modified mesenchymal stem cell into a genetically modified mature cell or precursor thereof, and administering the genetically modified mature cell or precursor thereof into the subject.
  • iPSC induced pluripotent stem cell
  • the iPSC or the mesenchymal stem cell is an autologous or an allogeneic cell. In some embodiments, the iPSC or the mesenchymal stem cell is derived from a donor that is a perfect human leukocyte antigen (HLA) match for the subject. In some embodiments, the subject is administered a myeloablative therapy prior to administration of the modified cells.
  • HLA human leukocyte antigen
  • the creating step can comprise: a) isolating a somatic cell, such as a skin cell or fibroblast, from the subject; and b) introducing a set of pluripotency-associated genes into the somatic cell in order to induce the cell to become a pluripotent stem cell.
  • a somatic cell such as a skin cell or fibroblast
  • the set of pluripotency-associated genes can be one or more of the genes selected from the group consisting of OCT4, SOX1, SOX2, SOX3, SOX15, SOX18, NANOG, KLF1, KLF2, KLF4, KLF5, c-MYC, n-MYC, REM2, TERT and LIN28.
  • Mesenchymal stem cells can be isolated according to any method known in the art, such as from a patient’s bone marrow or peripheral blood. For example, marrow aspirate can be collected into a syringe with heparin. Cells can be washed and centrifuged on a Percoll.
  • the cells can be cultured in Dulbecco’s modified Eagle’s medium (DMEM) (low glucose) containing 10% fetal bovine serum (FBS) (Pittinger M F, Mackay A M, Beck S C et al., Science 1999; 284: 143-147).
  • DMEM Dulbecco’s modified Eagle’s medium
  • FBS fetal bovine serum
  • Allogeneic cells refer to cells that are from a donor or donors (i.e., an individual or individuals from which the genetically modified cells are derived).
  • Autologous cells refer to cells that are from the subject undergoing treatment (i.e., the recipient of the genetically modified cells). Due to the risk of transplant rejection, an effort is made to optimize the degree of major histocompatibility complex (MHC)/human leukocyte antigen (HLA) matching between donor tissue and recipient.
  • MHC major histocompatibility complex
  • HLA human leukocyte antigen
  • HLA typing of donor tissue and the recipient concerns determining the genotype of six HLA antigens or alleles between a donor(s) and recipient to assess the degree to which the six HLA match.
  • HLA alleles usually refer to two each at the loci HLA-A, HLA-B and HLA-DR, or one each at the loci HLA-A, HLA-B and HLA-C and one each at the loci HAL-DRB1, HLA-DQB1 and HLA-DPB1 (see e.g., Kawase et al., 2007, Blood 110:2235-2241).
  • 4 of 6 HLA matching between donor(s) and recipient are sufficient for administration to the recipient of cells derived from a donor.
  • 5 of 6 HLA matching between donor(s) and recipient are sufficient for administration to the recipient of cells derived from a donor(s).
  • 6 of 6 HLA are matched between donor(s) and recipient for administration to the recipient of cells derived from the donor(s).
  • a 4/6, 5/6, or a 6/6 HLA match is the standard of clinical care. When all 6 HLA match between donor(s) and recipient, the match is referred to as being a perfect match.
  • treatment or “treating,” or “palliating” or “ameliorating” are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and/or a prophylactic benefit.
  • therapeutic benefit is meant any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment.
  • the compositions may be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more of the physiological symptoms of a disease, even though the disease, condition, or symptom may not have yet been manifested.
  • treatment may be administered after one or more symptoms have developed and/or after a disease has been diagnosed.
  • treatment may be administered in the absence of symptoms, e.g., to prevent or delay onset of a symptom or inhibit onset or progression of a disease.
  • treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example, to prevent or delay their prevention or recurrence.
  • an effective amount refers to the amount of an agent that is sufficient to effect beneficial or desired results.
  • the therapeutically effective amount may vary depending upon one or more of: the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.
  • the specific dose may vary depending on one or more of: the particular agent chosen, the dosing regimen to be followed, whether it is administered in combination with other compounds, timing of administration, and the delivery system in which it is carried.
  • administering refers to the placement of an active ingredient into a subject, by a method or route that results in at least partial localization of the introduced active ingredient at a desired site, such as a site of injury or repair, such that a desired effect(s) is produced.
  • the disclosure provides methods comprising delivering any of the RGN polypeptides, base editors, PEs, nucleic acid molecules, ribonucleoprotein complexes, vectors, pharmaceutical compositions and/or gRNAs described herein.
  • the disclosure further provides cells produced by such methods, and organisms (such as animals or plants) comprising or produced from such cells.
  • a RGN polypeptide (or base editor or PE comprising the same) and/or nucleic acid molecules as described herein in combination with (and optionally complexed with) a guide sequence is delivered to a cell.
  • the cells can be administered by any appropriate route that results in delivery to a desired location in the subject where at least a portion of the implanted cells or components of the cells remain viable.
  • the period of viability of the cells after administration to a subject can be as short as a few hours, e.g., twenty-four hours, to a few days, to as long as several years, or even the life time of the patient, i.e., long-term engraftment.
  • an effective amount of photoreceptor cells or retinal progenitor cells is administered via a systemic route of administration, such as an intraperitoneal or intravenous route.
  • the administering comprises administering by viral delivery.
  • Viral vectors comprising a nucleic acid encoding the RGN polypeptides, base editors, PEs, nucleic acid molecules, ribonucleoprotein complexes, or vectors disclosed herein may be administered directly to patients (i.e., in vivo) or they may be used to treat cells in vitro, and the modified cells may optionally be administered to patients (i.e., ex vivo).
  • Conventional viral based systems may include, without limitation, retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer.
  • Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers.
  • adenoviral based systems may be used. Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division.
  • a therapeutically effective dosage to a human can be in the range of from about 20 to about 50 ml of saline solution containing from about 1 x IO 10 to about 1 x IO 50 functional AAV/ml solution.
  • the dosage may be adjusted to balance the therapeutic benefit against any side effects.
  • the AAV dose can be from about 1 x 10 5 to 1 x IO 50 genomes AAV, from about 1 x 10 8 to 1 x IO 20 genomes AAV, from about 1 x IO 10 to about 1 x 10 16 genomes, or about 1 x 10 n to about 1 x 10 16 genomes AAV.
  • a human dosage may be about 1 x 10 13 genomes AAV.
  • Such concentrations may be delivered in from about 0.001 ml to about 100 ml, about 0.05 to about 50 ml, or about 10 to about 25 ml of a carrier solution.
  • Other effective dosages can be readily established by one of ordinary skill in the art through trials establishing dose response curves. See, for example, U.S. Patent No. 8,404,658 B2.
  • the administering comprises administering by other non-viral delivery of nucleic acids.
  • exemplary non-viral delivery methods include RNP complexes, lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipidmucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA.
  • Lipofection is described in e.g., U.S. Pat. Nos.5, 049, 386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., TransfectamTM and LipofectinTM).
  • Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO1991/17424; WO 1991/16024. Delivery can be to cells (e.g., in vitro or ex vivo administration) or target tissues (e.g., in vivo administration).
  • administration of a pharmaceutical composition of the disclosure includes daily intravenous injections of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg/kg/day, or more of an active ingredient in a pharmaceutical composition comprising a liposome or LNP.
  • administration of a pharmaceutical composition comprising a liposome or LNP includes doses of about 0.01 to 1 mg per kg of body weight.
  • administration of a pharmaceutical composition comprising a liposome or LNP includes doses of about 1 to 10 mg per kg of body weight.
  • the administering comprises administering by a method selected from the group consisting of: intravenously, subcutaneously, intramuscularly, orally, rectally, by aerosol, parenterally, ophthalmicly, pulmonarily, transdermally, vaginally, otically, nasally, and by topical administration, or any combination thereof.
  • administration by injection or infusion is used for the delivery of cells.
  • the term "subject" refers to any individual for whom diagnosis, treatment or therapy is desired.
  • the subject is an animal.
  • the subject is a mammal.
  • the subject is a human being.
  • Efficacy of a treatment can be determined by the skilled clinician. However, a treatment is considered an "effective treatment," if any one or all of the signs or symptoms of a disease or disorder are altered in a beneficial manner (e.g., decreased by at least 10%), or other clinically accepted symptoms or markers of disease are improved or ameliorated. Efficacy can also be measured by failure of an individual to worsen as assessed by hospitalization or need for medical interventions (e.g., progression of the disease is halted or at least slowed). Methods of measuring these indicators are known to those of skill in the art. Treatment includes: (1) inhibiting the disease, e.g., arresting, or slowing the progression of symptoms; or (2) relieving the disease, e.g., causing regression of symptoms; and (3) preventing or reducing the likelihood of the development of symptoms.
  • compositions comprising the presently disclosed variant LPG10145 RGN polypeptides or polynucleotides encoding the same, the presently disclosed base editors or polynucleotides encoding the same, the presently disclosed PEs or polynucleotides encoding the same, the presently disclosed gRNAs or polynucleotides encoding the same, the presently disclosed systems (e.g., RGN, base editing, or PE), the presently disclosed ribonucleoprotein complexes or cells comprising any of the RGN polypeptides or RGN-encoding polynucleotides, base editors, PEs, gRNA or gRNA-encoding polynucleotides, or the systems (e.g., RGN, base editing, or PE), and a pharmaceutically acceptable carrier are provided.
  • a “pharmaceutically acceptable carrier” refers to a material that does not cause significant irritation to an organism and does not abrogate the activity and properties of the active ingredient (e.g., an RGN polypeptide or nucleic acid molecule encoding the same). Carriers must be of sufficiently high purity and of sufficiently low toxicity to render them suitable for administration to a subject being treated.
  • the carrier can be inert, or it can possess pharmaceutical benefits.
  • a pharmaceutically acceptable carrier comprises one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration to a human or other vertebrate animal.
  • the pharmaceutical composition comprises a pharmaceutically acceptable carrier that is non-naturally occurring.
  • the pharmaceutically acceptable carrier and the active ingredient are not found together in nature and are thus, heterologous.
  • compositions used in the presently disclosed methods can be formulated with suitable carriers, excipients, and other agents that provide suitable transfer, delivery, tolerance, and the like.
  • suitable carriers excipients, and other agents that provide suitable transfer, delivery, tolerance, and the like.
  • suitable carriers excipients, and other agents that provide suitable transfer, delivery, tolerance, and the like.
  • Non-limiting examples include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents
  • antibacterial agents such as benzyl alcohol or methyl parabens
  • antioxidants such as ascorbic acid or sodium bisulfite
  • chelating agents such as ethylenediaminetetraacetic acid
  • buffers such as acetates
  • compositions for oral or parenteral use may be prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients.
  • dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.
  • These compositions also may contain adjuvants including preservative agents, wetting agents, emulsifying agents, and dispersing agents.
  • Prevention of the action of microorganisms may be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It also may be desirable to include isotonic agents, for example, sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form may be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • cells comprising or modified with the presently disclosed variant LPG10145 RGNs, base editors, PEs, gRNAs, systems (e.g., RGN, base editing, or PE), or polynucleotides encoding the same are administered to a subject
  • the cells are administered as a suspension with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier to be used in a cell composition will not include buffers, compounds, cryopreservation agents, preservatives, or other agents in amounts that substantially interfere with the viability of the cells to be delivered to the subject.
  • a formulation comprising cells can include e.g., osmotic buffers that permit cell membrane integrity to be maintained, and optionally, nutrients to maintain cell viability or enhance engraftment upon administration.
  • osmotic buffers that permit cell membrane integrity to be maintained, and optionally, nutrients to maintain cell viability or enhance engraftment upon administration.
  • Such formulations and suspensions are known to those of skill in the art and/or can be adapted for use with the cells described herein using routine experimentation.
  • a cell composition can also be emulsified or presented as a liposome composition, provided that the emulsification procedure does not adversely affect cell viability.
  • the cells and any other active ingredient can be mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient, and in amounts suitable for use in the therapeutic methods described herein.
  • Additional agents included in a cell composition can include pharmaceutically acceptable salts of the components therein.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids, such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases, such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.
  • Suitable routes of administering the pharmaceutical compositions described herein include, without limitation: topical, subcutaneous, transdermal, intradermal, intralesional, intraarticular, intraperitoneal, intravesical, transmucosal, gingival, intradental, intracochlear, transtympanic, intraorgan, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseus, periocular, intratumoral, intracerebral, and intracerebroventricular administration.
  • a pharmaceutical composition described herein is administered locally to a diseased site (e.g., the lung).
  • the pharmaceutical composition described herein is administered to a subject by injection, inhalation (e.g., of an aerosol), by means of a catheter, by means of a suppository, or by means of an implant, the implant being of a porous, non-porous, or gelatinous material, including a membrane, such as a sialastic membrane, or a fiber.
  • the pharmaceutical composition is formulated for delivery to a subject, e.g., for gene editing.
  • the pharmaceutical composition is formulated in accordance with routine procedures as a composition adapted for intravenous or subcutaneous administration to a subject, e.g., a human.
  • pharmaceutical composition for administration by injection are solutions in sterile isotonic aqueous buffer.
  • the pharmaceutical can also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • the pharmaceutical is to be administered by infusion
  • it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.
  • the pharmaceutical composition can be contained within a lipid particle or vesicle, such as a liposome or microcrystal, which is also suitable for parenteral administration.
  • compositions are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals or organisms of all sorts.
  • Hurler Syndrome also known as MPS-1, is the result of a deficiency of a-L-iduronidase (IDUA) resulting in a lysosomal storage disease characterized at the molecular level by the accumulation of dermatan sulfate and heparan sulfate in lysosomes.
  • IDUA a-L-iduronidase
  • This disease is generally an inherited genetic disorder caused by mutations in the IDUA gene encoding a-L-iduronidase.
  • Common IDUA mutations are W402X and Q70X, both nonsense mutations resulting in premature termination of translation.
  • PGE precise genome editing
  • reversion of a single nucleotide for example by a base-editing approach, would restore the wild-type coding sequence and result in protein expression controlled by the endogenous regulatory mechanisms of the genetic locus.
  • heterozygotes are known to be asymptomatic, a PGE therapy that targets one of these mutations would be useful to a large proportion of patients with this disease, as only one of the mutated alleles needs to be corrected (Bunge et al. (1994) Hum. Mol. Genet. 3(6): 861-866, herein incorporated by reference).
  • Hurler Syndrome Current treatments for Hurler Syndrome include enzyme replacement therapy and bone marrow transplants (Vellodi et al. (1997) Arch. Dis. Child. 76(2): 92-99; Peters et al. (1998) Blood 91(7): 2601- 2608, herein incorporated by reference). While enzyme replacement therapy has had a dramatic effect on the survival and quality of life of Hurler Syndrome patients, this approach requires costly and timeconsuming weekly infusions. Additional approaches include the delivery of the IDUA gene on an expression vector or the insertion of the gene into a highly expressed locus such as that of serum albumin (U.S. Patent No. 9,956,247, herein incorporated by reference). However, these approaches do not restore the original IDUA locus to the correct coding sequence.
  • a genome-editing strategy would have a number of advantages, most notably that regulation of gene expression would be controlled by the natural mechanisms present in healthy individuals. Additionally, using base editing does not necessitate causing a double stranded DNA breaks, which could lead to large chromosomal rearrangements, cell death, or oncogenecity by the disruption of tumor suppression mechanisms.
  • a general strategy may be directed toward using RGN- base editor fusion proteins or PEs of the invention, for example those comprising variant EPG10145 RGNs, to target and correct certain disease-causing mutations in the human genome. It will be appreciated that similar approaches to target diseases that can be corrected by base-editing or polymerase editing may also be pursued.
  • Common household pets and livestock include dogs, cats, horses, pigs, cows, sheep, chickens, donkeys, snakes, ferrets, and fish including salmon and shrimp.
  • RGNs of the invention could also be useful in human therapeutic approaches where the causal mutation is more complicated.
  • some diseases such as Friedreich’s Ataxia and Huntington’s Disease are the result of a significant increase in repeats of a three nucleotide motif at a particular region of a gene, which affects the ability of the expressed protein to function or to be expressed.
  • Friedreich’s Ataxia FRDA
  • FRDA Friedreich’s Ataxia
  • FXN frataxin
  • the reduced FXN expression has been linked to a GAA triplet expansion within the intron 1 of the somatic and germline FXN gene.
  • the GAA repeat frequently consists of more than 70, sometimes even more than 1000 (most commonly 600-900) triplets, whereas unaffected individuals have about 40 repeats or less (Pandolfo et al. (2012) Handbook of Clinical Neurology 103: 275-294; Campuzano et al. (1996) Science 271: 1423-1427; Pandolfo (2002) Adv. Exp. Med. Biol. 516: 99-118; all herein incorporated by reference).
  • RNA guided nucleases may be used for excising the instability region in FRDA patient cells. This approach requires 1) an RGN and guide RNA sequence that can be programmed to target the allele in the human genome; and 2) a delivery approach for the RGN and guide sequence.
  • SpCas9 Cas9 nuclease from .S' pyogenes
  • AAV adeno-associated viral
  • Variant LPG10145 RNA guided nucleases of the invention are well suited for packaging into an AAV vector along with a guide RNA.
  • the present invention encompasses a strategy using RGNs of the invention in which a region of genomic instability is removed. Such a strategy is applicable to other diseases and disorders which have a similar genetic basis, such as Huntington’s Disease. Similar strategies using RGNs of the invention may also be applicable to similar diseases and disorders in non-human animals of agronomic or economic importance, including dogs, cats, horses, pigs, cows, sheep, chickens, donkeys, snakes, ferrets, and fish including salmon and shrimp.
  • Variant LPG10145 RGNs of the invention could also be to introduce disruptive mutations that may result in a beneficial effect.
  • Genetic defects in the genes encoding hemoglobin, particularly the beta globin chain (the HBB gene) can be responsible for a number of diseases known as hemoglobinopathies, including sickle cell anemia and thalassemias.
  • hemoglobin In adult humans, hemoglobin is a heterotetramer comprising two alpha (a)-like globin chains and two beta (P)-like globin chains and 4 heme groups. In adults the a2p2 tetramer is referred to as Hemoglobin A (HbA) or adult hemoglobin.
  • HbA Hemoglobin A
  • the alpha and beta globin chains are synthesized in an approximate 1: 1 ratio and this ratio seems to be critical in terms of hemoglobin and red blood cell (RBC) stabilization.
  • fetal hemoglobin In a developing fetus, a different form of hemoglobin, fetal hemoglobin (HbF), is produced which has a higher binding affinity for oxygen than Hemoglobin A such that oxygen can be delivered to the baby's system via the mother's blood stream.
  • Fetal hemoglobin also contains two a globin chains, but in place of the adult P- globin chains, it has two fetal gamma (y)-globin chains (i.e., fetal hemoglobin is a2y2).
  • the regulation of the switch from production of gamma- to beta-globin is quite complex, and primarily involves a downregulation of gamma globin transcription with a simultaneous up-regulation of beta globin transcription.
  • Sickle cell disease is caused by a V6E mutation in the P globin gene (HBB) (a GAG to GTG at the DNA level), where the resultant hemoglobin is referred to as “hemoglobins” or “HbS.”
  • HBB P globin gene
  • HbS molecules aggregate and form fibrous precipitates. These aggregates cause the abnormality or ‘sickling’ of the RBCs, resulting in a loss of flexibility of the cells.
  • the sickling RBCs are no longer able to squeeze into the capillary beds and can result in vaso-occlusive crisis in sickle cell patients.
  • sickled RBCs are more fragile than normal RBCs, and tend towards hemolysis, eventually leading to anemia in the patient.
  • Thalassemias are also diseases relating to hemoglobin and typically involve a reduced expression of globin chains. This can occur through mutations in the regulatory regions of the genes or from a mutation in a globin coding sequence that results in reduced expression or reduced levels or functional globin protein.
  • Treatment of thalassemias usually involves blood transfusions and iron chelation therapy.
  • Bone marrow transplants are also being used for treatment of people with severe thalassemias if an appropriate donor can be identified, but this procedure can have significant risks.
  • SCD sickle cell disease
  • beta thalassemias Treatment of SCD patients with hydroxyurea is thought to be successful in part due to its effect on increasing gamma globin expression (DeSimone (1982) Proc Nat'l Acad Sci USA 79(14):4428-31; Ley, et al., (1982) N. Engl. J.
  • HbF HbF-derived gamma globin expression.
  • BCL11A encodes a zinc finger protein that expressed in adult erythroid precursor cells, and down-regulation of its expression leads to an increase in gamma globin expression (Sankaran et at (2008) Science 322: 1839, herein incorporated by reference).
  • RNA targeted to the BCL11A gene has been proposed (e.g., U.S. Patent Publication 2011/0182867, herein incorporated by reference) but this technology has several potential drawbacks, including that complete knock down may not be achieved, delivery of such RNAs may be problematic, and the RNAs must be present continuously, requiring multiple treatments for life.
  • Variant LPG10145 RGNs of the invention may be used to target the BCL11A enhancer region to disrupt expression of BCL11A, thereby increasing gamma globin expression.
  • This targeted disruption can be achieved by non-homologous end joining (NHEJ), whereby an RGN of the invention targets to a particular sequence within the BCL11A enhancer region, makes a double-stranded break, and the cell’s machinery repairs the break, typically simultaneously introducing deleterious mutations.
  • NHEJ non-homologous end joining
  • RGNs of the invention may have advantages over other known RGNs due to their relatively small size, which enables packaging expression cassettes for the RGN and its guide RNA into a single AAV vector for in vivo delivery. Similar strategies using RGNs of the invention may also be applicable to similar diseases and disorders in both humans and in non-human animals of agronomic or economic importance.
  • the RGN comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions differs from the corresponding amino acid residue in SEQ ID NO: 1: 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975.
  • the RGN comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions is a positively charged amino acid residue: 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975.
  • the RGN comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at one or more of the following amino acid positions is an R: 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975.
  • the RGN comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at amino acid position 778 and/or 969 differs from the corresponding amino acid residue in SEQ ID NO: 1. In some of these embodiments, the RGN comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at amino acid position 778 and/or 969 is a positively charged amino acid residue. In some of these embodiments, the RGN comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein the amino acid residue at amino acid position 778 and/or 969 is an R.
  • the RGN comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein amino acid residues at positions 778 and 856 differ from the corresponding amino acid residues in SEQ ID NO: 1. In some of these embodiments, the RGN comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein amino acid residues at positions 778 and 856 are positively charged amino acid residues.
  • the RGN comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein amino acid residues at positions 55, 647, 778, and 969 differ from the corresponding amino acid residues in SEQ ID NO: 1. In some of these embodiments, the RGN comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, wherein amino acid residues at positions 55, 647, 778, and 969 are positively charged amino acid residues.
  • the RGN comprises the amino acid sequence selected from: (a) the amino acid sequence set forth as SEQ ID NO: 1, wherein E at amino acid position 52 is an R; (b) the amino acid sequence set forth as SEQ ID NO: 1, wherein S at amino acid position 55 is an R; (c) the amino acid sequence set forth as SEQ ID NO: 1, wherein G at amino acid position 86 is an R; (d) the amino acid sequence set forth as SEQ ID NO: 1, wherein Y at amino acid position 472 is an R; (e) the amino acid sequence set forth as SEQ ID NO: 1, wherein K at amino acid position 533 is an R; (f) the amino acid sequence set forth as SEQ ID NO: 1, wherein K at amino acid position 541 is an R; (g) the amino acid sequence set forth as SEQ ID NO: 1, wherein Y at amino acid position 643 is substituted by an R; (h) the amino acid sequence set forth as SEQ ID NO: 1, wherein S at amino acid position 647 is an R; (i) the amino acid sequence set
  • the RGN comprises the amino acid sequence selected from: (a) the amino acid sequence set forth as SEQ ID NO: 2; (b) the amino acid sequence set forth as SEQ ID NO: 3; (c) the amino acid sequence set forth as SEQ ID NO: 4; (d) the amino acid sequence set forth as SEQ ID NO: 5; (e) the amino acid sequence set forth as SEQ ID NO: 6; (f) the amino acid sequence set forth as SEQ ID NO: 7; (g) the amino acid sequence set forth as SEQ ID NO: 8; (h) the amino acid sequence set forth as SEQ ID NO: 9; (i) the amino acid sequence set forth as SEQ ID NO: 10; (j) the amino acid sequence set forth as SEQ ID NO: 11; (k) the amino acid sequence set forth as SEQ ID NO: 12; (1) the amino acid sequence set forth as SEQ ID NO: 13; (m) the amino acid sequence set forth as SEQ ID NO: 14; (n) the amino acid sequence set forth as SEQ ID NO: 15; and
  • the guide RNA comprises a CRISPR repeat sequence comprising the nucleotide sequence set forth as SEQ ID NO: 33, 244, or 245, or an active variant or fragment thereof.
  • the guide RNA comprises a tracrRNA comprising the nucleotide sequence set forth as SEQ ID NO:34, 246, 247, or 248, or an active variant or fragment thereof.
  • the guide RNA of the system can be a single guide RNA or a dual-guide RNA.
  • the modified cells can be eukaryotic (e.g., mammalian, plant, insect, avian cell) or prokaryotic.
  • organelles and embryos comprising at least one target nucleotide sequence that has been modified by a process utilizing an RGN, base editor, PE, crRNA, tracrRNA and/or gRNA, or systems comprising such, as described herein.
  • the genetically modified cells, organisms, organelles, and embryos can be heterozygous or homozygous for the modified nucleotide sequence.
  • the chromosomal modification of the cell, organism, organelle, or embryo can result in altered expression (up-regulation or down-regulation), inactivation, or the expression of an altered protein product or an integrated sequence.
  • the genetically modified cell, organism, organelle, or embryo is referred to as a “knock out”.
  • the knock out phenotype can be the result of a deletion mutation (z. e. , deletion of at least one nucleotide), an insertion mutation (i. e. , insertion of at least one nucleotide), or a nonsense mutation (z'.e., substitution of at least one nucleotide such that a stop codon is introduced).
  • the chromosomal modification of a cell, organism, organelle, or embryo can produce a “knock in”, which results from the chromosomal integration of a nucleotide sequence that encodes a protein.
  • the coding sequence is integrated into the chromosome such that the chromosomal sequence encoding the wild-type protein is inactivated, but the exogenously introduced protein is expressed.
  • the chromosomal modification using the presently disclosed RGNs, base editors, PEs, gRNAs, or systems comprising such results in the production of a variant protein product.
  • the expressed variant protein product can have at least one amino acid substitution and/or the addition or deletion of at least one amino acid.
  • the variant protein product encoded by the altered chromosomal sequence can exhibit modified characteristics or activities when compared to the wild-type protein, including but not limited to altered enzymatic activity or substrate specificity.
  • the chromosomal modification can result in an altered expression pattern of a protein.
  • chromosomal alterations in the regulatory regions controlling the expression of a protein product can result in the overexpression or downregulation of the protein product or an altered tissue or temporal expression pattern.
  • the mutation(s) introduced as a result of RGNs, base editors, PEs, gRNAs, or systems comprising such described herein yields a reduction or elimination in expression of a gene.
  • Cells that have been modified may be introduced into an organism. These cells could have originated from the same organism (e.g., person) in the case of autologous cellular transplants, wherein the cells are modified in an ex vivo approach. Alternatively, the cells originated from another organism within the same species (e.g., another person) in the case of allogeneic cellular transplants.
  • the cells that have been modified can be grown into an organism, such as a plant, in accordance with conventional ways. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants may then be grown, and either pollinated with the same modified strain or different strains, and the resulting hybrid having the genetic modification.
  • the present invention provides genetically modified seed. Progeny, variants, and mutants of the regenerated plants are also included within the scope of the invention, provided that these parts comprise the genetic modification. Further provided is a processed plant product or byproduct that retains the genetic modification, including for example, soymeal.
  • the methods provided herein may be used for modification of any plant species, including, but not limited to, monocots and dicots.
  • plants of interest include, but are not limited to, com (maize), sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed rape, Brassica sp., alfalfa, rye, millet, safflower, peanuts, sweet potato, cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond, oats, vegetables, ornamentals, and conifers.
  • Vegetables include, but are not limited to, tomatoes, lettuce, green beans, lima beans, peas, and members of the genus Curcumis such as cucumber, cantaloupe, and musk melon. Ornamentals include, but are not limited to, azalea, hydrangea, hibiscus, roses, tulips, daffodils, petunias, carnation, poinsettia, and chrysanthemum.
  • plants of the present invention are crop plants (for example, maize, sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, oilseed rape, etc.).
  • the methods provided herein can also be used to genetically modify any prokaryotic species, including but not limited to, archaea and bacteria (e.g., Bacillus sp., Klebsiella sp. Streptomyces sp., Rhizobium sp., Escherichia sp., Pseudomonas sp., Salmonella sp., Shigella sp., Vibrio sp., Yersinia sp., Mycoplasma sp., Agrobacterium, Lactobacillus sp.).
  • archaea and bacteria e.g., Bacillus sp., Klebsiella sp. Streptomyces sp., Rhizobium sp., Escherichia sp., Pseudomonas sp., Salmonella sp., Shigella sp., Vibrio sp., Yers
  • the methods provided herein can be used to genetically modify any eukaryotic species or cells therefrom, including but not limited to animals (e.g., mammals, insects, fish, birds, and reptiles), fungi, amoeba, algae, and yeast.
  • the cell that is modified by the presently disclosed methods include cells of hematopoietic origin, such as cells of the immune system including but not limited to B cells, T cells, natural killer (NK) cells, pluripotent stem cells, induced pluripotent stem cells, chimeric antigen receptor T (CAR-T) cells, monocytes, macrophages, and dendritic cells.
  • Cells that have been modified may be introduced into an organism. These cells could have originated from the same organism (e.g., person) in the case of autologous cellular transplants, wherein the cells are modified in an ex vivo approach. Alternatively, the cells originated from another organism within the same species (e.g., another person) in the case of allogeneic cellular transplants.
  • a polypeptide means one or more polypeptides.
  • a nucleic acid molecule comprising a polynucleotide encoding an RNA-guided nuclease (RGN) polypeptide, wherein said RGN polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, and wherein the amino acid residue at one or more of amino acid positions 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975 of said amino acid sequence differs from the corresponding amino acid residue in SEQ ID NO: 1.
  • RGN RNA-guided nuclease
  • RGN polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, and wherein the amino acid residue at one or more of amino acid positions 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975 of said amino acid sequence is a positively charged amino acid residue.
  • nucleic acid molecule of embodiment 1 or 2 wherein said RGN polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, and wherein the amino acid residue at one or more of amino acid positions 52, 55, 86, 472, 533, 541, 643, 647, 653, 745, 774, 778, 780, 795, 822, 843, 856, 871, 872, 900, 911, 913, 954, 958, 968, 969, 973, 974, and 975 of said amino acid sequence is an R.
  • a nucleic acid molecule comprising a polynucleotide encoding an RNA-guided nuclease (RGN) polypeptide, wherein said RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence selected from:
  • (nn) the amino acid sequence set forth as SEQ ID NO: 12, wherein E at amino acid position 778 is an R, Q at amino acid position 822 is an R, S at amino acid position 55 is an R, and E at amino acid position 969 is an R;
  • RGN polypeptide comprises an amino acid sequence selected from:
  • amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence selected from the group consisting of: (a) the amino acid sequence set forth as SEQ ID NO: 1, wherein E at amino acid position 52 is an R;
  • (nn) the amino acid sequence set forth as SEQ ID NO: 12, wherein E at amino acid position 778 is an R, Q at amino acid position 822 is an R, S at amino acid position 55 is an R, and E at amino acid position 969 is an R;
  • nucleic acid molecule of any one of embodiments 1-5, wherein said polynucleotide encoding an RGN polypeptide comprises an mRNA comprising a 5 ’ untranslated region (UTR) and/or a 3 ’ UTR, wherein the 5’ UTR, the 3’ UTR, or both are heterologous to the mRNA.
  • UTR untranslated region
  • nucleic acid molecule of any one of embodiments 1-5 wherein said polynucleotide encoding an RGN polypeptide is operably linked to a promoter heterologous to said polynucleotide.
  • RGN polypeptide is capable of binding a target polynucleotide sequence in an RNA-guided sequence specific manner when bound to a guide RNA (gRNA) capable of hybridizing to said target polynucleotide sequence.
  • gRNA guide RNA
  • nucleic acid molecule of embodiment 8 wherein said RGN polypeptide recognizes a protospacer adjacent motif (PAM) that is 3' of said target polynucleotide sequence.
  • PAM protospacer adjacent motif
  • RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 182- 196, and 271-285.
  • RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 182-196, and 271-285.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Mycology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne des compositions et des procédés de liaison à une séquence cible d'intérêt. Les compositions trouvent une utilisation dans le clivage ou la modification d'une séquence cible d'intérêt, la visualisation d'une séquence cible d'intérêt et la modification de l'expression d'une séquence d'intérêt. Les compositions comprennent des polypeptides de nucléase guidée par ARN, des ARN CRISPR, des ARN CRISPR de trans-activation, des ARN guides et des molécules d'acide nucléique codant pour ceux-ci. L'invention concerne également des vecteurs et des cellules hôtes comprenant les molécules d'acide nucléique. L'invention concerne en outre des systèmes RGN permettant de lier une séquence cible d'intérêt, le système RGN comprenant un polypeptide de nucléase guidée par ARN et un ou plusieurs ARN guides.
PCT/IB2024/057272 2023-07-27 2024-07-27 Nucléases guidées par arn et fragments actifs, variants associés et méthodes d'utilisation Ceased WO2025022367A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP24758895.7A EP4750890A2 (fr) 2023-07-27 2024-07-27 Nucléases guidées par arn et fragments actifs, variants associés et méthodes d'utilisation

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363516127P 2023-07-27 2023-07-27
US63/516,127 2023-07-27

Publications (2)

Publication Number Publication Date
WO2025022367A2 true WO2025022367A2 (fr) 2025-01-30
WO2025022367A3 WO2025022367A3 (fr) 2025-04-17

Family

ID=92494648

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2024/057272 Ceased WO2025022367A2 (fr) 2023-07-27 2024-07-27 Nucléases guidées par arn et fragments actifs, variants associés et méthodes d'utilisation

Country Status (3)

Country Link
EP (1) EP4750890A2 (fr)
TW (1) TW202519651A (fr)
WO (1) WO2025022367A2 (fr)

Citations (104)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4186183A (en) 1978-03-29 1980-01-29 The United States Of America As Represented By The Secretary Of The Army Liposome carriers in chemotherapy of leishmaniasis
US4196265A (en) 1977-06-15 1980-04-01 The Wistar Institute Method of producing antibodies
US4217344A (en) 1976-06-23 1980-08-12 L'oreal Compositions containing aqueous dispersions of lipid spheres
US4235871A (en) 1978-02-24 1980-11-25 Papahadjopoulos Demetrios P Method of encapsulating biologically active materials in lipid vesicles
US4261975A (en) 1979-09-19 1981-04-14 Merck & Co., Inc. Viral liposome particle
US4485054A (en) 1982-10-04 1984-11-27 Lipoderm Pharmaceuticals Limited Method of encapsulating biologically active materials in multilamellar lipid vesicles (MLV)
US4501728A (en) 1983-01-06 1985-02-26 Technology Unlimited, Inc. Masking of liposomes from RES recognition
US4774085A (en) 1985-07-09 1988-09-27 501 Board of Regents, Univ. of Texas Pharmaceutical administration systems containing a mixture of immunomodulators
US4797368A (en) 1985-03-15 1989-01-10 The United States Of America As Represented By The Department Of Health And Human Services Adeno-associated virus as eukaryotic expression vector
US4837028A (en) 1986-12-24 1989-06-06 Liposome Technology, Inc. Liposomes with enhanced circulation time
US4853331A (en) 1985-08-16 1989-08-01 Mycogen Corporation Cloning and expression of Bacillus thuringiensis toxin gene toxic to beetles of the order Coleoptera
US4897355A (en) 1985-01-07 1990-01-30 Syntex (U.S.A.) Inc. N[ω,(ω-1)-dialkyloxy]- and N-[ω,(ω-1)-dialkenyloxy]-alk-1-yl-N,N,N-tetrasubstituted ammonium lipids and uses therefor
US4946787A (en) 1985-01-07 1990-08-07 Syntex (U.S.A.) Inc. N-(ω,(ω-1)-dialkyloxy)- and N-(ω,(ω-1)-dialkenyloxy)-alk-1-yl-N,N,N-tetrasubstituted ammonium lipids and uses therefor
US5039523A (en) 1988-10-27 1991-08-13 Mycogen Corporation Novel Bacillus thuringiensis isolate denoted B.t. PS81F, active against lepidopteran pests, and a gene encoding a lepidopteran-active toxin
US5049386A (en) 1985-01-07 1991-09-17 Syntex (U.S.A.) Inc. N-ω,(ω-1)-dialkyloxy)- and N-(ω,(ω-1)-dialkenyloxy)Alk-1-YL-N,N,N-tetrasubstituted ammonium lipids and uses therefor
WO1991016024A1 (fr) 1990-04-19 1991-10-31 Vical, Inc. Lipides cationiques servant a l'apport intracellulaire de molecules biologiquement actives
WO1991017424A1 (fr) 1990-05-03 1991-11-14 Vical, Inc. Acheminement intracellulaire de substances biologiquement actives effectue a l'aide de complexes de lipides s'auto-assemblant
EP0480762A2 (fr) 1990-10-12 1992-04-15 Mycogen Corporation Nouveaux isolats de Bacillus thuringiensis actifs contre les diptères
US5173414A (en) 1990-10-30 1992-12-22 Applied Immune Sciences, Inc. Production of recombinant adeno-associated virus vectors
US5268463A (en) 1986-11-11 1993-12-07 Jefferson Richard A Plant promoter α-glucuronidase gene construct
WO1993024641A2 (fr) 1992-06-02 1993-12-09 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Virus adeno-associe a sequences terminales inversees utilisees comme promoteur
US5364780A (en) 1989-03-17 1994-11-15 E. I. Du Pont De Nemours And Company External regulation of gene expression by inducible promoters
WO1994026877A1 (fr) 1993-05-17 1994-11-24 The Regents Of The University Of California Therapie genique ribozymique contre l'infection par le vih et contre le sida
US5380831A (en) 1986-04-04 1995-01-10 Mycogen Plant Science, Inc. Synthetic insecticidal crystal protein gene
US5399680A (en) 1991-05-22 1995-03-21 The Salk Institute For Biological Studies Rice chitinase promoter
US5436391A (en) 1991-11-29 1995-07-25 Mitsubishi Corporation Synthetic insecticidal gene, plants of the genus oryza transformed with the gene, and production thereof
US5466785A (en) 1990-04-12 1995-11-14 Ciba-Geigy Corporation Tissue-preferential promoters
US5569597A (en) 1985-05-13 1996-10-29 Ciba Geigy Corp. Methods of inserting viral DNA into plant material
US5604121A (en) 1991-08-27 1997-02-18 Agricultural Genetics Company Limited Proteins with insecticidal properties against homopteran insects and their use in plant protection
US5605793A (en) 1994-02-17 1997-02-25 Affymax Technologies N.V. Methods for in vitro recombination
US5608149A (en) 1990-06-18 1997-03-04 Monsanto Company Enhanced starch biosynthesis in tomatoes
US5608144A (en) 1994-08-12 1997-03-04 Dna Plant Technology Corp. Plant group 2 promoters and uses thereof
US5608142A (en) 1986-12-03 1997-03-04 Agracetus, Inc. Insecticidal cotton plants
US5659026A (en) 1995-03-24 1997-08-19 Pioneer Hi-Bred International ALS3 promoter
US5789156A (en) 1993-06-14 1998-08-04 Basf Ag Tetracycline-regulated transcriptional inhibitors
US5814618A (en) 1993-06-14 1998-09-29 Basf Aktiengesellschaft Methods for regulating gene expression
US5837458A (en) 1994-02-17 1998-11-17 Maxygen, Inc. Methods and compositions for cellular and metabolic engineering
WO1999043838A1 (fr) 1998-02-24 1999-09-02 Pioneer Hi-Bred International, Inc. Promoteurs de synthese
US6177611B1 (en) 1998-02-26 2001-01-23 Pioneer Hi-Bred International, Inc. Maize promoters
US20030087817A1 (en) 1999-01-12 2003-05-08 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
EP1519714A1 (fr) 2002-06-28 2005-04-06 Protiva Biotherapeutics Inc. Appareil liposomal et procedes de fabrication
US20050222064A1 (en) 2002-02-20 2005-10-06 Sirna Therapeutics, Inc. Polycationic compositions for cellular delivery of polynucleotides
EP1664316A1 (fr) 2003-09-15 2006-06-07 Protiva Biotherapeutics Inc. Composes conjugues lipidiques polyethyleneglycol modifies et utilisations de ces composes
EP1766035A1 (fr) 2004-06-07 2007-03-28 Protiva Biotherapeutics Inc. Arn interferant encapsule dans des lipides
EP1781593A2 (fr) 2004-06-07 2007-05-09 Protiva Biotherapeutics Inc. Lipides cationiques et leurs procédés d'utilisation
US7404969B2 (en) 2005-02-14 2008-07-29 Sirna Therapeutics, Inc. Lipid nanoparticle based compositions and methods for the delivery of biologically active molecules
WO2008103276A2 (fr) 2007-02-16 2008-08-28 Merck & Co., Inc. Compositions et méthodes de potentialisation de l'activité de molécules biologiquement actives
US7534939B2 (en) 1999-09-15 2009-05-19 Monsanto Technology Llc Plant transformed with polynucleotide encoding lepidopteran-active Bacillus thuringiensis δ-endotoxin
US7541517B2 (en) 2003-12-22 2009-06-02 Pioneer Hi-Bred International, Inc. Bacillus thuringiensis CRY9 nucleic acids
US20100036115A1 (en) 1997-07-23 2010-02-11 Sirna Therapeutics, Inc. Novel Compositions for the Delivery of Negatively Charged Molecules
WO2010021865A1 (fr) 2008-08-18 2010-02-25 Merck Sharp & Dohme Corp. Nouvelles nanoparticules lipidiques et nouveaux composants pour l'administration d'acides nucléiques
US7692068B2 (en) 2003-10-14 2010-04-06 Athenix Corporation AXMI-010, a delta-endotoxin gene and methods for its use
WO2010080724A1 (fr) 2009-01-12 2010-07-15 Merck Sharp & Dohme Corp. Nanoparticules lipidiques inédites et composants inédits utilisables pour l'administration d'acides nucléiques
US7772369B2 (en) 1999-05-04 2010-08-10 Monsanto Technology Llc Coleopteran-toxic polypeptide compositions and insect-resistant transgenic plants
US7838658B2 (en) 2005-10-20 2010-11-23 Ian Maclachlan siRNA silencing of filovirus gene expression
US7893302B2 (en) 2005-02-14 2011-02-22 Sirna Therapeutics, Inc. Lipid nanoparticle based compositions and methods for the delivery of biologically active molecules
WO2011022460A1 (fr) 2009-08-20 2011-02-24 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques avec différents groupes de tête pour délivrance d’oligonucléotide
US7915399B2 (en) 2006-06-09 2011-03-29 Protiva Biotherapeutics, Inc. Modified siRNA molecules and uses thereof
WO2011043913A2 (fr) 2009-10-08 2011-04-14 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques à chaînes lipidiques courtes pour une administration d'oligonucléotides
US7982027B2 (en) 2003-07-16 2011-07-19 Protiva Biotherapeutics, Inc. Lipid encapsulated interfering RNA
US20110182867A1 (en) 2008-09-15 2011-07-28 Children's Medical Center Corporation Modulation of bcl11a for treatment of hemoglobinopathies
WO2011090965A1 (fr) 2010-01-22 2011-07-28 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques pour transfert d'oligonucléotide
WO2011127255A1 (fr) 2010-04-08 2011-10-13 Merck Sharp & Dohme Corp. Préparation de nanoparticules de lipide
US8058069B2 (en) 2008-04-15 2011-11-15 Protiva Biotherapeutics, Inc. Lipid formulations for nucleic acid delivery
WO2011149733A2 (fr) 2010-05-24 2011-12-01 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques alcools aminés pour l'administration d'oligonucléotides
WO2011153120A1 (fr) 2010-06-04 2011-12-08 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques de faible poids moléculaire pour l'administration d'oligonucléotides
US8101741B2 (en) 2005-11-02 2012-01-24 Protiva Biotherapeutics, Inc. Modified siRNA molecules and uses thereof
WO2012040184A2 (fr) 2010-09-20 2012-03-29 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques de faible poids moléculaire pour l'administration d'oligonucléotides
US8147856B2 (en) 2006-06-14 2012-04-03 Athenix Corp. AXMI-031, AXMI-039, AXMI-040 and AXMI-049, a family of novel delta-endotoxin genes and methods for their use
WO2012044638A1 (fr) 2010-09-30 2012-04-05 Merck Sharp & Dohme Corp. Lipides cationiques de faible masse moléculaire utilisables en vue de l'administration d'oligonucléotides
WO2012054365A2 (fr) 2010-10-21 2012-04-26 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques à faible poids moléculaire destinés à une administration d'oligonucléotides
WO2012061259A2 (fr) 2010-11-05 2012-05-10 Merck Sharp & Dohme Corp. Amine cyclique inédite de faible masse moléculaire contenant des lipides cationiques en vue de l'administration d'oligonucléotides
WO2012099755A1 (fr) 2011-01-11 2012-07-26 Alnylam Pharmaceuticals, Inc. Lipides pégylés et leur utilisation pour une administration de médicament
US8236943B2 (en) 2009-07-01 2012-08-07 Protiva Biotherapeutics, Inc. Compositions and methods for silencing apolipoprotein B
US8283333B2 (en) 2009-07-01 2012-10-09 Protiva Biotherapeutics, Inc. Lipid formulations for nucleic acid delivery
US8404658B2 (en) 2007-12-31 2013-03-26 Nanocor Therapeutics, Inc. RNA interference for the treatment of heart failure
US8575425B2 (en) 2009-07-02 2013-11-05 Athenix Corporation AXMI-205 pesticidal gene and methods for its use
US8586832B2 (en) 2009-12-21 2013-11-19 Pioneer Hi Bred International Inc Bacillus thuringiensis gene with Lepidopteran activity
US20140068797A1 (en) 2012-05-25 2014-03-06 University Of Vienna Methods and compositions for rna-directed target dna modification and for rna-directed modulation of transcription
US8802934B2 (en) 2010-08-19 2014-08-12 Pioneer Hi Bred International Inc Bacillus thuringiensis gene with lepidopteran activity
US20170121693A1 (en) 2015-10-23 2017-05-04 President And Fellows Of Harvard College Nucleobase editors and uses thereof
US20170252461A1 (en) 2013-03-09 2017-09-07 Modernatx, Inc. Heterologous untranslated regions for mrna
US20170275648A1 (en) 2014-08-28 2017-09-28 North Carolina State University Novel cas9 proteins and guiding features for dna targeting and genome editing
US9790490B2 (en) 2015-06-18 2017-10-17 The Broad Institute Inc. CRISPR enzymes and systems
US9840699B2 (en) 2013-12-12 2017-12-12 President And Fellows Of Harvard College Methods for nucleic acid editing
WO2018027078A1 (fr) 2016-08-03 2018-02-08 President And Fellows Of Harard College Éditeurs de nucléobases d'adénosine et utilisations associées
US9956247B2 (en) 2012-07-11 2018-05-01 Sangamo Therapeutics, Inc. Method of treating lysosomal storage diseases
WO2020139783A2 (fr) 2018-12-27 2020-07-02 Lifeedit, Inc. Polypeptides utiles pour l'édition de gènes et procédés d'utilisation
WO2020156575A1 (fr) 2019-02-02 2020-08-06 Shanghaitech University Inhibition de mutations non intentionnelles dans l'édition de gènes
WO2021042047A1 (fr) 2019-08-30 2021-03-04 The General Hospital Corporation Éditeurs de bases d'adn à transversion de c-à-g
WO2021072328A1 (fr) 2019-10-10 2021-04-15 The Broad Institute, Inc. Procédés et compositions pour le prime editing d'arn
WO2021226558A1 (fr) 2020-05-08 2021-11-11 The Broad Institute, Inc. Méthodes et compositions d'édition simultanée des deux brins d'une séquence nucléotidique double brin cible
US11193123B2 (en) 2020-03-19 2021-12-07 Rewrite Therapeutics, Inc. Methods and compositions for directed genome editing
WO2022015969A1 (fr) 2020-07-15 2022-01-20 LifeEDIT Therapeutics, Inc. Protéines stabilisant l'uracile et fragments actifs et variants de celles-ci et procédés d'utilisation
WO2022056254A2 (fr) 2020-09-11 2022-03-17 LifeEDIT Therapeutics, Inc. Enzymes modifiant l'adn et fragments actifs et variants de celles-ci et procédés d'utilisation
WO2022150485A1 (fr) 2021-01-06 2022-07-14 Oncorus, Inc. Polynucléotides d'arn encapsulés et procédés d'utilisation
WO2022173531A1 (fr) 2021-02-10 2022-08-18 Oncorus, Inc. Composés, compositions et leurs méthodes d'utilisation
US11447770B1 (en) 2019-03-19 2022-09-20 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
WO2022204093A1 (fr) 2021-03-22 2022-09-29 LifeEDIT Therapeutics, Inc. Enzymes de modification de l'adn et fragments actifs et variants associés et méthodes d'utilisation
WO2022261394A1 (fr) 2021-06-11 2022-12-15 LifeEDIT Therapeutics, Inc. Promoteurs d'arn polymérase iii et procédés d'utilisation
US20230050143A1 (en) 2015-12-09 2023-02-16 Modernatx, Inc. Heterologous UTR Sequences for Enhanced mRNA Expression
WO2023139557A1 (fr) 2022-01-24 2023-07-27 LifeEDIT Therapeutics, Inc. Nucléases guidées par arn et fragments actifs, variants associés et procédés d'utilisation
WO2024042489A1 (fr) 2022-08-25 2024-02-29 LifeEDIT Therapeutics, Inc. Modification chimique d'arn guides avec un acide nucléique verrouillé pour l'édition de gènes à médiation par nucléase guidée par l'arn
WO2024095245A2 (fr) 2022-11-04 2024-05-10 LifeEDIT Therapeutics, Inc. Désaminases d'adénine évoluées et protéines de fusion de nucléase guidée par arn avec des sites d'insertion internes et procédés d'utilisation

Patent Citations (112)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4217344A (en) 1976-06-23 1980-08-12 L'oreal Compositions containing aqueous dispersions of lipid spheres
US4196265A (en) 1977-06-15 1980-04-01 The Wistar Institute Method of producing antibodies
US4235871A (en) 1978-02-24 1980-11-25 Papahadjopoulos Demetrios P Method of encapsulating biologically active materials in lipid vesicles
US4186183A (en) 1978-03-29 1980-01-29 The United States Of America As Represented By The Secretary Of The Army Liposome carriers in chemotherapy of leishmaniasis
US4261975A (en) 1979-09-19 1981-04-14 Merck & Co., Inc. Viral liposome particle
US4485054A (en) 1982-10-04 1984-11-27 Lipoderm Pharmaceuticals Limited Method of encapsulating biologically active materials in multilamellar lipid vesicles (MLV)
US4501728A (en) 1983-01-06 1985-02-26 Technology Unlimited, Inc. Masking of liposomes from RES recognition
US5049386A (en) 1985-01-07 1991-09-17 Syntex (U.S.A.) Inc. N-ω,(ω-1)-dialkyloxy)- and N-(ω,(ω-1)-dialkenyloxy)Alk-1-YL-N,N,N-tetrasubstituted ammonium lipids and uses therefor
US4946787A (en) 1985-01-07 1990-08-07 Syntex (U.S.A.) Inc. N-(ω,(ω-1)-dialkyloxy)- and N-(ω,(ω-1)-dialkenyloxy)-alk-1-yl-N,N,N-tetrasubstituted ammonium lipids and uses therefor
US4897355A (en) 1985-01-07 1990-01-30 Syntex (U.S.A.) Inc. N[ω,(ω-1)-dialkyloxy]- and N-[ω,(ω-1)-dialkenyloxy]-alk-1-yl-N,N,N-tetrasubstituted ammonium lipids and uses therefor
US4797368A (en) 1985-03-15 1989-01-10 The United States Of America As Represented By The Department Of Health And Human Services Adeno-associated virus as eukaryotic expression vector
US5569597A (en) 1985-05-13 1996-10-29 Ciba Geigy Corp. Methods of inserting viral DNA into plant material
US4774085A (en) 1985-07-09 1988-09-27 501 Board of Regents, Univ. of Texas Pharmaceutical administration systems containing a mixture of immunomodulators
US4853331A (en) 1985-08-16 1989-08-01 Mycogen Corporation Cloning and expression of Bacillus thuringiensis toxin gene toxic to beetles of the order Coleoptera
US5380831A (en) 1986-04-04 1995-01-10 Mycogen Plant Science, Inc. Synthetic insecticidal crystal protein gene
US5268463A (en) 1986-11-11 1993-12-07 Jefferson Richard A Plant promoter α-glucuronidase gene construct
US5608142A (en) 1986-12-03 1997-03-04 Agracetus, Inc. Insecticidal cotton plants
US4837028A (en) 1986-12-24 1989-06-06 Liposome Technology, Inc. Liposomes with enhanced circulation time
US5039523A (en) 1988-10-27 1991-08-13 Mycogen Corporation Novel Bacillus thuringiensis isolate denoted B.t. PS81F, active against lepidopteran pests, and a gene encoding a lepidopteran-active toxin
US5364780A (en) 1989-03-17 1994-11-15 E. I. Du Pont De Nemours And Company External regulation of gene expression by inducible promoters
US5466785A (en) 1990-04-12 1995-11-14 Ciba-Geigy Corporation Tissue-preferential promoters
WO1991016024A1 (fr) 1990-04-19 1991-10-31 Vical, Inc. Lipides cationiques servant a l'apport intracellulaire de molecules biologiquement actives
WO1991017424A1 (fr) 1990-05-03 1991-11-14 Vical, Inc. Acheminement intracellulaire de substances biologiquement actives effectue a l'aide de complexes de lipides s'auto-assemblant
US5608149A (en) 1990-06-18 1997-03-04 Monsanto Company Enhanced starch biosynthesis in tomatoes
EP0480762A2 (fr) 1990-10-12 1992-04-15 Mycogen Corporation Nouveaux isolats de Bacillus thuringiensis actifs contre les diptères
US5173414A (en) 1990-10-30 1992-12-22 Applied Immune Sciences, Inc. Production of recombinant adeno-associated virus vectors
US5399680A (en) 1991-05-22 1995-03-21 The Salk Institute For Biological Studies Rice chitinase promoter
US5604121A (en) 1991-08-27 1997-02-18 Agricultural Genetics Company Limited Proteins with insecticidal properties against homopteran insects and their use in plant protection
US5436391A (en) 1991-11-29 1995-07-25 Mitsubishi Corporation Synthetic insecticidal gene, plants of the genus oryza transformed with the gene, and production thereof
WO1993024641A2 (fr) 1992-06-02 1993-12-09 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Virus adeno-associe a sequences terminales inversees utilisees comme promoteur
WO1994026877A1 (fr) 1993-05-17 1994-11-24 The Regents Of The University Of California Therapie genique ribozymique contre l'infection par le vih et contre le sida
US5789156A (en) 1993-06-14 1998-08-04 Basf Ag Tetracycline-regulated transcriptional inhibitors
US5814618A (en) 1993-06-14 1998-09-29 Basf Aktiengesellschaft Methods for regulating gene expression
US5837458A (en) 1994-02-17 1998-11-17 Maxygen, Inc. Methods and compositions for cellular and metabolic engineering
US5605793A (en) 1994-02-17 1997-02-25 Affymax Technologies N.V. Methods for in vitro recombination
US5608144A (en) 1994-08-12 1997-03-04 Dna Plant Technology Corp. Plant group 2 promoters and uses thereof
US5659026A (en) 1995-03-24 1997-08-19 Pioneer Hi-Bred International ALS3 promoter
US6072050A (en) 1996-06-11 2000-06-06 Pioneer Hi-Bred International, Inc. Synthetic promoters
US20100036115A1 (en) 1997-07-23 2010-02-11 Sirna Therapeutics, Inc. Novel Compositions for the Delivery of Negatively Charged Molecules
WO1999043838A1 (fr) 1998-02-24 1999-09-02 Pioneer Hi-Bred International, Inc. Promoteurs de synthese
US6177611B1 (en) 1998-02-26 2001-01-23 Pioneer Hi-Bred International, Inc. Maize promoters
US20030087817A1 (en) 1999-01-12 2003-05-08 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US7772369B2 (en) 1999-05-04 2010-08-10 Monsanto Technology Llc Coleopteran-toxic polypeptide compositions and insect-resistant transgenic plants
US7534939B2 (en) 1999-09-15 2009-05-19 Monsanto Technology Llc Plant transformed with polynucleotide encoding lepidopteran-active Bacillus thuringiensis δ-endotoxin
US20050222064A1 (en) 2002-02-20 2005-10-06 Sirna Therapeutics, Inc. Polycationic compositions for cellular delivery of polynucleotides
US7901708B2 (en) 2002-06-28 2011-03-08 Protiva Biotherapeutics, Inc. Liposomal apparatus and manufacturing methods
EP1519714A1 (fr) 2002-06-28 2005-04-06 Protiva Biotherapeutics Inc. Appareil liposomal et procedes de fabrication
US7982027B2 (en) 2003-07-16 2011-07-19 Protiva Biotherapeutics, Inc. Lipid encapsulated interfering RNA
EP1664316A1 (fr) 2003-09-15 2006-06-07 Protiva Biotherapeutics Inc. Composes conjugues lipidiques polyethyleneglycol modifies et utilisations de ces composes
US7803397B2 (en) 2003-09-15 2010-09-28 Protiva Biotherapeutics, Inc. Polyethyleneglycol-modified lipid compounds and uses thereof
US7692068B2 (en) 2003-10-14 2010-04-06 Athenix Corporation AXMI-010, a delta-endotoxin gene and methods for its use
US7790846B2 (en) 2003-12-22 2010-09-07 Pioneer Hi-Bred International, Inc. Bacillus thuringiensis Cry9 toxins
US7541517B2 (en) 2003-12-22 2009-06-02 Pioneer Hi-Bred International, Inc. Bacillus thuringiensis CRY9 nucleic acids
EP1781593A2 (fr) 2004-06-07 2007-05-09 Protiva Biotherapeutics Inc. Lipides cationiques et leurs procédés d'utilisation
EP1766035A1 (fr) 2004-06-07 2007-03-28 Protiva Biotherapeutics Inc. Arn interferant encapsule dans des lipides
US7745651B2 (en) 2004-06-07 2010-06-29 Protiva Biotherapeutics, Inc. Cationic lipids and methods of use
US7799565B2 (en) 2004-06-07 2010-09-21 Protiva Biotherapeutics, Inc. Lipid encapsulated interfering RNA
US7404969B2 (en) 2005-02-14 2008-07-29 Sirna Therapeutics, Inc. Lipid nanoparticle based compositions and methods for the delivery of biologically active molecules
US7893302B2 (en) 2005-02-14 2011-02-22 Sirna Therapeutics, Inc. Lipid nanoparticle based compositions and methods for the delivery of biologically active molecules
US7838658B2 (en) 2005-10-20 2010-11-23 Ian Maclachlan siRNA silencing of filovirus gene expression
US8101741B2 (en) 2005-11-02 2012-01-24 Protiva Biotherapeutics, Inc. Modified siRNA molecules and uses thereof
US8188263B2 (en) 2005-11-02 2012-05-29 Protiva Biotherapeutics, Inc. Modified siRNA molecules and uses thereof
US7915399B2 (en) 2006-06-09 2011-03-29 Protiva Biotherapeutics, Inc. Modified siRNA molecules and uses thereof
US8147856B2 (en) 2006-06-14 2012-04-03 Athenix Corp. AXMI-031, AXMI-039, AXMI-040 and AXMI-049, a family of novel delta-endotoxin genes and methods for their use
WO2008103276A2 (fr) 2007-02-16 2008-08-28 Merck & Co., Inc. Compositions et méthodes de potentialisation de l'activité de molécules biologiquement actives
US8404658B2 (en) 2007-12-31 2013-03-26 Nanocor Therapeutics, Inc. RNA interference for the treatment of heart failure
US8058069B2 (en) 2008-04-15 2011-11-15 Protiva Biotherapeutics, Inc. Lipid formulations for nucleic acid delivery
WO2010021865A1 (fr) 2008-08-18 2010-02-25 Merck Sharp & Dohme Corp. Nouvelles nanoparticules lipidiques et nouveaux composants pour l'administration d'acides nucléiques
US20110182867A1 (en) 2008-09-15 2011-07-28 Children's Medical Center Corporation Modulation of bcl11a for treatment of hemoglobinopathies
WO2010080724A1 (fr) 2009-01-12 2010-07-15 Merck Sharp & Dohme Corp. Nanoparticules lipidiques inédites et composants inédits utilisables pour l'administration d'acides nucléiques
US8236943B2 (en) 2009-07-01 2012-08-07 Protiva Biotherapeutics, Inc. Compositions and methods for silencing apolipoprotein B
US8283333B2 (en) 2009-07-01 2012-10-09 Protiva Biotherapeutics, Inc. Lipid formulations for nucleic acid delivery
US8575425B2 (en) 2009-07-02 2013-11-05 Athenix Corporation AXMI-205 pesticidal gene and methods for its use
WO2011022460A1 (fr) 2009-08-20 2011-02-24 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques avec différents groupes de tête pour délivrance d’oligonucléotide
WO2011043913A2 (fr) 2009-10-08 2011-04-14 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques à chaînes lipidiques courtes pour une administration d'oligonucléotides
US8586832B2 (en) 2009-12-21 2013-11-19 Pioneer Hi Bred International Inc Bacillus thuringiensis gene with Lepidopteran activity
WO2011090965A1 (fr) 2010-01-22 2011-07-28 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques pour transfert d'oligonucléotide
WO2011127255A1 (fr) 2010-04-08 2011-10-13 Merck Sharp & Dohme Corp. Préparation de nanoparticules de lipide
WO2011149733A2 (fr) 2010-05-24 2011-12-01 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques alcools aminés pour l'administration d'oligonucléotides
WO2011153120A1 (fr) 2010-06-04 2011-12-08 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques de faible poids moléculaire pour l'administration d'oligonucléotides
US8802934B2 (en) 2010-08-19 2014-08-12 Pioneer Hi Bred International Inc Bacillus thuringiensis gene with lepidopteran activity
WO2012040184A2 (fr) 2010-09-20 2012-03-29 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques de faible poids moléculaire pour l'administration d'oligonucléotides
WO2012044638A1 (fr) 2010-09-30 2012-04-05 Merck Sharp & Dohme Corp. Lipides cationiques de faible masse moléculaire utilisables en vue de l'administration d'oligonucléotides
WO2012054365A2 (fr) 2010-10-21 2012-04-26 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques à faible poids moléculaire destinés à une administration d'oligonucléotides
WO2012061259A2 (fr) 2010-11-05 2012-05-10 Merck Sharp & Dohme Corp. Amine cyclique inédite de faible masse moléculaire contenant des lipides cationiques en vue de l'administration d'oligonucléotides
WO2012099755A1 (fr) 2011-01-11 2012-07-26 Alnylam Pharmaceuticals, Inc. Lipides pégylés et leur utilisation pour une administration de médicament
US20140068797A1 (en) 2012-05-25 2014-03-06 University Of Vienna Methods and compositions for rna-directed target dna modification and for rna-directed modulation of transcription
US9956247B2 (en) 2012-07-11 2018-05-01 Sangamo Therapeutics, Inc. Method of treating lysosomal storage diseases
US20170252461A1 (en) 2013-03-09 2017-09-07 Modernatx, Inc. Heterologous untranslated regions for mrna
US9840699B2 (en) 2013-12-12 2017-12-12 President And Fellows Of Harvard College Methods for nucleic acid editing
US20170275648A1 (en) 2014-08-28 2017-09-28 North Carolina State University Novel cas9 proteins and guiding features for dna targeting and genome editing
US9790490B2 (en) 2015-06-18 2017-10-17 The Broad Institute Inc. CRISPR enzymes and systems
US20170121693A1 (en) 2015-10-23 2017-05-04 President And Fellows Of Harvard College Nucleobase editors and uses thereof
US20230050143A1 (en) 2015-12-09 2023-02-16 Modernatx, Inc. Heterologous UTR Sequences for Enhanced mRNA Expression
US20180073012A1 (en) 2016-08-03 2018-03-15 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
WO2018027078A1 (fr) 2016-08-03 2018-02-08 President And Fellows Of Harard College Éditeurs de nucléobases d'adénosine et utilisations associées
WO2020139783A2 (fr) 2018-12-27 2020-07-02 Lifeedit, Inc. Polypeptides utiles pour l'édition de gènes et procédés d'utilisation
WO2020156575A1 (fr) 2019-02-02 2020-08-06 Shanghaitech University Inhibition de mutations non intentionnelles dans l'édition de gènes
US11447770B1 (en) 2019-03-19 2022-09-20 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
WO2021042047A1 (fr) 2019-08-30 2021-03-04 The General Hospital Corporation Éditeurs de bases d'adn à transversion de c-à-g
WO2021072328A1 (fr) 2019-10-10 2021-04-15 The Broad Institute, Inc. Procédés et compositions pour le prime editing d'arn
US11193123B2 (en) 2020-03-19 2021-12-07 Rewrite Therapeutics, Inc. Methods and compositions for directed genome editing
WO2021226558A1 (fr) 2020-05-08 2021-11-11 The Broad Institute, Inc. Méthodes et compositions d'édition simultanée des deux brins d'une séquence nucléotidique double brin cible
WO2022015969A1 (fr) 2020-07-15 2022-01-20 LifeEDIT Therapeutics, Inc. Protéines stabilisant l'uracile et fragments actifs et variants de celles-ci et procédés d'utilisation
WO2022056254A2 (fr) 2020-09-11 2022-03-17 LifeEDIT Therapeutics, Inc. Enzymes modifiant l'adn et fragments actifs et variants de celles-ci et procédés d'utilisation
WO2022150485A1 (fr) 2021-01-06 2022-07-14 Oncorus, Inc. Polynucléotides d'arn encapsulés et procédés d'utilisation
WO2022173531A1 (fr) 2021-02-10 2022-08-18 Oncorus, Inc. Composés, compositions et leurs méthodes d'utilisation
WO2022204093A1 (fr) 2021-03-22 2022-09-29 LifeEDIT Therapeutics, Inc. Enzymes de modification de l'adn et fragments actifs et variants associés et méthodes d'utilisation
WO2022261394A1 (fr) 2021-06-11 2022-12-15 LifeEDIT Therapeutics, Inc. Promoteurs d'arn polymérase iii et procédés d'utilisation
WO2023139557A1 (fr) 2022-01-24 2023-07-27 LifeEDIT Therapeutics, Inc. Nucléases guidées par arn et fragments actifs, variants associés et procédés d'utilisation
WO2024042489A1 (fr) 2022-08-25 2024-02-29 LifeEDIT Therapeutics, Inc. Modification chimique d'arn guides avec un acide nucléique verrouillé pour l'édition de gènes à médiation par nucléase guidée par l'arn
WO2024095245A2 (fr) 2022-11-04 2024-05-10 LifeEDIT Therapeutics, Inc. Désaminases d'adénine évoluées et protéines de fusion de nucléase guidée par arn avec des sites d'insertion internes et procédés d'utilisation

Non-Patent Citations (179)

* Cited by examiner, † Cited by third party
Title
"Advanced Bacterial Genetics", 1980, COLD SPRING HARBOR LABORATORY PRESS
AHMAD ET AL., CANCER RES., vol. 52, 1992, pages 4817 - 4820
ALTSCHUL ET AL., NUCLEIC ACIDS RES., vol. 25, 1997, pages 3389 - 3402
ANDERSON, SCIENCE, vol. 256, 1992, pages 808 - 813
AUSUBEL ET AL.: "Current Protocols in Molecular Biology", 2003, WILEY-INTERSCIENCE
BALLAS ET AL., NUCLEIC ACIDS RES, vol. 17, 1989, pages 7891 - 7903
BARTLETT ET AL., PLANT METHODS, vol. 4, 2008, pages 1 - 12
BASHA ET AL., MOL. THER., vol. 19, 2011, pages 2186 - 2200
BASHA ET AL., MOLECULAR THERAPY, vol. 19, no. 12, 2011, pages 1286 - 2200
BATES, G.W., METHODS IN MOLECULAR BIOLOGY, vol. 111, 1999, pages 359 - 366
BENOIT ET AL., BIOMACROMOLECULES, vol. 12, 2011, pages 2708 - 2714
BINNSTHOMASHOW, ANNUAL REVIEWS IN MICROBIOLOGY, vol. 42, 1988, pages 575 - 606
BLAESE ET AL., CANCER GENE THER., vol. 2, 1995, pages 291 - 297
BRINER ET AL., MOLECULAR CELL, vol. 56, 2014, pages 333 - 339
BRINERBARRANGOU, COLD SPRING HARB PROTOC; DOI: 10.1101/PDB.TOP090902, 2016
BROWN ET AL.: "Serine recombinases as tools for genome engineering", METHODS, vol. 53, no. 4, 2011, pages 372 - 9, XP028165732, DOI: 10.1016/j.ymeth.2010.12.031
BUCHSCHER ET AL., J. VIRAL., vol. 66, 1992, pages 1635 - 1640
BUNGE ET AL., HUM. MOL. GENET., vol. 3, no. 6, 1994, pages 861 - 866
CAMPBELLGOWRI, PLANT PHYSIOL., vol. 92, 1990, pages 1 - 11
CAMPUZANO ET AL., SCIENCE, vol. 271, 1996, pages 1423 - 1427
CARRIE ET AL., FEBS J, vol. 276, 2009, pages 1187 - 1195
CARRIE ET AL., FEBSJ, vol. 276, 2009, pages 1187 - 1195
CARRIESMALL, BIOCHIM BIOPHYS ACTA, vol. 1833, 2013, pages 253 - 259
CHAVEZCALOS: "Therapeutic applications of the <DC3l integrase system", CURR. GENE THER., vol. 11, no. 5, 2011, pages 375 - 81
CHEN ET AL., J. AMER. CHEM. SOC., vol. 134, 2012, pages 6948 - 6951
CHEN PJ ET AL., CELL, 2021
CHRISTENSEN ET AL., PLANT MOL. BIOL., vol. 12, 1989, pages 619 - 632
CHRISTENSEN ET AL., PLANT MOL. BIOL., vol. 18, 1992, pages 675 - 689
CHRISTOU, P, EUPHYTICA, vol. 85, 1995, pages 13 - 27
CHRISTOU, P, THE PLANT JOURNAL, vol. 2, 1992, pages 275 - 281
CONSTANTOULAKIS ET AL., BLOOD, vol. 72, no. 6, 1988, pages 1961 - 1967
COSTA ET AL., NAT METH, vol. 2, 2005, pages 259 - 260
CRAMERI ET AL., NATURE BIOTECH, vol. 15, 1997, pages 436 - 438
CRAMERI ET AL., NATURE, vol. 391, 1998, pages 288 - 291
CRYSTAL, SCIENCE, vol. 270, 1995, pages 404 - 410
DAYHOFF ET AL.: "Atlas of Protein Sequence and Structure", vol. 5, 1978, NATL. BIOMED. RES. FOUND., article "A model of evolutionary change in proteins", pages: 345 - 352
DE LA LUNA ET AL., GENE, vol. 62, 1988, pages 121 - 126
DESIMONE, PROC NAT'L ACAD SCI USA, vol. 79, no. 14, 1982, pages 4428 - 31
FENSKECULLIS, EXPERT OPIN DRUG DELIV, vol. 5, 2008, pages 309 - 319
FENTON ET AL., ADVANCED MATERIALS, vol. 28, no. 15, 2016, pages 2939 - 2943
FINN ET AL., CELL REPORTS, vol. 22, 2018, pages 2227 - 2235
GAO ET AL., GENE THERAPY, vol. 2, 1995, pages 710 - 722
GAUDELLI ET AL., NATURE, vol. 551, 2017, pages 464 - 471
GEISBERT ET AL., LANCET, vol. 375, 2010, pages 1896 - 1905
GILPROUDFOOT, CELL, vol. 49, no. 3, 1987, pages 399 - 406
GLASER ET AL., PLANT MOL BIOL, vol. 38, 1998, pages 311 - 338
GOODWINROTTMAN, THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 267, no. 23, 1992, pages 16330 - 16334
GRUBER ET AL., CELL, vol. 106, no. 1, 2008, pages 23 - 24
GUERINEAU ET AL., MOL. GEN. GENET., vol. 227, 1991, pages 229 - 237
GUSCHIN ET AL., METHODS MOL BIOL, vol. 649, 2010, pages 247 - 256
GUTBIER ET AL., PULM PHARMACOL. THER., vol. 23, 2010, pages 334 - 344
HADDADA ET AL., CURRENT TOPICS IN MICROBIOLOGY AND IMMUNOLOGY, 1995
HAN ET AL., SCI ADV, vol. 8, no. 3, 2022, pages eabj6901
HARTMANMULLIGAN, PROC. NATL. ACAD. SCI. U.S.A., vol. 85, 1988, pages 8047 - 8051
HENIKOFF ET AL., PROC. NATL. ACAD. SCI. USA, vol. 89, 1992, pages 10915 - 10919
HERMONATMUZYCZKA, PNAS, vol. 81, 1984, pages 6466 - 6470
HERRMANNNEUPERT, IUBMB LIFE, vol. 55, 2003, pages 219 - 225
HIRANO ET AL.: "Site-specific recombinases as tools for heterologous gene integration", APPL. MICROBIOL. BIOTECHNOL., vol. 92, no. 2, 2011, pages 227 - 39, XP019957609, DOI: 10.1007/s00253-011-3519-5
JAYARAMAN ET AL., ANGEW CHEM INT ED ENGL, vol. 51, no. 34, 2012, pages 8529 - 8533
JONES ET AL., PLANT METHODS, vol. 1, no. 5, 2005, pages 5
JOSHI ET AL., NUCLEIC ACIDS RES, vol. 15, 1987, pages 9627 - 9639
JUDGE ET AL., J CLIN INVEST, vol. 119, 2009, pages 661 - 673
JUDGE ET AL., J. CLIN. INVEST., vol. 119, 2009, pages 661 - 673
KARPENSHIFBERNSTEIN: "From yeast to mammals: recent advances in genetic control of homologous recombination", DNA REPAIR (AMST, vol. 11, no. 10, 2012, pages 781 - 8
KARVELIS ET AL., GENOME BIOL, vol. 16, 2015, pages 253
KATIN, HUMAN GENE THERAPY, vol. 5, 1994, pages 793 - 801
KAUFMANN ET AL., MICROVASC RES, vol. 80, 2010, pages 286 - 293
KAWASE ET AL., BLOOD, vol. 110, 2007, pages 2235 - 2241
KIM ET AL., SCI ADV, vol. 7, no. 9, 2021, pages eabf4398
KIM, METHODS MOL BIOL, vol. 721, 2011, pages 339 - 353
KOLHATKAR ET AL., CURR DRUG DISCOV TECHNOL, vol. 8, 2011, pages 197 - 206
KREMERPERRICAUDET, BRITISH MEDICAL BULLETIN, vol. 51, no. 1, 1995, pages 31 - 44
KUNZEBERGER, FRONT PHYSIOL DX.DOI.ORG/10.3389/FPHYS.2015.00259, 2015
LAMTRUONG, ACS SYNTH. BIOL., vol. 9, no. 10, 2020, pages 2625 - 2631
LANGE ET AL., J. BIOL. CHEM., vol. 282, 2007, pages 5101 - 5105
LANOIXACHESON, EMBO J., vol. 7, no. 8, 1988, pages 2515 - 2522
LAST ET AL., THEOR. APPL. GENET., vol. 81, 1991, pages 581 - 588
LEY ET AL., BLOOD, vol. 62, 1983, pages 370 - 380
LEY ET AL., N. ENGL. J. MEDICINE, vol. 307, 1982, pages 1469 - 1475
LI ET AL., GENE THERAPY, vol. 19, 2012, pages 775 - 780
LITTLEFIELD, SCIENCE, vol. 145, 1964, pages 709 - 710
LOVE ET AL., PROC. NATL ACAD. SCI. USA, vol. 107, 2010, pages 1864 - 1869
MACKENZIE, TRENDS CELL BIOL, vol. 15, 2005, pages 548 - 554
MANTHORPE ET AL., HUM GENE THER, vol. 4, 1993, pages 419 - 431
MARNEF ET AL., J MOL BIOL, vol. 429, no. 9, 2017, pages 1277 - 1288
MCBRIDE ET AL., PROC. NATL. ACAD. SCI. USA, vol. 91, 1994, pages 10747 - 10751
MCCORMICK ET AL., PLANT CELL REPORTS, vol. 5, 1986, pages 81 - 84
MCELROY ET AL., PLANT CELL, vol. 2, 1990, pages 1261 - 1272
MCNELLIS ET AL., PLANT J, vol. 14, no. 2, 1998, pages 247 - 257
MEINKOTHWAHL, ANAL. BIOCHEM., vol. 138, 1984, pages 267 - 284
MILLER ET AL., 1. VIRAL., vol. 65, 1991, pages 2220 - 2224
MILLER, NATURE, vol. 357, 1992, pages 455 - 460
MILLETTI F, DRUG DISCOV TODAY, vol. 17, 2012, pages 850 - 860
MITANICASKEY, TIBTECH, vol. 11, 1993, pages 167 - 175
MIYAGISHI ET AL., NATURE BIOTECHNOLOGY, vol. 20, 2002, pages 497 - 500
MOORE ET AL., J. MOL. BIOL., vol. 272, 1997, pages 336 - 347
MORRISSEY ET AL., NATURE BIOTECHNOLOGY, vol. 23, no. 8, 2005, pages 1002 - 1007
MULLIGANBERG, PROC. NATL. ACAD. SCI. USA., vol. 78, 1981, pages 2072 - 2076
MUNROE ET AL., GENE, vol. 91, 1990, pages 151 - 158
MURCHA ET AL., J EXP BOT, vol. 65, 2014, pages 6301 - 6335
MURPHY: "Phage recombinases and their applications", ADV. VIRUS RES., vol. 83, 2012, pages 367 - 414
MURRAY ET AL., NUCLEIC ACIDS RES., vol. 17, 1989, pages 477 - 498
MUSACCHIOTORCHILIN, FRONT BIOSCI, vol. 16, 2011, pages 1388 - 1412
MUZYCZKA, 1. CLIN. INVEST., vol. 94, 1994, pages 1351
NASSOURYMORSE, BIOCHIM BIOPHYS ACTA, vol. 1743, 2005, pages 5 - 19
NELSON JW ET AL., NAT BIOTECHNOL, 2021
NGUYEN ET AL., J SURG RES, vol. 148, 2008, pages 60 - 66
NZALONE, AV ET AL., NATURE, 2019
ODELL, NATURE, vol. 313, 1985, pages 810 - 812
OELLIGSELIGER, J NEUROSCI RES, vol. 26, 1990, pages 390 - 396
PANDOLFO ET AL.: "Handbook of Clinical Neurology", vol. 103, 2012, pages: 275 - 294
PANDOLFO, ADV. EXP. MED. BIOL., vol. 516, 2002, pages 99 - 118
PASLEAU ET AL., GENE, vol. 38, 1985, pages 227 - 232
PATIL ET AL., CRIT REV THER DRUG CARRIER SYST, vol. 25, 2008, pages 1 - 61
PEER ET AL., PROC NATL ACAD SCI USA., vol. 104, 2007, pages 4095 - 4100
PEER ET AL., SCIENCE, vol. 322, 2008, pages 1839 - 630
PEER, D, J OF CONTROLLED RELEASE, vol. 148, no. 1, 2010, pages 63 - 68
PEERLIEBERMAN, GENE THER, vol. 18, 2011, pages 1127 - 1133
PEETERSSMALL, BIOCHIM BIOPHYS ACTA, vol. 1541, 2001, pages 54 - 63
PETERS ET AL., BLOOD, vol. 91, no. 7, 1998, pages 2601 - 2608
PINELLO ET AL., NATURE BIOTECH, vol. 34, 2016, pages 695 - 697
PITTINGER M FMACKAY A MBECK S C ET AL., SCIENCE, vol. 284, 1999, pages 143 - 147
PROUDFOOT, CELL, vol. 64, 1991, pages 671 - 674
RAKOCZY-TROJANOWSKA, M, CELL MOL BIOL LETT, vol. 7, 2002, pages 849 - 858
RAY ET AL., BIOCONJUG CHEM, vol. 26, no. 6, 2015, pages 1004 - 7
REMY ET AL., BIOCONJUGATE CHEM, vol. 5, 1994, pages 647 - 654
RIVERA ET AL., PHYSICS OF LIFE REVIEWS, vol. 9, 2012, pages 308 - 345
SAMBROOKRUSSELL: "A Laboratory Manual", 1988, COLD SPRING HARBOR LABORATORY PRESS, article "Molecular Cloning"
SAMULSKI ET AL., 1. VIRAL., vol. 63, 1989, pages 03822 - 3828
SANFACON ET AL., GENES DEV, vol. 5, 1991, pages 141 - 149
SANTEL ET AL., GENE THER, vol. 13, 2006, pages 1360 - 1370
SCHEK ET AL., MOLECULAR AND CELLULAR BIOLOGY, vol. 12, no. 12, 1992, pages 5386 - 5393
SCHENA ET AL., PROC. NATL. ACAD. SCI. USA, vol. 88, 1991, pages 10421 - 10425
SILVA-FILHO, CURR OPIN PLANT BIOL, vol. 6, 2003, pages 589 - 595
SIMONSENLEVINSON, PROC. NATL. ACAD. SCI. U.S.A., vol. 80, 1983, pages 2495 - 2499
SOLL, CURR OPIN PLANT BIOL, vol. 5, 2002, pages 529 - 535
SOMMNERFELT ET AL., VIRAL, vol. 176, 1990, pages 58 - 59
SONG ET AL., NAT BIOTECHNOL, vol. 23, 2005, pages 709 - 717
SONG, NAT BIOTECHNOL., vol. 23, 2005, pages 709 - 717
SPUCHNAVARRO, JOURNAL OF DRUG DELIVERY, 2011
SRINIVASAN ET AL., METHODS MOL BIOL, vol. 757, 2012, pages 497 - 507
STEMMER, NATURE, vol. 370, 1994, pages 389 - 391
SUBRAMANYA ET AL., MOL THER, vol. 18, 2010, pages 2028 - 2037
SVAB ET AL., PROC. NAIL. ACAD. SCI. USA, vol. 87, 1990, pages 8526 - 8530
SVABMALIGA, EMBO J., vol. 12, 1993, pages 601 - 606
SVABMALIGA, PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 913 - 917
TAKAHASHIYAMANAKA, CELL, vol. 126, no. 4, 2006, pages 663 - 76
TENG ET AL., NAT COMMUN, vol. 9, no. 1, 2018, pages 4115
TRATSCHIN ET AL., MOL. CELL. BIOL., vol. 4, 1984, pages 2072 - 2081
TRATSCHIN ET AL., MOL. CELL. BIOL., vol. 5, 1985, pages 3251 - 3260
TROELSTRA ET AL., CELL, vol. 71, 1992, pages 939 - 953
TURANBODE: "Site-specific recombinases: from tag-and-target- to tag-and-exchange-based genomic modifications", FASEB J, vol. 25, no. 12, 2011, pages 4088 - 107
TZFIRA ET AL., TRENDS IN GENETICS, vol. 20, 2004, pages 375 - 383
VAN BRUNT, BIOTECHNOLOGY, vol. 6, no. 10, 1988, pages 1149 - 1154
VAN DEN BOOM ET AL., J CELL BIOL, vol. 166, no. 1, 2004, pages 27 - 36
VAN GOOL ET AL., EMBO J, vol. 16, no. 19, 1997, pages 5955 - 65
VELLODI ET AL., ARCH. DIS. CHILD., vol. 76, no. 2, 1997, pages 92 - 99
VELTEN ET AL., EMBO J, vol. 3, 1984, pages 2723 - 2730
VENKENBELLEN: "Genome-wide manipulations of Drosophila melanogaster with transposons, Flp recombinase, and <DC3l integrase", METHODS MOL. BIOL., vol. 859, 2012, pages 203 - 28
VIGNE, RESTORATIVE NEUROLOGY AND NEUROSCIENCE, vol. 8, 1995, pages 35 - 36
WEI ET AL., NATURE COMMUNICATIONS, vol. 11, 2020, pages 3232
WEI ET AL., PNAS USA, vol. 112, no. 27, 2015, pages E3495 - 504
WEST ET AL., VIROLOGY, vol. 160, 1987, pages 38 - 47
WILSON ET AL., J. VIRAL., vol. 63, 1989, pages 2374 - 2378
XIA, NUCLEIC ACIDS RES, vol. 31, no. 17, 2003, pages e100 - e100
XU ET AL., GENE, vol. 272, 2001, pages 149 - 156
YAO ET AL., JOURNAL OF EXPERIMENTAL BOTANY, vol. 57, 2006, pages 3737 - 3746
YEW ET AL., HUM GENE THER, vol. 8, 1997, pages 575 - 584
YU ET AL., GENE THERAPY, vol. 1, 1994, pages 13 - 26
YU ET AL., MOL MEMBR BIOL, vol. 27, 2010, pages 286 - 298
ZHANG ET AL., ACS NANO, vol. 2, no. 8, 2008, pages 1696 - 1702
ZHANG ET AL., CHEM. SCI., vol. 7, 2016, pages 4951 - 4957
ZHANG ET AL., LANGMUIR, vol. 27, no. 5, 2011, pages 1907 - 1914
ZHANG ET AL., PROC. NATL. ACAD. SCI. USA, vol. 94, 1997, pages 4504 - 4509
ZHANG ET AL.: "Conditional gene manipulation: Cre-ating a new biological era", J. ZHEJIANG UNIV. SCI. B., vol. 13, no. 7, 2012, pages 511 - 24, XP035080787, DOI: 10.1631/jzus.B1200042
ZHOU ET AL., PROC. NATL ACAD. SCI. USA, vol. 113, 2016, pages 520 - 525
ZIMMERMAN ET AL., NATURE, vol. 441, 2006, pages 111 - 114
ZUKERSTIEGLER, NUCLEIC ACIDS RES, vol. 9, 1981, pages 133 - 148
ZUPANZAMBRYSKI, PLANT PHYSIOLOGY, vol. 107, 1995, pages 1041 - 1047

Also Published As

Publication number Publication date
WO2025022367A3 (fr) 2025-04-17
EP4750890A2 (fr) 2026-06-03
TW202519651A (zh) 2025-05-16

Similar Documents

Publication Publication Date Title
US12252706B2 (en) RNA-guided nucleases and active fragments and variants thereof and methods of use
US12398385B2 (en) RNA-guided nucleases and active fragments and variants thereof and methods of use
CA3173953A1 (fr) Promoteurs de l&#39;arn polymerase iii et methodes d&#39;utilisation
TW202424186A (zh) Rna引導核酸酶中介基因編輯用之具鎖核酸引導 rna之化學修飾
AU2023323547A1 (en) Rna-guided nucleases and active fragments and variants thereof and methods of use
WO2025174908A1 (fr) Nouvelles nucléases guidées par arn et protéines pour l&#39;édition de polymérase
TW202519651A (zh) Ｒｎａ引導之核酸酶及其活性片段及變異體以及使用方法
WO2026003754A1 (fr) Nouvelles transcriptases inverses et leurs utilisations
WO2025083619A1 (fr) Nucléases guidées par arn et fragments actifs, variants associés et méthodes d&#39;utilisation
HK40119698A (en) Rna-guided nucleases and active fragments and variants thereof and methods of use
HK40099508A (en) Rna-guided nucleases and active fragments and variants thereof and methods of use

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 2024758895

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24758895

Country of ref document: EP

Kind code of ref document: A2