WO2025019675A1 - Système d'édition primaire et ses utilisations - Google Patents

Système d'édition primaire et ses utilisations Download PDF

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WO2025019675A1
WO2025019675A1 PCT/US2024/038551 US2024038551W WO2025019675A1 WO 2025019675 A1 WO2025019675 A1 WO 2025019675A1 US 2024038551 W US2024038551 W US 2024038551W WO 2025019675 A1 WO2025019675 A1 WO 2025019675A1
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editing
pegrna
sequence
seq
cells
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Brittany ADAMSON
Jun Yan
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Princeton University
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Princeton University
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • 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]
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
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    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present disclosure relates to compositions and systems for genome editing and uses thereof.
  • the present disclosure shows modifying synthetic pegRNAs to allow SSB protein binding and overexpression of small RNA binding exonuclease protection factor La (SSB) protein can improve prime editing.
  • SSB small RNA binding exonuclease protection factor La
  • a system comprising a Cas9 nickase; a reverse transcriptase; a small RNA binding exonuclease protection factor La (SSB) protein; and a prime editing guide RNA (pegRNA).
  • SSB small RNA binding exonuclease protection factor La
  • pegRNA prime editing guide RNA
  • the pegRNA comprises a 3 ’-polyuridine domain.
  • the 3 ’-polyuridine domain comprises at least one chemically modified uridine (for example, at least two, three, four, or five chemically modified uridines).
  • the 3 ’-polyuridine domain comprises at least one unmodified uridine (for example, at least two, three, four, or five unmodified uridines).
  • the 3’- polyuridine domain comprises at least one chemically modified uridine (for example, at least two, three, four, or five chemically modified uridines) and at least one unmodified uridine (for example, at least one, two, three, four, or five unmodified uridines).
  • the at least one unmodified uridine locates at the 3’ end of the pegRNA.
  • the chemical modification is 2’ -O-m ethylation and/or replacement of a phosphodiester bond to a phosphorothioate bond.
  • the 3 ’-polyuridine domain comprises at least one (for example, at least two, three, four, or five) uridine with unmodified 2'-hydroxyl (OH) group.
  • the at least one uridine with unmodified 2'- OH group locates at the 3’ end of the pegRNA.
  • the SSB protein of the system of any preceding aspect comprises a sequence at least 80% identical to SEQ ID NO: 34 or a fragment thereof.
  • the SSB protein comprises a La motif and/or an RNA recognition motif (RRM) (e.g., amino acid residues 1-194 or 2-194 of SEQ ID NO: 34).
  • RRM RNA recognition motif
  • the SSB protein comprises a sequence at least 80% identical to SEQ ID NO: 33 or SEQ ID NO: 35 or a fragment thereof.
  • the Cas9 nickase comprises a sequence at least 80% identical to SEQ ID NO: 26 or 27, or a fragment thereof.
  • the SSB protein is operatively linked to the Cas9 nickase and the reverse transcriptase.
  • the system of any preceding aspect comprises a recombinant polypeptide, wherein the recombinant polypeptide comprises a sequence at least 80% identical to any of SEQ ID NOs: 1-12 or a fragment thereof.
  • a system comprising a first polynucleotide encoding a Cas9 nickase; a second polynucleotide encoding a reverse transcriptase; and a third polynucleotide encoding a small RNA binding exonuclease protection factor La (SSB) protein; and a prime editing guide RNA (pegRNA).
  • SSB small RNA binding exonuclease protection factor La
  • pegRNA prime editing guide RNA
  • the third polynucleotide encodes a La motif and/or an RNA recognition motif (RRM) of the SSB protein.
  • the third polynucleotide comprises a sequence at least 80% identical to SEQ ID NO: 50 or a fragment thereof.
  • the third polynucleotide comprises a sequence at least 80% identical to SEQ ID NO: 49 or 51 or a fragment thereof.
  • the first, second, and third polynucleotides are operatively linked thereby forming one recombinant polynucleotide.
  • the recombinant polynucleotide comprises a sequence at least 80% identical to any of SEQ ID NOs: 13-24.
  • the first, second, and third polynucleotides are located on a same or different vectors.
  • composition comprising the system, polypeptides, or polynucleotides of any preceding aspect.
  • Also disclosed herein is a method of treating a genetic disorder in a subject in need, comprising administering to the subject a therapeutically effective amount of the system, polypeptides, or polynucleotides of any preceding aspect or a pharmaceutical composition of any preceding aspect. Also disclosed herein is a method for altering expression of a gene product in a cell, comprising introducing into the cell an effective amount of the system, polypeptides, or polynucleotides of any preceding aspect.
  • FIGS. 1A-1H show Genome-scale CRISPRi screens identify La as a determinant of prime editing.
  • FIG. 1 A shows schematic of prime editing.
  • FIG. IB shows a schematic of FACS reporter that expresses GFP upon installation of +7 GGto CA substitution prime edit (mCherry marker constitutively expressed).
  • FIG. 1C shows flow cytometry analysis of GFP expression in K562 CRISPRi cells with integrated FACS reporter with and without prime editing (+7 GG to CA, PE3 with a +50 complementary strand nick) and with and without transduction of an MSH2-targeting sgRNA.
  • FIG. ID shows the gene-level phenotypes from genome-scale CRISPRi screen performed in FACS reporter cells with +7 GGto CA edit using PE3 approach.
  • Phenotypes represent enrichment of normalized sgRNA counts in GFP+ over GFP- populations after prime editing. Genes identified as hits using CRISPhieRmix (FDR ⁇ 0.01) and pseudogene controls generated from randomly selected non-targeting sgRNAs are indicated.
  • FIG. IE shows the quantification of CRISPRi-mediated La depletion. RT-qPCR data collected from K562 CRISPRi cells with integrated MCS reporter. Data are normalized to ACTB and presented relative to a non-targeting sgRNA.
  • FIG. 1G show a comparison of prime editing outcomes using a pegRNA (left) or an epegRNA (right, tevopreQi) with the PE2 approach (plasmid delivery) at stably integrated FACS reporter in K562 CRISPRi cells after transduction of a La-targeting or non-targeting sgRNA.
  • FIG. 1H shows a comparison of prime editing outcomes with the indicated edit and approach (plasmid delivery) at integrated MCS reporter with and without depletion of La in K562 CRISPRi cells.
  • FIGS. 2A-2H show that La promotes prime editing across edit types and genomic loci in multiple cell lines. FIG.
  • FIG. 2A shows western blot analysis of K562 cells constitutively expressing PEmax (K562 PEmax cell line) and derived clones with genetic disruption of La (La-kol through La-ko5). Whole-cell lysates were sequentially immunoblotted for La, GAPDH, and prime editor protein (PEmax) with corresponding antibodies. Asterisks denote La knockout cell lines used in this study.
  • FIG. 2B shows percentages of intended prime editing and indels at multiple genomic loci in K562 PEmax and La-ko4 cells. pegRNAs and epegRNAs (evopreQi) were delivered as plasmids without or with MLHldn (PE2 or PE4, respectively).
  • FIG. 2c shows percentages of intended prime editing and indels at the endogenous DNMT1 locus in K562 PEmax and La-ko4/5 cells with or without ectopic expression of La.
  • Expression plasmids for La or an mRFP control were delivered alongside plasmids encoding pegRNA or epegRNA (evopreQi) specifying a +5 G to T edit.
  • FIG. 2D shows quantification of siRNA-mediated La depletion. RT-qPCR data were collected from HEK293T cells at specified time points during prime editing.
  • FIG. 2E shows fold changes in indicated editing outcomes across ten PE3 edits (substitutions, insertions, and deletions) at five genomic loci in HEK293T cells with or without La depletion by siRNAs. Editing components delivered by plasmid transfection. Editing percentages presented in FIG. 8F.
  • FIG. 2F shows effects of La depletion on DSB repair. Schematic of the MCS reporter (top), with distances between predicted SaCas9 cut site and sequences required for GFP expression indicated.
  • FIG. 2G shows fold changes in SaPE2, PE4 approach-, SaCas9-, SaBE4- and SaABE8e-induced editing outcomes in La-ko4 relative to parental controls (intended edits only).
  • the same pegRNA expression plasmid was used for the editing systems at each of the four genomic loci. Editing percentages presented in FIGS. 9C-9F.
  • FIGS. 9C-9F show fold changes in SaCas9-, SaBE4-, and SaABE8e-induced editing outcomes in La-ko4 relative to parental controls (intended edits only).
  • the same sgRNA expression plasmid was used for the editing systems at each of the four genomic loci. Editing percentages presented in FIGS. 9C-9F.
  • Indel frequency for each sample included adjacent to corresponding intending editing efficiency in FIGS. 2B-2C.
  • FIGS. 3A-3J show that La functionally interacts with the 3' ends of polyuridylated pegRNAs.
  • FIG. 3 A shows schematics of La domain architecture (top) and five La mutants used in FIG. 3B (bottom).
  • FIG. 3B shows percentages of intended prime editing and indels at the endogenous DNMT1 locus with or without ectopic expression of La or La mutants in a.
  • Expression plasmids (La, mutants, or mRFP control) were delivered to K562 PEmax and La- ko4 cells alongside plasmids encoding one pegRNA (+5 G to T edit).
  • FIG. 1 shows schematics of La domain architecture (top) and five La mutants used in FIG. 3B (bottom).
  • FIG. 3B shows percentages of intended prime editing and indels at the endogenous DNMT1 locus with or without ectopic expression of La or La mutants in a.
  • Expression plasmids (La, mutants, or
  • FIG. 3C shows the chemical structure of RNA with phosphorothioate bonds (*) and 2'-O-methylation modifications (m).
  • FIG. 3D shows percentages of intended prime editing and indels at the endogenous DNMT1 locus in K562 PEmax and La-ko4 cells using 100 pmole of synthetic pegRNAs with indicated 3' end sequences and chemical modifications.
  • FIG. 3E shows fold changes in average intended prime editing achieved at four genomic loci in La-ko4 cells relative to parental controls using 100 pmole synthetic pegRNAs with indicated no-polyU, blocked, or La-accessible end configurations. Editing percentages presented in FIG. 10E.
  • FIG. 3F shows a model of La interaction with 3' ends of polyuridylated pegRNAs promotes prime editing.
  • FIG. 3G shows a schematic of pegRNA specifying RUNX1 +5 G to T with the minimum sequence defining each class of small RNA-seq fragments highlighted (c/.s-active, orange; /ra//.s-active, purple). The edit-encoding nucleotide and cryptic terminators are also indicated (white base and green asterisks, respectively).
  • FIGS. 3H-3I show coverage plots of small RNA-seq fragments aligned to pegRNA (h) or epegRNA (i) encoding RUNX1 +5 G to T.
  • Alignment categories are indicated (human small RNA, gray; cv.s-active, orange; trans-active, purple; premature termination, green) and genes with adjusted /z-values ⁇ 0.05 are highlighted in light gray (calculated by DESeq2 using the Wald test).
  • Nucleotide position 0 denotes the 5' end of the RNA, and positions of the edit-encoding nucleotide (vertical solid line) and the start of PBS (vertical dashed line) are indicated. Shaded areas represent sgRNA sequence, Pol III terminator for pegRNA, and linker plus evopreQi and Pol III terminator for epegRNA.
  • FIGS. 4A-4I show fusion of La RNA-binding N-terminal domain to PEmax improves prime editing.
  • FIG. 4A shows architectures of prime editors. Medium gray NLS, bpNLS SV40 ; Dark gray NLS, NLS c ‘ Myc ; A, 32 amino acid linker; B, 34 amino acid linker; C, SGGS linker; La, full length La or the N-terminal domain of La (La 1-194 ); MMLV-RT, human codon- optimized MMLV-RT.
  • FIG. 4A shows architectures of prime editors.
  • FIG. 4B shows percentages of intended prime edits and indels produced with the indicated editors (from a) and pegRNA or epegRNA (evopreQi) at the endogenous DNMT1 and VEGFA loci in HEK293T and U2OS cells, respectively. Editing components (PE2) delivered by plasmid transfection.
  • FIG. 4C shows percentages of intended prime editing and indels at the endogenous DNMT1 and VEGFA loci in HEK293T, HeLa, and U2OS cells. Editing components delivered by plasmid transfection.
  • FIG. 1 shows percentages of intended prime edits and indels produced with the indicated editors (from a) and pegRNA or epegRNA (evopreQi) at the endogenous DNMT1 and VEGFA loci in HEK293T and U2OS cells, respectively. Editing components (PE2) delivered by plasmid transfection.
  • FIG. 4C shows percentages of intended prime editing and ind
  • FIG. 4D shows percentages of intended prime editing and indels at eight endogenous loci in U2OS cells using pegRNAs or epegRNAs (mpknot: HEK3, tevopreQi: HEK4, evopreQi: all other loci). Editing components delivered by plasmid transfection. pegRNA data also presented in FIG. 15B.
  • FIG. 4E shows percentages of intended prime editing and indels at the endogenous HEK3 locus in HEK293T cells. Editing components delivered by plasmid transfection.
  • FIG. 4F shows fold changes in intended prime editing (left) and ratios of intended editing to indel frequency (right) for each indicated condition. Editing percentages presented in FIG. 4D.
  • FIG. 4G shows a schematic of interactions between La N-terminal domain and RNA with 3 '-UUUOH. Four residues were mutated in PE7 mutant to disrupt 3' polyU binding (Q20, Y23, Y24 and F35; indicated in red).
  • FIG. 4H shows a schematic of PE7 mutant harboring four mutations in La 1-194 to disrupt 3’ polyU binding (Q20A, Y23A, Y24F and F35A; indicated by red lines).
  • FIG. 41 shows percentages of intended prime edits and indels produced with PEmax, PE7 or PE7 mutant at the endogenous RUNX1 and VEGFA loci in U2OS cells. Editing components delivered by plasmid transfection.
  • FIGS. 4C-4E, and 41 Indel frequency for each sample included adjacent to corresponding intending editing efficiency in FIGS. 4C-4E, and 41.
  • One-tailed unpaired Student’s /-test (FIGS. 4B-4E, and 41). *P ⁇ 0.05.
  • FIGS. 5A-5H show that PE7 enhances prime editing at disease-related targets and primary human cells.
  • FIG. 5A-5H show that PE7 enhances prime editing at disease-related targets and primary human cells.
  • FIG. 5A Percentages of intended prime editing and indels at six endogenous loci in U2OS cells using pegRNAs and epegRNAs (tevopreQi). Editing components delivered by plasmid transfection. pegRNA data also presented in FIG. 5C.
  • FIG. 5B Fold changes in intended prime editing for each indicated condition. Editing percentages presented in FIG. 5A.
  • FIG. 5C Summary plot of intended prime edit and indel frequencies produced with indicated editor and prime editing approaches at genomic loci. Data for PE2 and PE4 from six loci indicated in a plus HBG1/2. Data for PE3 and PE5 from a subset of those targets (HBB, PRNP, IL2RB, CXCR4).
  • FIG. 5D Percentages of intended prime editing and indels at four genomic loci in K562 cells using indicated editor and synthetic pegRNAs with no-polyU, blocked or La-accessible end configurations.
  • FIG. 5E Fold changes in average intended prime editing in K562 cells using PE7 mRNA relative to PEmax mRNA for synthetic pegRNAs with each indicated end configuration. Editing percentages presented in FIG. 5D.
  • FIG. 5F Percentages of intended prime editing and indels in primary human T cells using PEmax or PE7 mRNA and synthetic pegRNAs with indicated end configurations.
  • FIG. 5G Fold changes in intended prime editing in primary human T cells using PE7 mRNA and synthetic pegRNAs with La-accessible end configuration relative to intended editing with PEmax mRNA and the same pegRNAs at eight genomic loci.
  • Data in FIG. 5G indicate ratios of values for individual edits and donors (4 different T cell donors) and horizontal bar in FIG. 5G indicate median. Fold changes included for select comparisons in FIGS. 5A, 5D and 5F.
  • FIGS. 6A-6I show characterization of prime editing reporters before and during genome-scale CRISPRi screens.
  • FIG. 6A shows a schematic of isolating prime edited cells with intended edit using the FACS reporter.
  • the reporter expresses GFP upon installation of select prime edits, thus enabling separation of cells into mostly edited or mostly unedited populations using flow cytometry.
  • the complete FACS reporter is depicted in FIG. IB.
  • FIG. 6B shows a schematic of isolating prime edited cells with intended edit using the MCS reporter.
  • the reporter expresses a synthetic cell surface marker (IgK-hlgGl-Fc-PDGFRP) upon installation of select prime edits, thus enabling facile separation of cells into mostly edited or mostly unedited populations using magnetic Protein G beads.
  • IgK-hlgGl-Fc-PDGFRP synthetic cell surface marker
  • FIG. 6C shows three prime edits capable of ‘switching on’ the FACS and MCS reporters (depicted with the former).
  • FIG. 6D shows flow cytometry analysis of GFP expression from the FACS reporter after prime editing with each of the edits depicted in FIG. 6C.
  • FIG. 6E shows percentages of prime editing outcomes in GFP+ or GFP- cell populations sorted by flow cytometry after editing with each of the edits depicted in FIG. 6C. Outcomes quantified by sequencing the FACS reporter target site.
  • FIG. 6F shows percentages of prime editing outcomes in bead-bound or unbound cell populations isolated by magnetic separation after editing with each of the edits depicted in c.
  • FIG. 6G shows flow cytometry analysis of GFP expression in the FACS reporter cells (ie., K562 CRISPRi cells with stably integrated FACS reporter) after transduction with genome-scale CRISPRi library (hCRISPRi-v2) and prime editing by plasmid transfection (+7 GG to CA, PE3). Data from repeat measurements of each replicate of the genome-scale screen.
  • FIG. 6H shows percentages of prime editing outcomes observed in GFP+ or GFP- cell populations for each replicate of the genome-scale FACS screen. Outcomes quantified by sequencing the FACS reporter target site.
  • FIG. 6G shows flow cytometry analysis of GFP expression in the FACS reporter cells (ie., K562 CRISPRi cells with stably integrated FACS reporter) after transduction with genome-scale CRISPRi library (hCRISPRi-v2) and prime editing by plasmid transfection (+7 GG to CA, PE3). Data from repeat measurements of each replicate of the genome-scale screen.
  • FIG. 6H shows
  • FIG. 61 shows sequences and frequencies of alleles observed at the FACS reporter target site in cell populations sorted for replicate 1 of the genome-scale FACS screen.
  • FIGS. 7A-7I show results of genome-scale CRISPRi screens performed with FACS and MCS reporters.
  • FIG. 7A Pearson correlations of read counts per sgRNA between each pair of samples isolated from genome-scale FACS screen.
  • FIG. 7B sgRNA-level phenotypes from each replicate of the genome-scale FACS screen.
  • FIG. 7C Gene-level phenotypes (average of replicates) and per gene FDRs from the genome-scale FACS screen, as determined by CRISPhieRmix analysis.
  • FIG. 7D Pearson correlations of read counts per sgRNA between each pair of samples isolated from the genome-scale MCS screen performed with the PE3 approach.
  • FIG. 7A Pearson correlations of read counts per sgRNA between each pair of samples isolated from genome-scale FACS screen.
  • FIG. 7F Gene-level phenotypes (average of replicates) from genome- scale FACS and MCS screens performed with the PE3 approach.
  • FIGS. 7G-7I Gene-level phenotypes from each replicate of MCS reporter screens performed with the PE3 (FIG. 7G), PE4 (FIG. 7H) and PE5 (FIG. 71) approaches.
  • FIGS. 7B-7E sgRNAs targeting genes identified as hits (FDR ⁇ 0.01) using CRISPhieRmix are highlighted in red.
  • FIGS. 7C, 7F- 7G genes identified as hits (FDR ⁇ 0.01) in the indicated screen using CRISPhieRmix are highlighted in red.
  • FIGS. 8A-8F show validation of La phenotypes with various genetic perturbation modalities.
  • FIG. 8A shows a schematic of workflow used to engineer K562 clonal cell lines with PEmax expressed constitutively from the AAVS1 safe-harbor locus (K562 PEmax cell line).
  • FIG. 8B shows sequences and frequencies of alleles observed at the La locus in the La- knockout clones used in this study (La-ko3 through La-ko5).
  • FIG. 8C shows full images of western blot presented in FIG. 2 A.
  • FIG. 8D shows cumulative population doublings of La-ko4 and La-ko5 cells compared to K562 PEmax parental cells.
  • FIG. 8A shows a schematic of workflow used to engineer K562 clonal cell lines with PEmax expressed constitutively from the AAVS1 safe-harbor locus (K562 PEmax cell line).
  • FIG. 8B shows sequences and frequencies of alleles observed at the La locus
  • FIG. 8E shows flow cytometry analysis of GFP expressed from the PEmax construct at the AAVS1 locus in K562 PEmax parental, La-ko3, La-ko4 and La-ko5 cells prior to transfection in FIG. 2C.
  • FIG. 8F shows percentages of intended prime editing and indels across ten edits with pegRNAs (top) or epegRNAs (bottom, mpknot: HEK3, evopreQi : all other loci) at five genomic loci in HEK293T cells with and without depletion of La by siRNAs.
  • Prime editing components PE3 were delivered as expression plasmids. Percentages in FIG.
  • FIGS. 9A-9F show that La has a stronger impact on prime editing than other editing modalities.
  • FIG. 9A shows flow cytometry analysis of GFP expression from a stably integrated MCS reporter in K562 CRISPRi cells after transduction of indicated sgRNAs and editing with SaCas9 nuclease. Editing components (SaCas9, +7 GG to CA pegRNA) delivered by plasmid transfection.
  • FIG. 9B shows quantification of SaCas9-induced indels at stably integrated MCS reporter described in FIG. 9 A.
  • FIGS. 9A-9F show that La has a stronger impact on prime editing than other editing modalities.
  • FIG. 9A shows flow cytometry analysis of GFP expression from a stably integrated MCS reporter in K562 CRISPRi cells after transduction of indicated sgRNAs and editing with SaCas9 nuclease. Editing components (SaCas9, +7 GG to CA peg
  • FIGS. 9C-9F show percentages of intended editing achieved in K562 PEmax parental and La-knockout cells using SaPE2 with PE4 approach, SaCas9, SaBE4, and SaABE8e across four genomic loci, HEK3 (FIG. 9C), EMX1 (FIG. 9D), FANCF (FIG. 9E) and HBB (FIG. 9F).
  • the same pegRNA or sgRNA expression plasmid was used for the editing systems at each target, with select combinations excluded (ie., SaPE2 with PE4 approach with any sgRNA and SaBE4 at EMX1).
  • FIGS. 10A-10E show prime editing with synthetic pegRNAs designed to block or allow La binding reveals functional interaction between La and polyuridylated 3' ends.
  • FIG. 10A shows chemical structures of ribonucleotides linked by a phosphorothioate bond (left) or with substitution of ribose 2'-OH for 2'-O-methyl groups (2'-0Me) (right).
  • FIG. 10B shows percentages of intended prime editing and indels at the endogenous DNMT1 locus in K562 PEmax parental cells using synthetic pegRNA with indicated 3' end configuration. Input was titrated from 0 to 500 pmole at 100 pmole increments.
  • FIGS. 10A shows chemical structures of ribonucleotides linked by a phosphorothioate bond (left) or with substitution of ribose 2'-OH for 2'-O-methyl groups (2'-0Me) (right).
  • FIG. 10B shows percentages of intended prime editing and
  • FIG. 10C-10D shows percentages of intended prime editing and indels at the endogenous HEK3 (c, PE3) and DNMT1 (b, PE2) loci in K562 PEmax parental, La-ko4, and La-ko5 (where indicated) cells using 100 pmole of synthetic pegRNAs with specified 3' end sequences and chemical modifications.
  • FIG. 10E shows percentages of intended prime editing and indels at endogenous DNMT1, CXCR4, VEGFA and RUNX1 loci in K562 PEmax parental and La-ko4 cells using 100 pmole of synthetic pegRNAs with indicated 3' end configurations.
  • FIGS. 11 A-l ID show details of small RNA-seq experiment performed with two sets of (e)pegRNAs.
  • FIG. 11A shows composition of small RNA-seq libraries from K562 PEmax parental or La-ko4 cells. Data from samples collected one and two days after transfection of eleven (e)pegRNAs in two sets.
  • FIG. 1 IB shows fold changes in normalized counts of indicated biotypes in La-ko4 cells relative to parental controls, from samples collected one and two days after transfection of eleven (e)pegRNAs in two sets. Counts were calculated per replicate independently for each set of (e)pegRNAs as the sum of properly aligned fragments classified as each biotype and normalized by total RNA counts.
  • FIG. 11A shows composition of small RNA-seq libraries from K562 PEmax parental or La-ko4 cells. Data from samples collected one and two days after transfection of eleven (e)pegRNAs in two sets.
  • FIG. 1 IB shows fold changes in normalized
  • Data are from samples collected one day after pegRNA plasmid transfection and normalized by counts of fragments from total human small RNA (top) or those within the corresponding bins (bottom). Plotted data represent coverages of indicated bins (c/.s-active, /ra/z.s-active or inactive) in specified cell lines.
  • FIGS. 12A-12C show additional coverage plots of (e)pegRNAs from small RNA-seq experiment performed with two sets of (e)pegRNAs.
  • FIGS. 12A-12C Coverage plots of small RNA-seq fragments aligned to pegRNA (left) or epegRNA (right) encoding EMX1 +5 G to T (FIG. 12 A), HEK3 +1 T to A (FIG. 12B) or DNMT1 +5 G to T (FIG. 12C).
  • Data are from samples collected one day after (e)pegRNA plasmid transfection and normalized by counts of fragments from total human small RNA (top) or those within the corresponding bins (bottom).
  • Nucleotide position 0 denotes the 5' end of the RNA, and positions of the edit-encoding nucleotide (vertical solid line) and the start of PBS (vertical dashed line) are indicated.
  • Shaded areas represent sgRNA sequence, Pol III terminator for pegRNAs, and linker plus evopreQi/mpknot and Pol III terminator for epegRNAs.
  • FIGS. 13A-13E show details of small RNA-seq experiment performed with nontargeting pegRNA and epegRNA, each specifying a +6 G to C edit in the Mus musculus DNMT1 gene.
  • FIG. 13A shows the composition of small RNA-seq libraries from K562 PEmax parental or La-ko4 cells. Data from samples collected one and two days after transfection of plasmids encoding a pegRNA or an epegRNA specifying mouse DNMT1 +6 Gto C.
  • FIG. 13A shows the composition of small RNA-seq libraries from K562 PEmax parental or La-ko4 cells. Data from samples collected one and two days after transfection of plasmids encoding a pegRNA or an epegRNA specifying mouse DNMT1 +6 Gto C.
  • FIGS. 13C-13D show coverage plots of small RNA-seq fragments aligned to the pegRNA (left) or the epegRNA (right) specifying mouse DNMT1 +6 G to C edit. Data are from cells without the (e)pegRNA target collected one (FIG.
  • FIGS. 14A-14I show PE7 has no or negligible effects on cell viability, cell growth, and mRNA abundance compared to PEmax and PE7 mutant.
  • FIG. 14A shows percentages of intended prime editing and indels at the endogenous HEK3 and PRNP loci in K562 cells with PEmax, PE7 or PE7 mutant. Editing components delivered by plasmid transfection. Cells from this experiment were used for analyses in FIGS. 14B-14I.
  • FIG. 14B shows percentages of viable K562 cells quantified by flow cytometry one, two and three days after transfection of PEmax, PE7 or PE7 mutant editor plasmid and pegRNA plasmid specifying either HEK3 +1 T to A or PRNP +6 G to T.
  • FIG. 14A shows percentages of intended prime editing and indels at the endogenous HEK3 and PRNP loci in K562 cells with PEmax, PE7 or PE7 mutant. Editing components delivered by plasmid transfection. Cells from
  • FIGS. 14G-14I shows venn diagrams of differentially expressed genes (p ⁇ 0.05) in K562 cells edited at two different loci across three comparisons: PE7 relative to PEmax (FIG. 14g), PE7 relative to PE7 mutant (FIG. 14H), and PEmax relative to PE7 mutant (FIG. 141). Indel frequency for each sample included adjacent to corresponding intending editing efficiency in a.
  • FIGS. 15A-15G show PE7 improves prime editing with different approaches and delivery strategies without substantially increasing off-target effect.
  • FIG. 15A-15G show PE7 improves prime editing with different approaches and delivery strategies without substantially increasing off-target effect.
  • FIG. 15A shows percentages of editing outcomes produced by PEmax or PE7 with the PE2 approach at on- and off-target sites using pegRNAs targeting the EMX1 (left), FANCF (middle left), HEK3 (middle right), and HEK4 (right) loci in U2OS cells.
  • On-target editing data also presented in FIG. 15B and FIG. 4D.
  • FIG. 15B shows a summary plot of intended prime edit and indel frequencies observed at genomic loci with indicated editor and prime editing approaches.
  • Data for PE2 and PE4 from eight loci indicated in FIG. 4D.
  • Data for PE3 and PE5 from a subset of those targets (RNF2, HEK3, DNMT1 and VEGFA).
  • FIG. 15C shows percentages of intended prime editing and indels at endogenous HEK3 (top) and DNMT1 (bottom) loci after lentiviral transduction of pegRNAs or (e)pegRNAs (tevopreQi) and transfection of PEmax or PE7 editor encoded on mRNA or plasmid in HeLa (left) and U2OS (right) cells. (e)pegRNAs use a modified sgRNA scaffold.
  • FIG. 15D shows percentages of intended prime editing and indels at endogenous DNMT1 (left) and HEK3 (right) loci after lentiviral transduction of editing components in K562 cells. Two different editor expression constructs (as indicated) were tested.
  • FIG. 15E shows percentages of intended prime editing and indels at three genomic loci in U2OS cells using indicated editor mRNA and synthetic pegRNAs with no-polyU, blocked or La-accessible end configurations.
  • FIG. 15F shows fold changes in average intended prime editing in U2OS cells using PE7 mRNA relative to PEmax mRNA for synthetic pegRNAs with each indicated end configuration. Editing percentages presented in FIG. 15E.
  • FIG. 15G shows percentages of intended prime editing and indels at five genomic loci in primary human T cells using PEmax or PE7 mRNA and synthetic pegRNAs with La-accessible end configuration.
  • administering to a subject includes any route of introducing or delivering to a subject an agent. Administration can be carried out by any suitable route, including oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation, via an implanted reservoir, or via a transdermal patch, and the like. Administration includes self-administration and the administration by another.
  • beneficial agent and “active agent” are used interchangeably herein to refer to a chemical compound or composition that has a beneficial biological effect.
  • beneficial biological effects include both therapeutic effects, i.e., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, i.e., prevention of a disorder or other undesirable physiological condition.
  • the terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, prodrugs, active metabolites, isomers, fragments, analogs, and the like.
  • “Complementary” or “substantially complementary” refers to the hybridization or base pairing or the formation of a duplex between nucleotides or nucleic acids, such as, for instance, between the two strands of a double stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single stranded nucleic acid.
  • Complementary nucleotides are, generally, A and T/U, or C and G.
  • Cas9 recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus non-self.
  • Cas9 nuclease sequences and structures are well known to those of skill in the art (see, e.g., “Complete genome sequence of an Ml strain of Streptococcus pyogenes.” Ferretti et al., J. J., McShan W. M., Ajdic D. J., Savic D. J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A. N., Kenton S., Lai H. S., Lin S. P., Qian Y., Jia H.
  • an “effective amount” of an agent can also refer to an amount covering both therapeutically effective amounts and prophylactically effective amounts.
  • An “effective amount” of an agent necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • Encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom, Thus, a gene encodes a protein if transcription and translation of mRNA occurs.
  • a polynucleotide such as a gene, a cDNA, or an mRNA
  • an expression cassette refers to a nucleic acid construct, which when introduced into a host cell, results in transcription and/or translation of a RNA or polypeptide, respectively.
  • an expression cassette comprising a promoter operably linked to a second nucleic acid (e.g. polynucleotide) may include a promoter that is heterologous to the second nucleic acid (e.g.
  • an expression cassette comprising a terminator (or termination sequence) operably linked to a second nucleic acid may include a terminator that is heterologous to the second nucleic acid (e.g., polynucleotide) as the result of human manipulation.
  • the expression cassette comprises a promoter operably linked to a second nucleic acid (e.g., polynucleotide) and a terminator operably linked to the second nucleic acid (e.g., polynucleotide) as the result of human manipulation.
  • the expression cassette comprises an endogenous promoter.
  • the expression cassette comprises an endogenous terminator.
  • the expression cassette comprises a synthetic (or non-natural) promoter.
  • the expression cassette comprises a synthetic (or non-natural) terminator.
  • fragments can include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the fragment is not significantly altered or impaired compared to the nonmodified peptide or protein. These modifications can provide for some additional property, such as to remove or add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc.
  • gene refers to the coding sequence or control sequence, or fragments thereof.
  • a gene may include any combination of coding sequence and control sequence, or fragments thereof.
  • a “gene” as referred to herein may be all or part of a native gene.
  • a polynucleotide sequence as referred to herein may be used interchangeably with the term “gene”, or may include any coding sequence, non-coding sequence or control sequence, fragments thereof, and combinations thereof.
  • the term “gene” or “gene sequence” includes, for example, control sequences upstream of the coding sequence.
  • genetically engineered cell or “genetically modified cell” as used herein refers to a cell modified by means of genetic engineering.
  • engineered or “modified” thereof may refer to one or more changes of nucleic acids, such as nucleic acids within the genome of an organism.
  • engineered or “modified” may refer to a change, addition and/or deletion of a gene.
  • Engineered cells or modified cells can also refer to cells that contain added, deleted, and/or changed genes.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 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% or higher identity over a specified region when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see,
  • sequences are then said to be “substantially identical.”
  • This definition also refers to, or may be applied to, the compliment of a test sequence.
  • the definition also includes sequences that have deletions and/or additions, as well as those that have substitutions.
  • the preferred algorithms can account for gaps and the like.
  • identity exists over a region that is at least about 10 amino acids or 20 nucleotides in length, or more preferably over a region that is 10-50 amino acids or 20-50 nucleotides in length.
  • percent (%) amino acid sequence identity is defined as the percentage of amino acids in a candidate sequence that are identical to the amino acids in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.
  • Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.
  • sequence comparisons typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence algorithm program parameters Preferably, default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • HSPs high scoring sequence pairs
  • T is referred to as the neighborhood word score threshold (Altschul et al. (1990) J. Mol. Biol. 215:403-410). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score.
  • Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Set. USA 90:5873-5787).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01.
  • “increased” or “increase” as used herein generally means an increase by a statically significant amount; for the avoidance of any doubt, “increased” means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level so long as the increase is statistically significant.
  • the terms “may,” “optionally,” and “may optionally” are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur.
  • the statement that a formulation “may include an excipient” is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient.
  • Nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence or amino acid sequence.
  • DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence;
  • a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • “operably linked” means that the DNA sequences being linked are near each other, and, in the case of a secretory leader, contiguous and in reading phase.
  • operably linked nucleic acids do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
  • a promoter is operably linked with a coding sequence when it is capable of affecting (e.g., modulating relative to the absence of the promoter) the expression of a protein from that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter).
  • promoter refers to a region or sequence determinants located upstream or downstream from the start of transcription and which are involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. Promoters need not be of bacterial origin, for example, promoters derived from viruses or from other organisms can be used in the compositions, systems, or methods described herein.
  • regulatory element is intended to include promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences).
  • Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences).
  • tissue-specific promoter may direct expression primarily in a desired tissue of interest, such as muscle, neuron, bone, skin, blood, specific organs, or particular cell types.
  • Regulatory elements may also direct expression in a temporal-dependent manner, such as in a cell-cycle dependent or developmental stage-dependent manner, which may or may not also be tissue or cell-type specific.
  • a vector comprises one or more pol III promoter (e.g., 1, 2, 3, 4, 5, or more pol I promoters), one or more pol II promoters (e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof.
  • pol III promoters include, but are not limited to, U6 and Hl promoters.
  • pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al, Cell, 41 :521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the P-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFla promoter.
  • RSV Rous sarcoma virus
  • CMV cytomegalovirus
  • PGK phosphoglycerol kinase
  • enhancer elements such as WPRE; CMV enhancers; the R-U5' segment in LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit P-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981).
  • WPRE WPRE
  • CMV enhancers the R-U5' segment in LTR of HTLV-I
  • SV40 enhancer SV40 enhancer
  • the intron sequence between exons 2 and 3 of rabbit P-globin Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981.
  • recombinant refers to a human manipulated nucleic acid (e.g., polynucleotide) or a copy or complement of a human manipulated nucleic acid (e.g., polynucleotide), or if in reference to a protein (i.e, a “recombinant protein”), a protein encoded by a recombinant nucleic acid (e.g., polynucleotide).
  • a recombinant expression cassette comprising a promoter operably linked to a second nucleic acid (e.g., polynucleotide) may include a promoter that is heterologous to the second nucleic acid (e.g.
  • a recombinant expression cassette may comprise nucleic acids (e.g., polynucleotides) combined in such a way that the nucleic acids (e.g., polynucleotides) are extremely unlikely to be found in nature.
  • “Pharmaceutically acceptable” component can refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a pharmaceutical formulation of the invention and administered to a subject as described herein without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained.
  • the term When used in reference to administration to a human, the term generally implies the component has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.
  • nucleic acid as used herein means a polymer composed of nucleotides, e.g., deoxyribonucleotides or ribonucleotides.
  • nucleobase refers to the part of a nucleotide that bears the Watson/Crick base-pairing functionality.
  • the most common naturally-occurring nucleobases, adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T) bear the hydrogen-bonding functionality that binds one nucleic acid strand to another in a sequence specific manner.
  • ribonucleic acid and “RNA” as used herein mean a polymer composed of ribonucleotides.
  • deoxyribonucleic acid and “DNA” as used herein mean a polymer composed of deoxyribonucleotides.
  • polynucleotide refers to a single or double stranded polymer composed of nucleotide monomers.
  • the term “prime editing system” involves a Cas9 nickase (for example, a Cas9 H840A nickase or a Cas9 R221K N394K H840A nickase) and a reverse transcriptase, in combination with a guide RNA (herein referred as “prime editing guide RNA” or “pegRNA”).
  • the pegRNA is a sgRNA with a primer binding site (PBS) and a DNA synthesis template appended to the 3’ end containing the desired nucleic acid sequence.
  • PBS primer binding site
  • the primer binding site allows the 3’ end of a nicked DNA strand to hybridize to the pegRNA, while the RT template serves as a template for the synthesis of edited genetic information.
  • the pegRNA encodes the new sequence and allows DNA synthesis to introduce the desired mutations.
  • the prime editing systems and pegRNAs are those described in U.S. Patent Nol, 1447,770, which is incorporated herein by reference in its entirety.
  • the pegRNA comprises an engineered pegRNA (epegRNA).
  • a pegRNA can be longer than standard sgRNAs commonly used for CRISPR gene editing.
  • the pegRNA disclosed herein can be at least 60 nt, 70 nt, 80 nt, 90 nt, 100 nt, 120 nt, 140 nt, 160 nt, 180 nt, 200 nt, 250 nt, 300 nt, 350 nt, 400 nt, or 500 nt in length.
  • guide RNA refers to the polynucleotide sequence comprising the guide sequence, the tracrRNAand the crRNA.
  • guide sequence refers to the about 20 bp sequence within the guide RNA that specifies the target site and may be used interchangeably with the term “guide” or “spacer”.
  • a “crRNA” is a bacterial RNA that confers target specificity and requires tracrRNA to bind to Cas9.
  • a “tracrRNA” is a bacterial RNA that links the crRNA to the Cas9 nuclease and typically can bind any crRNA.
  • the sequence specificity of a Cas DNA-binding protein is determined by gRNAs, which have nucleotide base-pairing complementarity to target DNA sequences.
  • the pegRNA comprises a 3 ’-polyuridine domain (which comprises, for example, at least 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, 60, 70, 80, 90, or 100 uridines).
  • the 3 ’-polyuridine domain can locate at the 3’- end of the pegRNA or near the 3’ - end of the pegRNA.
  • the 3 ’-polyuridine domain comprises at least one chemically modified uridine (for example, at least one, two, three, four, or five chemically modified uridines).
  • the 3’- polyuridine domain comprises at least one unmodified uridine (for example, at least one, two, three, four, or five unmodified uridines).
  • the 3 ’-polyuridine domain comprises at least one chemically modified uridine (for example, at least one, two, three, four, or five chemically modified uridines) and at least one unmodified uridine (for example, at least one, two, three, four, or five unmodified uridines).
  • the at least one unmodified uridine locates at the 3’ end of the pegRNA.
  • the pegRNA disclosed herein can have two unmodified uridines locating at its 3’ end, downstream of at least one, two, three, four, or five chemically modified uridines.
  • the 3 ’-polyuridine domain comprises the sequence UU*mU*mU*mUU.
  • the 3’- polyuridine domain comprises a 3’ sequence fragment selected from the sequences in Table 3.
  • the 3 ’-polyuridine domain comprises a 3’ sequence fragment selected from SEQ ID NOs: 63-108.
  • the chemical modification is 2’ -O-m ethylation and/or replacement of a phosphodiester bond to a phosphorothioate bond.
  • the 3 ’-polyuridine domain comprises at least one (for example, at least two, three, four, or five) uridine with unmodified 2'-hydroxyl (OH) group.
  • the at least one uridine with unmodified 2'- OH group locates at the 3’ end of the pegRNA.
  • the SSB protein comprises a La motif and/or an RNA recognition motif (RRM) (e.g., amino acid residues 1-194 or 2-194 of SEQ ID NO: 34).
  • RRM RNA recognition motif
  • the SSB protein comprises a sequence at least 80% (e.g., at least 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 33 or a fragment thereof.
  • the SSB protein comprises a sequence at least 80% (e.g., at least 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 34 or a fragment thereof.
  • the SSB protein comprises a sequence at least 80% (e.g., at least 80%, 85%, 90%, 95%, 98%, or 99%) identical SEQ ID NO: 35 or a fragment thereof. In some embodiments, the SSB protein comprises a sequence at least 80% (e.g., at least 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 36 or a fragment thereof. In some embodiments, the Cas9 nickase of the system disclosed herein comprises a sequence at least 80% identical to SEQ ID NO: 26, or a fragment thereof. In some embodiments, the Cas9 nickase of the system disclosed herein comprises a sequence at least 80% identical to SEQ ID NO: 27, or a fragment thereof.
  • the reverse transcriptase of the system disclosed herein comprises a sequence at least 80% (e.g., at least 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 31 or a fragment thereof. In some embodiments, the reverse transcriptase of the system disclosed herein comprises a sequence at least 80% (e.g., at least 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 32 or a fragment thereof.
  • the Cas9 nickase is operatively linked to the reverse transcriptase (e.g., directly or through a linker).
  • the SSB protein is operatively linked to the Cas9 nickase and the reverse transcriptase (e.g., directly or through one or more linkers).
  • the system disclosed herein comprises a recombinant polypeptide that comprises the reverse transcriptase, the Cas9 nickase, and the SSB protein disclosed herein.
  • the recombinant polypeptide can further comprise one or more linkers and/or one or more nuclear localization sequences (NLS) (e.g., aNLS of SV40 or c-Myc).
  • the linker comprises a sequence at least 80% (e.g., at least 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 28, 29, 30, or 40, or a fragment thereof.
  • the NLS comprises a sequence at least 80% (e.g., at least 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 25, 37, 38, 39, or 40 or a fragment thereof.
  • the recombinant polypeptide comprising the reverse transcriptase, the Cas9 nickase, and the SSB protein comprises a sequence at least 80% (e.g., at least 80%, 85%, 90%, 95%, 98%, or 99%) identical to any of SEQ ID NOs: 1-12 or a fragment thereof. In some embodiments, the recombinant polypeptide comprising the reverse transcriptase, the Cas9 nickase, and the SSB protein comprises a sequence at least 80% (e.g., at least 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 5 or a fragment thereof.
  • the recombinant polypeptide comprising the reverse transcriptase, the Cas9 nickase, and the SSB protein comprises a sequence at least 80% (e.g., at least 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 1 or a fragment thereof. In some embodiments, the recombinant polypeptide comprising the reverse transcriptase, the Cas9 nickase, and the SSB protein comprises a sequence at least 80% (e.g., at least 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 2 or a fragment thereof.
  • the recombinant polypeptide comprising the reverse transcriptase, the Cas9 nickase, and the SSB protein comprises a sequence at least 80% (e.g., at least 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 3 or a fragment thereof. In some embodiments, the recombinant polypeptide comprising the reverse transcriptase, the Cas9 nickase, and the SSB protein comprises a sequence at least 80% (e.g., at least 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 4 or a fragment thereof.
  • the recombinant polypeptide comprising the reverse transcriptase, the Cas9 nickase, and the SSB protein comprises a sequence at least 80% (e.g., at least 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 5 or a fragment thereof. In some embodiments, the recombinant polypeptide comprising the reverse transcriptase, the Cas9 nickase, and the SSB protein comprises a sequence at least 80% (e.g., at least 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 6 or a fragment thereof.
  • the recombinant polypeptide comprising the reverse transcriptase, the Cas9 nickase, and the SSB protein comprises a sequence at least 80% (e.g., at least 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 7 or a fragment thereof. In some embodiments, the recombinant polypeptide comprising the reverse transcriptase, the Cas9 nickase, and the SSB protein comprises a sequence at least 80% (e.g., at least 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 8 or a fragment thereof.
  • the recombinant polypeptide comprising the reverse transcriptase, the Cas9 nickase, and the SSB protein comprises a sequence at least 80% (e.g., at least 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 9 or a fragment thereof. In some embodiments, the recombinant polypeptide comprising the reverse transcriptase, the Cas9 nickase, and the SSB protein comprises a sequence at least 80% (e.g., at least 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 10 or a fragment thereof.
  • the recombinant polypeptide comprising the reverse transcriptase, the Cas9 nickase, and the SSB protein comprises a sequence at least 80% (e.g., at least 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 11 or a fragment thereof. In some embodiments, the recombinant polypeptide comprising the reverse transcriptase, the Cas9 nickase, and the SSB protein comprises a sequence at least 80% (e.g., at least 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 12 or a fragment thereof.
  • a system comprising a Cas9 nickase; a reverse transcriptase; and a small RNA binding exonuclease protection factor La (SSB) protein.
  • the Cas9 nickase, reverse transcriptase, and the SSB protein can be on a same or different polypeptide.
  • the Cas9 nickase, reverse transcriptase, and the SSB protein can be on a same or different pharmaceutically acceptable carriers.
  • a system comprising a first polynucleotide encoding a Cas9 nickase; a second polynucleotide encoding a reverse transcriptase; and a third polynucleotide encoding a small RNA binding exonuclease protection factor La (SSB) protein; and a prime editing guide RNA (pegRNA).
  • SSB small RNA binding exonuclease protection factor La
  • pegRNA prime editing guide RNA
  • the third polynucleotide encodes a La motif and/or an RNA recognition motif (RRM) of the SSB protein.
  • the third polynucleotide comprises a sequence at least 80% (e.g., at least 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 50 or a fragment thereof.
  • the third polynucleotide comprises a sequence at least 80% (e.g., at least 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 49 or a fragment thereof.
  • the first, second, and third polynucleotides are operatively linked thereby forming one recombinant polynucleotide.
  • the system disclosed herein comprises a recombinant polynucleotide encoding a recombinant polypeptide that comprises the reverse transcriptase, the Cas9 nickase, and the SSB protein disclosed herein.
  • the recombinant polypeptide can further comprise one or more linkers and/or one or more nuclear localization sequences (NLS) (e.g., a NLS of SV40 or c-Myc).
  • NLS nuclear localization sequences
  • the polynucleotide encoding the linker comprises a sequence at least 80% (e.g., at least 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 44, 45, 46, or 56, or a fragment thereof.
  • the polynucleotide encoding the NLS comprises a sequence at least 80% (e.g., at least 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 41, 53, 54, 55, 56 or a fragment thereof.
  • the recombinant nucleotide comprises a sequence at least 80% (e.g., at least 80%, 85%, 90%, 95%, 98%, or 99%) identical to any of SEQ ID NOs: 13-24 or a fragment thereof. In some embodiments, the recombinant nucleotide comprises a sequence at least 80% (e.g., at least 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 17 or a fragment thereof.
  • compositions disclosed herein may be in solution, suspension (for example, incorporated into microparticles (such as exosomes) or liposomes). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands.
  • the compositions disclosed herein may be in exosomes.
  • exosome refers to a cell-derived membranous vesicle. They refer to extracellular vesicles, which are generally of between 30 and 200 nm in size, for example in the range of 50-100 nm in size.
  • the exosomes can be engineered to express one or more ligands or molecules for cell-targeting delivery.
  • compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid
  • organic acids such as formic acid, acetic acid, propionic acid, glyco
  • compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.
  • dosing frequency for the composition disclosed herein includes, but is not limited to, no more than once every 30 years, every 25 years, every 20 years, every 15 years, every 10 years, every 5 years, every 4 years, every 3 years, every 2 years, every 12 months, or every 6 months.
  • the interval between each administration is less than about 4 months, less than about 3 months, less than about 2 months, less than about a month, less than about 3 weeks, less than about 2 weeks, or less than less than about a week, such as less than about any of 6, 5, 4, 3, 2, or 1 day.
  • the dosing frequency for the composition includes, but is not limited to, at least once a day, twice a day, or three times a day.
  • the interval between each administration is less than about 48 hours, 36 hours, 24 hours, 22 hours, 20 hours, 18 hours, 16 hours, 14 hours, 12 hours, 10 hours, 9 hours, 8 hours, or 7 hours.
  • the interval between each administration is less than about 24 hours, 22 hours, 20 hours, 18 hours, 16 hours, 14 hours, 12 hours, 10 hours, 9 hours, 8 hours, 7 hours, or 6 hours. In some embodiments, the interval between each administration is constant. For example, the administration can be carried out daily, every two days, every three days, every four days, every five days, or weekly. Administration can also be continuous and adjusted to maintaining a level of the compound within any desired and specified range. It should be understood and herein contemplated that the compositions disclosed herein can be used in combination with a pain reliever and, in some examples, reduce the dosing frequency of the pain reliever.
  • the therapeutically effective amount typically will vary from about 0.001 mg/kg to about 1000 mg/kg, from about 0.01 mg/kg to about 750 mg/kg, from about 100 mg/kg to about 500 mg/kg, from about 1 mg/kg to about 250 mg/kg, from about 10 mg/kg to about 150 mg/kg in one or more dose administrations daily, for one or several days (depending of course of the mode of administration and the factors discussed above).
  • Other suitable dose ranges include 1 mg to 10,000 mg per day, 100 mg to 10,000 mg per day, 500 mg to 10,000 mg per day, and 500 mg to 1,000 mg per day.
  • the amount is less than 10,000 mg per day with a range of 750 mg to 9,000 mg per day.
  • the doses of the compositions disclosed herein for gene editing in a cell or a subject is less (e.g., about 2-fold less, about 3-fold less, about 4-fold less, about 5-fold less, about 6-fold less, about 7-fold less, about 8-fold less, about 9-fold less, about 10- fold less, about 15-fold less, about 20-fold less, about 30-fold less, about 40-fold less, about 50-fold less, about 100-fold less, or about 1000-fold less) than the doses commonly known in the art for gene editing.
  • Parenteral administration of the composition is generally characterized by injection.
  • Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions.
  • a more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein.
  • Example 1 Genome-scale CRISPRi screens identify La (SSB) as a strong mediator of prime editing
  • Prime editing allows precise modification of genomes.
  • scalable prime editing reporters were developed and performed genome-scale CRISPR-interference screens. From these screens, a single factor emerged as the strongest mediator of prime editing: the small RNA-binding exonuclease protection factor La (SSB).
  • La small RNA-binding exonuclease protection factor
  • La binds polyuridine tracts at the 3' ends of RNA polymerase III transcripts and protects those transcripts from cellular exonucleases. Accordingly, functional interaction were observed between La and the 3' ends of polyuridylated prime editing guide RNAs. Guided by these insights, a strategy was developed to improve prime editing, namely fusing the RNA-binding, N-terminal domain of La to the prime editor protein PEmax. Application of the editor dramatically increased prime editing efficiencies. The results provide key insights into how prime editing components interact with the cellular environment and suggest general strategies for stabilizing exogenous small RNAs therein.
  • Prime editing minimally consists of an engineered Cas9 protein (Cas9 H840A nickase fused to a reverse transcriptase) and a prime editing guide RNA (pegRNA) that specifies both the DNA target and the intended edit (FIG. 1 A).
  • the editor protein binds its cognate guide RNA and, directed by the spacer sequence, finds a complementary DNA target. Once bound to the target, the editing complex nicks the displaced DNA strand and releases a 3' DNA end. This end can then hybridize to the 3' extension of the pegRNA and prime reverse transcription of the pegRNA-encoded edit, which is ultimately incorporated into the genome or removed by DNA mismatch repair (MMR).
  • MMR DNA mismatch repair
  • Prime editing Several features that impact prime editing have already been reported, including the expression, stability, localization and activity of editing components, as well as chromatin context of target loci. Additionally, demonstrating that mechanistic understanding can reveal avenues for technological improvement, Previous results showed that small prime edits can be installed with higher efficiency and precision when MMR is suppressed or evaded. Studies of prime editing to date, however, have been limited in focus, with inquiry restricted to optimization of editing components or examination of inferred cellular determinants (e.g., DNA repair). By interrogating prime editing with unbiased, genome-scale CRISPR-interference (CRISPRi) screens, an unanticipated mediator of prime editing was identified: the small RNA-binding protein La (SSB). Subsequent characterization of this factor, then showed how exploiting an interaction between La and pegRNAs can dramatically enhance prime editing.
  • CRISPRi genome-scale CRISPR-interference
  • Prime editing reporter system in which installation of an intended edit ‘switches on’ a reporter gene was developed (FIG. IB). By design, this system expresses a single bicistronic mRNA but, due to lack of a properly positioned start codon, produces only a constitutive marker protein (driven by an internal ribosome entry site) until an upstream, inframe ATG is edited into a defined target site to induce expression of a different reporter gene.
  • the system was designed for use with an orthogonal Staphylococcus aureus Cas9 (SaCas9)-based prime editor (SaPE2).
  • SaPE2 protospacers in the target site were included: one for ATG installation and another at which a +50 complementary strand nick can be introduced.
  • Such nicks have been shown to enhance prime editing, and their inclusion, by use of additional single guide RNAs (sgRNAs), constitutes the PE3 approach.
  • sgRNAs single guide RNAs
  • Two versions of the reporter system were built: one that uses the fluorescent protein EGFP to report on editing and another that uses a synthetic cell surface protein (IgK-hlgGl-Fc- PDGFRP) (FIGS. 6A-6B).
  • the gene products encoded by each of these reporters were chosen to allow efficient isolation of successfully edited, marker-positive cells: GFP through fluorescence-activated cell sorting (FACS reporter) and the surface protein via magnetic cell separation with protein G beads (MCS reporter).
  • FACS reporter fluorescence-activated cell sorting
  • MCS reporter magnetic cell separation with protein G beads
  • Each of these reporters were transduced into K562 cells constitutively expressing CRISPRi machinery (K562 CRISPRi cells) and, to validate their performance, edited the resulting cells with substitution or insertion edits designed to install one or more start codons (FIG. 6C).
  • the FACS reporter After editing, the FACS reporter produced a clear population of GFP+ cells (FIG. 1C). Two observations also demonstrated that that the percentage of those marker-positive cells faithfully reports intended prime editing efficiency: (1) perturbation of MSH2, an MMR gene known to suppress small substitution edits, increased GFP+ percentage (FIG. 1C) and (2) PE3-based editing, which is more efficient than PE2, showed higher GFP+ percentage (FIG. 6D). Additionally, confirming reporter accuracy, quantification of editing outcomes from GFP+ and GFP- populations of FACS reporter cells separated by flow cytometry, and from MCS reporter cells that either bound protein G beads or did not, revealed enrichment of intended edits in GFP+ and bead-bound cells, respectively (FIGS. 6E-6F).
  • FACS reporter cells were transduced with the hCRISPRi-v2 library (18,905 targeted genes, 5 sgRNAs per gene), introduced prime editing components by plasmid transfection (SaPE2, +7 GG to CA pegRNA, +50 nicking sgRNA), and separated resulting GFP+/- populations.
  • Flow cytometry analysis prior to sorting confirmed reasonable editing efficiencies (FIG. 6G) and sequencing of the target site showed expected enrichment in sorted populations (FIGS. 6H-6I).
  • Example 2 La promotes prime editing across pegRNA and editor designs, programmed edits, endogenous targets, and cell types
  • La a ubiquitously expressed eukaryotic protein, is involved in diverse aspects of RNA metabolism, but one of its most characterized roles is binding polyuridine (polyU) tracts at the 3' ends of nascent RNA polymerase III (Pol III) transcripts and protecting them from exonucleases.
  • polyU polyuridine
  • Pol III nascent RNA polymerase III
  • the La phenotypes observed therefrom may represent an interaction between La and the pegRNA used for screening.
  • pegRNAs and epegRNAs were tested.
  • the latter contain structured motifs at their 3' ends and have been shown to enhance prime editing, with improvements loosely attributed to pegRNA stabilization.
  • This difference fits a model wherein La promotes editing by interacting with the 3' ends of (e) pegRNAs but has a stronger effect on pegRNAs, which may be less stable or more accessible to La due to less structured 3’ ends.
  • Example 3 The effect of La on prime editing does not extend to other editing modalities
  • Prime editing relies on pegRNA 3' extensions, which encode intended edits, but other editing modalities such as nuclease-mediated gene disruption and base editing do not. This difference was a prompt to examine the effects of La on other genome editing approaches.
  • SaCas9 was used to induce DNA double-strand breaks (DSBs) in the MCS prime editing reporter using the +7 GG to CA pegRNA, which targets SaCas9 to a locus near a transduced GFP marker gene but not directly within sequences required for expression (FIG. 2F). Because Cas9-induced DSBs often generate large deletions, such breaks can disrupt nearby reporter genes, even when those genes are distant from the target site and especially when targeting transduced lentiviral constructs.
  • Example 4 La promotes prime editing by interacting with the 3' ends of polyuridylated pegRNAs
  • La has been tenuously implicated in Pol Ill-mediated transcription, with phosphorylation of a single residue (S336) potentially involved in transcriptional modulation via Pol III recycling.
  • S336 phosphorylation of a single residue
  • La mutants were examined.
  • La is a 408-residue protein consisting of a highly conserved La motif, two RNA recognition motifs (RRMs), and a flexible region with a nuclear localization signal (NLS) at the C-terminus (FIG. 3 A).
  • the N-terminal domain of La (La 1-194 ), which contains the La motif and RRM1, is necessary and sufficient for high-affinity binding to 3' polyU, while regulation of Pol III recycling has been attributed to the phosphorylation status of Ser366 (S366). It was reasoned that if La promotes prime editing through transcription, truncation of the C-terminal domain or mutation of S366 could abolish or alter its impact, but if La promotes prime editing by binding to the 3' ends of pegRNAs, La 1-194 alone should be sufficient for that activity.
  • Example 7 PE7 enhances prime editing of therapeutic-relevant targets and cell types
  • 2.1E8 MCS reporter cells were transduced with hCRISPRi-v2 viruses at a 0.16 MOI (15% infection) for the screen conditions and were selected by 3 pg mL' 1 puromycin 48 hours after transduction. 7 days after transduction, 1E8 fully selected cells were nucleofected for each replicate of each edit with SE Cell Line 4D- Nucleofector X Kit L (Lonza V4XC-1024) and pulse code FF120, according to the manufacturer’s protocol. Each nucleofection consists of 1E7 cells and varying amounts of plasmids encoding prime editing components.
  • PE3 7500 ng pCMV-SaPE2, 2500 ng +7 GG to CA pegRNA plasmid, 833 ng +50 nicking sgRNA plasmid (PE3) were used per nucleofection.
  • PE4 and PE5 6000 ng pCMV-SaPE2, 3000 ng pEFla- hMLHldn (Addgene #174823), 2000 ng +7 GG to CA pegRNA plasmid and 667 ng +50 nicking sgRNA plasmid (PE5) were used. 4 days post nucleofection, cells from each replicate and condition were magnetically separated into bound and unbound fractions as previously described.
  • the lysis buffer consisted of 10 mM Tris pH 8.0 (Gibco AM9855G), 0.05% SDS (Invitrogen 15553027), 25 pg mL 1 proteinase K (Invitrogen AM2546) and Nuclease-Free Water (AM9939).
  • the genomic DNA extract was incubated at 37 °C for 90 minutes and transferred into PCR strips (USA Scientific 1402-4700) for 80°C inactivation of proteinase K for 30 minutes in Bio-Rad T100 Thermal Cycler.
  • 1E6 cells were nucleofected with specified amounts of plasmids or synthetic guide RNAs using SE Cell Line 4D-Nucleofector X Kit S (Lonza V4XC-1032) and program FF-120, according to the manufacturer’s protocol.
  • 900 ng pCMV-SaPE2 300 ng pegRNA plasmid, 100 ng nicking sgRNA plasmid (PE3/5) and 450 ng pEFla-hMLHldn (PE4/5) were nucleofected.
  • 500 ng pegRNA plasmid was nucleofected.
  • 500 ng pegRNA plasmid and 1000 ng plasmid encoding La, La mutants or mRFP control were nucleofected.
  • 800 ng pX600 (Addgene #61592) and 400 ng +7 GG to CA pegRNA plasmid were nucleofected.
  • Synthetic pegRNAs and nicking sgRNAs with specified sequences and modifications were ordered as Custom Alt-R gRNA from Integrated DNA Technologies (Table 3). According to an incremental titration of a DNMT1 +5 G to T synthetic pegRNA with standard chemical modifications in K562 PEmax parental cells, intended editing efficiencies were already saturated at 100 pmole input (FIG. 10B). Therefore, 100 pmole synthetic pegRNA and 50 pmole nicking sgRNA (PE3) were used for nucleofection unless otherwise specified.
  • 1E6-2E6 cells were harvested in 1.5 mL tubes (Eppendorf 0030123611), washed with 1 mL DPBS (Gibco 14190144) and resuspended in 100 pL freshly prepared lysis buffer described above.
  • the genomic DNA extract was incubated at 37 °C for 120 minutes and transferred into PCR strips (USA Scientific 1402-4700) for 80°C inactivation of proteinase K for 40 minutes in Bio-Rad T100 Thermal Cycler.
  • 1E6 K562 and 1E5 U2OS cells were nucleofected with 1 pg editor mRNA and 50 pmole synthetic pegRNA using SE Cell Line 4D-Nucleofector X Kit S (Lonza V4XC-1032) with program FF-120 and DN-100, respectively, according to the manufacturer’s protocols. After nucleofection, cells were cultured for 72 hours and harvested for genomic DNA extract.
  • cells were transduced with lentiviruses expressing (e)pegRNAs (20-40% infection) and were fully selected by 3 pg mL' 1 puromycin.
  • Stably transduced HeLa and U2OS cells were nucleofected with 750 ng editor plasmid or 1 pg editor mRNA using SE Cell Line 4D-Nucleofector X Kit S (Lonza V4XC-1032) with program CN- 114 and DN-100, respectively, according to the manufacturer’s protocols. After nucleofection, cells were cultured for 72 hours and harvested for genomic DNA extract.
  • K562 cells were transduced with lentiviruses expressing PEmax or PE7 (with IRES2-driven EGFP or EGFP-T2A-NeoR as selectable marker).
  • the transduced populations (EGFP+, 20- 30%) were isolated by BD FACSAria Fusion Flow Cytometer 9 days post transduction, further transduced with lentiviruses expressing (e)pegRNAs (approximately 50% infection), fully selected by 3 pg mL' 1 puromycin and harvested 11 days after second transduction for genomic DNA extract.
  • Genomic DNA sequences containing target sites were amplified through two rounds of PCR reactions (PCR1 and 2). In PCR1, genomic regions of interest were amplified with primers containing forward and reverse adapters for Illumina sequencing (Integrated DNA Technologies).
  • Each PCR1 reaction consisted of 1 pL genomic DNA extract, 0.1 pL of each 100 pM forward and reverse primer (0.5 pM final concentration), 10 pL Phusion U Green Multiplex PCR Master Mix (Thermo Scientific F564L) and 8.8 pL Nuclease-Free Water (AM9939) and was performed with the following cycling conditions: 98 °C for 2 min, 28 cycles of [98 °C for 10 s, 61 °C for 20 s, and 72 °C for 30 s], followed by 72 °C for 2 min.
  • PCR1 amplification was confirmed by 1% agarose (Goldbio A-201-100) gel electrophoresis before proceeding to PCR2 to uniquely index each sample with both forward and reverse Illumina barcoding primers.
  • Each 14 pL PCR2 reaction consisted of 1 pL unpurified PCR 1 reaction, 0.5 pM of each forward and reverse Illumina barcoding primer, 7 pL Phusion U Green Multiplex PCR Master Mix (Thermo Scientific F564L) and Nuclease-Free Water (AM9939) and was performed with the following cycling conditions: 98 °C for 2 min, 9 cycles of [98 °C for 10 s, 61 °C for 20 s, and 72 °C for 30 s], followed by 72 °C for 2 min.
  • the gel purified PCR2 products were quantified by Qubit IX dsDNA High Sensitivity kit (Invitrogen Q33231) and a high sensitivity DNA chip (Agilent Technologies 5067-4626) on an Agilent 2100 Bioanalyzer and sequenced with the MiSeq Reagent Micro Kit v2 300 cycles (Illumina MS- 103 -1002) or Nano Kit v2 300 cycles (Illumina MS-103-1001) with 300 cycles for R1 read, 8 cycles i7 index read and 8 cycles i5 index read.
  • Sequencing reads were demultiplexed through HTSEQ (Princeton University High Throughput Sequencing Database) or bcl2fastq2 (Illumina) and sequencing adapters were trimmed using Cutadapt with the parameter “-a AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC” (SEQ ID NO: 61).
  • amplicon sequencing reads were aligned to corresponding reference sequences with CRISPResso2 in HDR batch mode using the intended editing outcome as the expected allele (-e) with the parameters “-q 30” and “- discard indel reads TRUE”.
  • the CRISPResso2 quantification window was centered at the pegRNA nick (“-wc -3”) and the window size (“-w”) was set to 10 + the distance between nicks generated by the pegRNA and the nicking sgRNA.
  • the same parameters were used for PE2, PE3, PE4 and PE5 conditions.
  • the frequency of intended editing without indels was calculated as: (number of non-discarded HDR-aligned reads)/(number of reads that aligned all amplicons).
  • the frequency of intended editing with indels was calculated as: (number of discarded HDR-aligned reads)/(number of reads that aligned all amplicons).
  • the frequency of total intended editing (with or without indels) was calculated as (number of HDR-aligned reads)/(number of reads that aligned all amplicons).
  • the frequency of total indels was calculated as: (number of discarded reads)/( number of reads that aligned all amplicons).
  • the frequency of indels without intended editing was calculated as (number of discarded reference- aligned reads)/(number of reads that aligned all amplicons).
  • the intended prime editing efficiencies referred to frequencies of intended editing without indels and the indel efficiencies referred to frequencies of total indels in this study unless otherwise specified.
  • Each off-target amplicon sequence was compared to the 3' DNA flap sequence encoded by the pegRNA extension starting from the nucleotide 3 ' of Cas9 nick to the downstream until reaching the first nucleotide on the off-target amplicon that is different from the 3' DNA flap. Any reads with this nucleotide converted to that on the 3 ' DNA flap were considered off-target reads and the number of such reads can be found in the output file “Nucleotide frequency summary around sgRNA”. Off-target editing efficiencies were calculated as (number of off-target reads + number of indel-containing reads)/(number of reads that aligned all amplicons).
  • CRISPResso2 was run in standard batch mode with the parameters “-q 30” and “-discard indel reads TRUE”.
  • the intended editing efficiency referred to the frequency of indels which was calculated as (number of discarded reference- aligned reads)/ (number of reads that aligned all amplicons). Base editing outcomes were quantified by CRISPResso2 as previously described.
  • Each 20 pL qPCR consists of 2 pL cDNA, 0.3 pM of each forward and reverse primer, 10 pL S YBR Green PCR Master Mix (Applied Biosystems 4309155) and Nuclease-Free Water (AM9939) and were performed in technical triplicate on a ViiA 7 Real-Time PCR System (Applied Biosystems) with the following cycling conditions: 50 °C for 2 min, 95 °C for 10 min, and 40 cycles of [95 °C for 15 s, 60 °C for 1 min].
  • Relative La expression levels were calculated using the 2' AACT method with ACTB (a housekeeping gene) as the internal control in comparison to a non-targeting sgRNA or a non-targeting control siRNA pool.
  • La knock-out K562 PEmax cells 122 pmole Alt-R S .p. Cas9 Nuclease V3 (Integrated DNA Technologies 1081058) and 200 pmole Alt-R CRISPR-Cas9 sgRNA targeting La (Integrated DNA Technologies Hs.Cas9.SSB. l.AA) were complexed for 20 minutes at room temperature and were nucleofected into 5E5 K562 PEmax parental cells using the SE Cell Line 4D-Nucleofector X Kit (Lonza V4XC-1032) and program FF-120, according to the manufacturer’s protocol.
  • cells were sorted by BD FACSAria Fusion Flow Cytometer into 96-well plates at 1 cell per well with 150 pL conditioned culture medium. Single cells were grown and expanded for 2-3 weeks into clonal lines. Clones with high EGFP+ cell% according to AttueNXT flow cytometry analysis were selected for further characterization by targeted sequencing at genomic La locus and CRISPResso2 analysis.
  • Antibodies were diluted in 5% Blotto (5% nonfat dry milk in TBST) and incubated with the membrane for 1 hour at room temperature.
  • the following primary antibodies were used: anti-La mouse monoclonal antibody (1 :5000; Abeam ab75927); anti-GAPDH rabbit monoclonal antibody (1 :5000; Abeam abl81602); Guide-it Cas9 rabbit Polyclonal Antibody (1 : 1000; Takara 632607).
  • the following secondary antibodies were used: HRP-conjugated sheep anti-mouse polyclonal antibody (1 :2000; VWR 95017-332) and HRP-conjugated donkey anti-rabbit polyclonal antibody (1 :2000; VWR 95017-556).
  • the membrane was washed with TBST and immersed into Lumi-LightPLUS Western Blotting Substrate (Sigma 12015196001) for 3 minutes in dark prior to exposure with Azure Biosystems 600.
  • the Restore Western Blot Stripping Buffer (Thermo Scientific 21059) was applied to strip the membrane before reprobing.
  • Small RNA sequencing Small RNA sequencing.
  • the small RNA sequencing (small RNA-seq) with targeting (e)pegRNAs was performed in triplicate and for each replicate, 5E6 K562 PEmax parental or La-ko4 cells were nucleofected with 2500 ng either one of the two (e)pegRNA plasmid sets (Set 1 and 2) using the SE Cell Line 4D-Nucleofector X Kit L (Lonza V4XC-1024) and pulse code FF120, according to the manufacturer’s protocol.
  • Set 1 consists of plasmids encoding FANCF +5 G to T pegRNA, HEK3 +1 T to A pegRNA, DNMT1 +5 G to T pegRNA, RUNX1 +5 G to T epegRNA (evopreQi), VEGFA +5 G to T pegRNA and EMX1 +5 G to T epegRNA (mpknot).
  • Set 2 consists of plasmids encoding RNF2 +1 C to A pegRNA, HEK3 +1 T to A epegRNA (mpknot), DNMT1 +5 G to T epegRNA (evopreQi), RUNX1 +5 G to T pegRNA, VEGFA +5 G to T pegRNA and EMX1 +5 G to T pegRNA.
  • the VEGFA +5 G to T pegRNA plasmid was shared by both sets and served as the internal control for potential cross-set normalization.
  • the FANCF +5 G to T pegRNA plasmid and the RNF2 +1 C to A pegRNA were specific to set 1 and 2 respectively.
  • each set has the pegRNA plasmid while the other has the epegRNA plasmid encoding the same prime edit.
  • Each set only had one evopreQi epegRNA plasmid and one mpknot epegRNA plasmid.
  • the sets were formulated so that each (e)pegRNA transcript from cells nucleofected with one set can be aligned uniquely to the corresponding (e)pegRNA in that set, based on the observation in preliminary experiments that few fragments were solely mapped to the sgRNA scaffold shared by different (e)pegRNAs.
  • RNA-seq with non-targeting mDNMTl +6 G to C pegRNA and epegRNA were performed in quadruplicate and for each replicate, 5E6 K562 PEmax parental or La-ko4 cells were nucleofected with 5000 ng (e)pegRNA plasmid using the SE Cell Line 4D-Nucleofector X Kit L (Lonza V4XC-1024) and pulse code FF120, according to the manufacturer’s protocol.
  • a small RNA library was constructed with 1 pg total RNA as the input using NEBNext Multiplex Small RNA Library Prep Set for Illumina (Set 1) (New England Biolabs E7300S) and NEBNext Multiplex Oligos for Illumina Index Primers Set 3 (New England Biolabs E7710S) and Set 4 (New England Biolabs E7730S) according to the manufacturer’s protocol.
  • K562 PEmax parental and La-ko4 cells were transduced with lentiviruses harboring the mDNMTl target.
  • 1E6 each transduced cells were nucleofected with 500 or 1000 ng pegRNA or epegRNA plasmid using the SE Cell Line 4D-Nucleofector X Kit (Lonza V4XC-1032) and program FF-120, according to the manufacturer’s protocol. 14 the amount of Cells from each nucleofection were harvested 1, 2, 3 and 4 days after nucleofection and the editing outcomes were quantified by high-throughput DNA sequencing and CRISPResso2 analysis.
  • the trimmed reads were then aligned to the sequence(s) of (e)pegRNA(s) the sample was nucleofected with, using Bowtie2 with default alignment options. Reads that did not align to the (e)pegRNA references were then aligned to the human genome (GRCh38 primary assembly from Ensembl release 107) using Bowtie2 with default alignment parameters. Downstream analysis of the alignments used only reads mapped in a proper pair, ensuring both ends of the sequenced fragment were properly mapped.
  • Quantifications of human small RNA including assigning fragments to human transcripts, genes, and biotypes as well as counting, were performed on properly paired alignments using a custom Python script available in the GitHub repository. To distinguish between overlapping annotations, each aligned fragment was assigned to the annotation that most closely matched the start and end point of the fragment.
  • the (e)pegRNA(s) were quantified for each sample by assigning each properly aligned fragment into one of three bins defined in the main text (c/.s-active, /ra/z.s-active and inactive) using Rsamtools and plyranges.
  • Differential expression was calculated using DESeq2 version 1.38.3 with a design consisting of two covariates: (e)pegRNA plasmid set nucleofected (set 1 or 2) and cell line (K562 PEmax or La-ko4). Default parameters were used to estimate library size factors, genewise dispersion, and fitting of the negative binomial GLM to determine log2 fold change values. Log fold change shrinkage was performed using the apeglm algorithm. The default two-sided Wald test was used to determine the p values and the Bonferroni Holm method was used for multiple test correction. Coverage plots were generated using ggplot2 on data organized using the readr, dplyr, tidyr, and stringr packages.
  • RNA-seq and data analysis. Each condition of RNA-seq was performed in quadruplicate and for each replicate, 1E6 K562 cells were nucleofected with 750 ng PEmax or PE7 editor plasmid and 250 ng pegRNA plasmid encoding HEK3 +1 T to A or PRNP + 6 G to T using SE Cell Line 4D-Nucleofector X Kit S (Lonza V4XC-1032) with program FF-120, according to the manufacturer’s protocols. Nucleofected cells were cultured in 6-well plates with 2.5 mL medium per well.
  • Sequencing libraries were pooled, quantified by Qubit IX dsDNA High Sensitivity kit (Invitrogen Q33231) and a high sensitivity DNA chip (Agilent Technologies 5067-4626) on an Agilent 2100 Bioanalyzer and sequenced with the NovaSeq 6000 SP Reagent kit vl.5 100 cycles (Illumina 20028401) with 112 cycles for R1 read, 10 cycles index read.
  • Sequencing reads were demultiplexed through HTSEQ (Princeton University High Throughput Sequencing Database). Alignment, quantification, and differential expression were performed using a Snakemake workflow and R scripts available on GitHub github.com/Princeton-LSI-ResearchComputing/PE-mRNA-seq-diffexp. The reads were aligned to the GRCh38 genome from Ensembl release 100 using STAR with default alignment parameters. Quantification was performed by STAR during alignment. Differential expression between editors was performed separately for each pegRNA. The standard DESeq2 procedure was performed to determine the differential expression between each editor within the set of samples for each pegRNA. Fold changes for lowly expressed genes were shrunken using the adaptive shrinkage estimator from the ashr package. Figures were generated using R packages ggplot2 and ggpubr.
  • T cell isolation culture and prime editing.
  • Human peripheral blood Leukopaks enriched for PBMCs were sourced from STEMCELL Technologies (catalog # 200-0092). No preference was given with regard to sex, gender, ethnicity or race.
  • T cells were isolated with the Easy Sep Human T cell isolation kit (STEMCELL Technologies 100-0695) according to manufacturer's instructions. Immediately after isolation, T cells were used directly for in vitro experiments.
  • T cells were cultured in complete X-VIVO 15 consisting of X- VI VO 15 (Lonza Bioscience 04-418Q) supplemented with 5% FBS (R&D systems), 4mM N-acetyl- cysteine (RPI A10040) and 55 pM 2-mercaptoethanol (GIbco 21985023).
  • Pan CD3+ T cells were thawed and activated with anti-CD3/anti-CD28 dynabeads (Gibco 40203D) at a 1 : 1 bead:cell ratio in presence of 500 IU mL’ 1 IL-2.
  • T cells were magnetically de-beaded and taken up in P3 buffer with supplement (Lonza Bioscience V4SP- 3096) at 37.5E6 cells mL' 1 .
  • 1.5 pg PEmax or PE7 mRNA mixed with 50 pmole synthetic pegRNA (IDT) was added per 20pL cells, not exceeding 25 pL total volume per reaction.
  • Cells were subsequently electroporated on a Lonza 4D Nucleofector using program DS-137.
  • PCR was performed with 25 uL of eluted genomic DNA per sample in an 100 pL PCR reaction with KAPA HiFi HotStart ReadyMix (Roche 9420398001) with the following cycling conditions: 95 °C for 3 min, 28 cycles of [98 °C for 20 s, 63 °C for 15 s, and 72 °C for 60 s], followed by 72 °C for 2 min.
  • PCR products were purified by SPRI selection (Beckman Coulter B23317) and 2 pL eluted product was used for 8 cycles of additional PCR with KAPA HiFi HotStart ReadyMix to add Illumina sequencing adapters and indices.
  • the final PCR products were purified by SPRI selection, quantified with Qubit IX dsDNA High Sensitivity (HS) assay kit (Invitrogen Q33230), equimolarly pooled, and sequenced with the MiSeq Reagent Kit v2 300 cycles (Illumina MS-102-2002) with 300 cycles for R1 read, 8 cycles i7 index read and 8 cycles i5 index read. Sequencing data were demultiplexed using BaseSpace and analyzed by CRISPResso2.
  • mRNA in vitro transcription template plasmids for HPSC experiments were constructed by cloning PEmax and PE7 into a previously described vector. mRNA was generated using HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs E2040S) and BbsI linearized plasmids as templates with UTP fully replaced by NkMethylpseudouridine-S '-triphosphate (TriLink Biotechnologies N-1081) and co-transcriptional capping by CleanCap AG (TriLink Biotechnologies N-7113).
  • mRNA was purified using the Monarch RNA Cleanup kit (500 pg) (NEB T2050S), eluted in IDTE pH 7.5 (Integrated DNA Technologies 11-05-01-15) and quantified using Qubit RNA High Sensitivity (HS) Assay Kit (Invitrogen Q32852).
  • Synthetic pegRNAs were ordered as Custom Alt-R gRNA from Integrated DNA Technologies and resuspended at 200 pM in IDTE pH 7.5.
  • Cryopreserved human CD34 + HSPCs from mobilized peripheral blood of deidentified healthy donors were obtained from the Fred Hutchinson Cancer Research Center (Seattle, Washington).
  • CD34 + HSPCs were cultured with X-Vivo-15 media supplemented with 100 ng mL' 1 human Stem Cell Growth Factor (SCF), 100 ng mL' 1 human thrombopoietin (TPO), and 100 ng mL' 1 recombinant human FMS-like Tyrosine Kinase 3 Ligand (Flt3-L).
  • CD34 + HSPCs were thawed and cultured for 24 hours in the presence of cytokines prior to nucleofection.
  • 2.5E5 CD34 + HSPCs were electroporated using the P3 Primary Cell X kit S (Lonza Bioscience V4SP-3096) according to manufacturer’s recommendations with 2000 ng PEmax or PE7 mRNA and 200 pmole synthetic pegRNA using pulse code DS-130. Genomic DNA was harvested 3 days post nucleofection with QuickExtract DNA Extraction Solution (LGC Biosearch Technologies QE09050) following manufacturer’s recommendations. Prime editing outcomes were quantified by high-throughput DNA sequencing and CRISPResso2 analysis as described earlier.

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Abstract

La présente divulgation concerne des systèmes d'édition primaire, des compositions et des procédés pour modifier des séquences de gènes cibles et/ou l'expression de séquences de gènes cibles.
PCT/US2024/038551 2023-07-18 2024-07-18 Système d'édition primaire et ses utilisations Pending WO2025019675A1 (fr)

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WO2023039586A1 (fr) * 2021-09-10 2023-03-16 Agilent Technologies, Inc. Arn guides avec modification chimique pour l'édition primaire
WO2023070062A2 (fr) * 2021-10-21 2023-04-27 Prime Medicine, Inc. Compositions d'édition de génome et méthodes de traitement du syndrome d'usher de type 3
WO2023070110A2 (fr) * 2021-10-21 2023-04-27 Prime Medicine, Inc. Compositions d'édition génomique et méthodes de traitement de la rétinite pigmentaire

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Publication number Priority date Publication date Assignee Title
WO2023039586A1 (fr) * 2021-09-10 2023-03-16 Agilent Technologies, Inc. Arn guides avec modification chimique pour l'édition primaire
WO2023070062A2 (fr) * 2021-10-21 2023-04-27 Prime Medicine, Inc. Compositions d'édition de génome et méthodes de traitement du syndrome d'usher de type 3
WO2023070110A2 (fr) * 2021-10-21 2023-04-27 Prime Medicine, Inc. Compositions d'édition génomique et méthodes de traitement de la rétinite pigmentaire

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ALFANO CATERINA, SANFELICE DOMENICO, BABON JEFF, KELLY GEOFF, JACKS AMANDA, CURRY STEPHEN, CONTE MARIA R: "Structural analysis of cooperative RNA binding by the La motif and central RRM domain of human La protein", NATURE STRUCTURAL & MOLECULAR BIOLOGY, NATURE PUBLISHING GROUP US, NEW YORK, vol. 11, no. 4, 1 April 2004 (2004-04-01), New York , pages 323 - 329, XP093268078, ISSN: 1545-9993, DOI: 10.1038/nsmb747 *
YAN ET AL.: "Improving prime editing with an endogenous small RNA-binding protein", NATURE, vol. 628, 3 April 2024 (2024-04-03), pages 639 - 647, XP038062744, DOI: 10.1038/s41586-024-07259-6 *

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