EP4623081A2 - Geteilte synthese von langen rnas - Google Patents

Geteilte synthese von langen rnas

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Publication number
EP4623081A2
EP4623081A2 EP23821097.5A EP23821097A EP4623081A2 EP 4623081 A2 EP4623081 A2 EP 4623081A2 EP 23821097 A EP23821097 A EP 23821097A EP 4623081 A2 EP4623081 A2 EP 4623081A2
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EP
European Patent Office
Prior art keywords
rna sequence
nucleotides
length
pegrna
rna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23821097.5A
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English (en)
French (fr)
Inventor
Ekambareswara Rao KANDIMALLA
Venubabu Kotikam
Meetu SETH
Cong Zhou
Andrew Doyle
David Wiley
Jeffrey HUSSMANN
Jonathan Levy
Seth Alexander
Andrew ANZALONE
Rohan KUMBHARE
Mallikarjuna Putta
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Prime Medicine Inc
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Prime Medicine Inc
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Application filed by Prime Medicine Inc filed Critical Prime Medicine Inc
Publication of EP4623081A2 publication Critical patent/EP4623081A2/de
Pending legal-status Critical Current

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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/344—Position-specific modifications, e.g. on every purine, at the 3'-end
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2330/00—Production
    • C12N2330/30—Production chemically synthesised

Definitions

  • the Sequence Listing XML file that has been electronically filed contains the information of the nucleotide and/or amino acid sequences disclosed in the patent application using the symbols and format in accordance with the requirements of 37 C.F.R. ⁇ 1.832 through 1.834. [3] The Sequence Listing XML filed herewith serves as the electronic copy required by ⁇ 1.834(b)(1). [4] The Sequence Listing XML is identified as follows: “272059-538182_Sequence- listing.xml” (196,614 bytes in size), which was created on November 21, 2023. BACKGROUND [5] Prime editing is a gene editing technology that allows researchers to make nucleotide substitutions, insertions, deletions, or combinations thereof in the DNA of cells.
  • PEgRNAs full-length prime editing guide RNAs
  • the disclosure provides methods for synthesizing a full-length prime editing guide RNA (PEgRNA) comprising a guide RNA (gRNA) core, the methods comprising, contacting a first RNA sequence with a second RNA sequence, wherein the first RNA sequence comprises a first chemical moiety at its 3’ end and the second RNA sequence comprises a second chemical moiety at its 5’ end; and ligating the first RNA sequence and the second RNA sequence to obtain the synthesized full-length PEgRNA, wherein the ligation forms a linker between the first chemical moiety of the first RNA sequence and the second chemical moiety of the second RNA sequence; and wherein the linker is present in the gRNA core of the synthesized full- length PEgRNA.
  • PEgRNA prime editing guide RNA
  • the ligation occurs via a chemical ligation. In some embodiments, the ligation occurs via an enzymatic ligation. [11] In some embodiments, the gRNA core is a modified gRNA core. In some embodiments, the gRNA core is a chemically-modified gRNA core.
  • the disclosure provides methods for synthesizing a full-length prime editing guide RNA (PEgRNA) comprising a chemically-modified guide RNA (gRNA) core, the methods comprising, providing a first RNA sequence, wherein the first RNA sequence comprises a first chemical moiety at its 3’ end; providing a second RNA sequence, wherein the second RNA sequence comprises a second chemical moiety at its 5’ end; providing a third RNA sequence, wherein the third RNA sequence comprises a third chemical moiety at its 5’ end and a fourth chemical moiety at its 3’ end; ligating the first, second, and third RNA sequences to obtain the synthesized full- length PEgRNA, wherein the ligation forms (i) a first linker between the first chemical moiety of the first RNA sequence and the third chemical moiety of the third RNA sequence, and (ii) a second linker between the second chemical moiety of the second RNA sequence and the fourth chemical moiety of the third RNA sequence; wherein the
  • the second linker is present in the chemically-modified gRNA core of the synthesized full-length PEgRNA. In some embodiments, the second linker is present in the junction of the synthesized full-length PEgRNA.
  • the disclosure provides methods for synthesizing a full-length prime editing guide RNA (PEgRNA) comprising a chemically-modified guide RNA (gRNA) core, the methods comprising, providing a first RNA sequence, wherein the first RNA sequence comprises an amino modified phosphodiester backbone at its 3’end; functionalizing the 3’ amino group of the first RNA sequence with a first chemical moiety; providing a second RNA sequence; wherein the second RNA sequence comprises an amino modified phosphodiester backbone at its 5’end; functionalizing the 5’ amino group of the second RNA sequence with a second chemical moiety; reacting the first chemical moiety of the first RNA sequence with the second chemical moiety of the second RNA sequence to form a linker; wherein
  • the disclosure provides methods for synthesizing a full-length prime editing guide RNA (PEgRNA) comprising a chemically-modified guide RNA (gRNA) core, the methods comprising, providing a first RNA sequence of Formula A: providing a second RNA sequence of Formula B: reacting M1 with M2 to form a linker, wherein forming the linker results in the formation of the synthesized full-length PEgRNA; wherein: each X 1 , X 2 , X 4 , and X 5 is independently absent or (O-CH 2 -CH 2 )n, (CH 2 -CH 2 -O)n, ((CH 2 )n-C(O)NH-CH 2 ), or an alkylene optionally substituted with one or more of –OH or – O–, or –O–; “n” is an integer between 1 and 12; M 1 is an amino, a halogen, a hydroxyl, a phosphate ion, a maleimi
  • the disclosure provides methods for synthesizing a full-length prime editing guide RNA (PEgRNA) comprising a chemically-modified guide RNA (gRNA) core, the methods comprising, providing a first RNA sequence of Formula C: ; providing a second RNA sequence of Formula D: contacting the first RNA sequence with the second RNA sequence to form a linker, wherein forming the linker results in the formation of the synthesized full-length PEgRNA of Formula E: wherein each X1 and X2 is independently absent or (O-CH 2 -CH 2 )n, (CH 2 -CH 2 -O)n, ((CH 2 )n-C(O)NH-CH 2 ), or an alkylene optionally substituted with one or more –OH or –O–, or –O–, “n” is an integer between 1 and 12; and wherein the linker is present in the chemically-modified gRNA core of the synthesized full-length PEgRNA.
  • PEgRNA prime editing guide
  • the disclosure provides methods for synthesizing a full-length prime editing guide RNA (PEgRNA) comprising a chemically-modified guide RNA (gRNA) core, the methods comprising, providing a first RNA sequence of Formula F: providing a second RNA sequence of Formula G: annealing the first and second RNA sequences to form a pre-annealed duplex; providing a BS3 reagent of the formula: contacting the pre-annealed duplex with the BS3 reagent to form a linker, wherein forming the linker results in the formation of the synthesized full-length PEgRNA of Formula H: wherein each X1 and X2 is independently absent or (O-CH 2 -CH 2 )n, (CH 2 -CH 2 -O)n, ((CH 2 )n-C(O)NH-CH 2 ), or an alkylene optionally substituted with one or more –OH or –O–, or –O–, “n” is an
  • the disclosure provides methods for synthesizing a full-length prime editing guide RNA (PEgRNA) comprising a chemically-modified guide RNA (gRNA) core, the methods comprising, providing a first RNA sequence of Formula I: providing a second RNA sequence of Formula J: annealing the first and second RNA sequences to form a pre-annealed duplex; providing a BMH reagent; contacting the pre-annealed duplex with the BMH reagent to form a linker, wherein forming the linker results in the formation of Formula P: wherein Formula P comprises an amine at its 5’ end; providing a third RNA sequence of Formula F: contacting Formula P with Formula F to form a second pre-annealed duplex; providing a BS3 reagent; contacting the second pre-annealed duplex with the BS3 reagent to form
  • the disclosure provides methods for synthesizing a full-length prime editing guide RNA (PEgRNA) comprising a chemically-modified guide RNA (gRNA) core, the methods comprising, providing a first RNA sequence of Formula R: providing a second RNA sequence of Formula S: contacting the first RNA sequence with the second RNA sequence to form a linker, wherein forming the linker results in the formation of the synthesized full-length PEgRNA of Formula T:
  • PEgRNA prime editing guide RNA
  • gRNA chemically-modified guide RNA
  • the disclosure provides methods for editing a gene, the method comprising contacting the gene with (i) the full-length PEgRNA prepared by the methods described herein and (ii) a prime editor comprising a DNA binding domain and a DNA polymerase domain, wherein the PEgRNA directs the prime editor to incorporate the intended nucleotide edit in the gene, thereby editing the gene.
  • the disclosure provides methods for editing a gene, the method comprising contacting the gene with the prime editing complex disclosed herein, wherein the PEgRNA directs the prime editor to incorporate the intended nucleotide edit in the gene, thereby editing the gene.
  • the disclosure provides an RNA sequence of Formula G: , wherein X1 is absent or (-O-CH 2 -CH 2 )n, (CH 2 -CH 2 -O)n, ((CH 2 )n-C(O)NH-CH 2 ), or an alkylene optionally substituted with one or more –OH or –O–, or –O–; and “n” is an integer between 1 and 12.
  • the disclosure provides an RNA sequence of Formula G: ; wherein X 2 is absent or (-O-CH 2 -CH 2 )n, (CH 2 -CH 2 -O)n, ((CH 2 )n-C(O)NH-CH 2 ), or an alkylene optionally substituted with one or more –OH or –O–, or –O–; and “n” is an integer between 1 and 12.
  • the disclosure provides an RNA sequence of Formula U: wherein X2 is absent or (O-CH 2 -CH 2 )n, (CH 2 -CH 2 -O)n, ((CH 2 )n-C(O)NH-CH 2 ), or an alkylene optionally substituted with one or more –OH or –O–, or –O–; and “n” is an integer between 1 and 12.
  • the disclosure provides an RNA sequence of Formula Y: wherein X2 is absent or (O-CH 2 -CH 2 )n, (CH 2 -CH 2 -O)n, ((CH 2 )n-C(O)NH-CH 2 ), or an alkylene optionally substituted with one or more –OH or –O–, or –O–; and “n” is an integer between 1 and 12.
  • the disclosure provides a method for synthesizing a full-length prime editing guide RNA (PEgRNA) comprising a guide RNA (gRNA) core, the method comprising, contacting a first RNA sequence with a second RNA sequence and a splint sequence, wherein the first RNA sequence comprises a free 3’ OH, the second RNA sequence comprises a 5’-monophosphate, and the splint sequence comprises a sequence partially complementary to both the first and second RNA sequences; ligating the first RNA sequence and the second RNA sequence with a nucleic acid ligase to obtain the synthesized full-length PEgRNA, wherein the ligation forms a linker between the 3’ OH and the 5’- monophosphate; and wherein the linker is present in the spacer, gRNA core, RTT, or PBS of the synthesized full-length PEgRNA.
  • PEgRNA prime editing guide RNA
  • gRNA guide RNA
  • the disclosure provides a method for synthesizing a full-length prime editing guide RNA (PEgRNA) comprising a guide RNA (gRNA) core, the method comprising, providing a first RNA sequence, wherein the first RNA sequence comprises a first chemical moiety at its 3’ end; providing a second RNA sequence, wherein the second RNA sequence comprises a second chemical moiety at its 5’ end and a free 3’ OH; providing a third RNA sequence, wherein the third RNA sequence comprises a 5’- monophosphate; ligating the first, second, and third RNA sequences to obtain the synthesized full- length PEgRNA, wherein the ligation forms (i) a first linker between the first chemical moiety of the first RNA sequence and the second chemical moiety of the second RNA sequence, and (ii) a second linker between the 3’ OH of the second RNA sequence and the 5’-monophosphate of the third RNA sequence; wherein the first linker is present in the
  • the method further comprises providing a splint sequence, wherein the splint sequence comprises a sequence partially complementary to both the second and third RNA sequences.
  • FIG.1 shows an exemplary schematic of a PEgRNA.
  • FIG.2 shows an exemplary PEgRNA gRNA core.
  • FIG.3A shows an exemplary PEgRNA with the split sites at the TL in the gRNA core for use in a two-part ligation method: “X” and “Y” represent chemical moieties.
  • FIG.3B shows an exemplary PEgRNA with the split sites at the SL2 in the gRNA core for use in a two-part ligation method: “X” and “Y” represent chemical moieties.
  • FIG.4A shows an exemplary PEgRNA with the split sites in the PEgRNA for use in a three-part ligation method. The figure shows a split site at the TL and a split site at the SL2 of the gRNA core and “X,” “Y,” “M,” and “N” represent chemical moieties.
  • FIG.4B shows an exemplary PEgRNA with the split sites in the PEgRNA for use in a three-part ligation method.
  • FIG.5A shows an exemplary gRNA core extension at the TL position.
  • An original TL is shown as “native,” and the extended TL is shown as “extension.”
  • the loop structure is shown in gray with underline font, the original base pairs are shown in gray, the extended 11- base pairs are shown in black with bold font.
  • the base pair sequences shown in the figure are arbitrary and meant to function mainly as an example.
  • the split site is shown in dash line. The rest of the sequences are abbreviated as gray dots.
  • FIG.5B shows an exemplary gRNA core extension at the SL2 position.
  • FIG.6 shows a denaturing PAGE (15%) analysis of the thioether ligation of Example 1.
  • FIG.11 shows an LC-MS analysis of PEGRNAL1005 purified ligation product.
  • FIG.12 shows In vitro prime editing of split synthesized PEgRNAs in primary human hepatocytes.
  • FIG.13 shows a denaturing PAGE (15%) analysis of the bisamide ligation. Lane 1: oligonucleotide ladder for length; Lane 2-4: Bisamide ligation of RNA010 and RNA011 to generate the ligation product RNA010_bisamide_RNA011 using different BS3 reagent equivalents (from high to low equivalents). The quantified yield is up to 70% (Lane 3).
  • FIG.14 shows a RP-HPLC analysis of the bisamide ligation.
  • the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value.
  • a protein that comprises amino acid sequences from different origins or naturally occurring proteins may be referred to as a fusion, or chimeric protein.
  • the term “polynucleotide,” “oligonucleotide,” or “nucleic acid molecule” can be any polymeric form of nucleotides, including DNA, RNA, a hybridization thereof, or RNA-DNA chimeric molecules.
  • a polynucleotide comprises cDNA, genomic DNA, mRNA, tRNA, rRNA, or microRNA.
  • a polynucleotide is double stranded, e.g., a double-stranded DNA in a gene.
  • a polynucleotide is single-stranded or substantially single-stranded, e.g., single-stranded DNA or an mRNA. In some embodiments, a polynucleotide is a cell-free nucleic acid molecule. In some embodiments, a polynucleotide circulates in blood. In some embodiments, a polynucleotide is a cellular nucleic acid molecule. In some embodiments, a polynucleotide is a cellular nucleic acid molecule in a cell circulating in blood. [96] Polynucleotides can have any three-dimensional structure.
  • polynucleotides a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), an exon, an intron, intergenic DNA (including, without limitation, heterochromatic DNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), a ribozyme, cDNA, a recombinant polynucleotide, a branched polynucleotide, a plasmid, a vector, isolated DNA, isolated RNA, sgRNA, guide RNA, a nucleic acid probe, a primer, an snRNA, a long non-coding RNA, a snoRNA, a siRNA, a miRNA, a tRNA-derived small RNA (tsRNA), an antisense RNA, an shRNA, or a small rDNA-derived RNA (srRNA).
  • a gene or gene fragment for example, a probe, primer, EST or SAGE tag
  • a polynucleotide comprises deoxyribonucleotides, ribonucleotides or analogs thereof.
  • a polynucleotide comprises modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide.
  • the sequence of nucleotides can be interrupted by non-nucleotide components.
  • a polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component.
  • a “chemically modified” polynucleotide includes modifications which introduce chemistries which differ from those seen in naturally occurring DNA or RNAs, for example, covalent modifications that introduce a group not naturally found in DNA or RNA molecules.
  • a “chemically modified” polynucleotide may be obtained by a chemical reaction with a polynucleotide precursor and a chemical reagent.
  • a “modified gRNA core” refers to a gRNA core that has been obtained by a chemical reaction with a gRNA core precursor and a chemical reagent.
  • the modified gRNA core may comprise chemical modifications in the base, sugar, and/or backbone.
  • the chemical moiety is structurally different from a natural nucleotide, a natural sugar, and/or a natural internucleoside linkage. In some embodiments, the chemical moiety is a chemical modification on the internucleoside linkage (e.g., phosphate backbone) of the first, second, and/or third RNA sequence.
  • a “linker” refers to a covalent bond or a chemical moiety which connects one chemical moiety to another. In some embodiments, a linker is a covalent bond. In some embodiments, a linker is a chemical linker.
  • a percentage of complementarity indicates the percentage of nucleotides in a polynucleotide molecule which can base pair with a second polynucleotide molecule (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary, respectively).
  • Perfectly complementary means that all the contiguous nucleotides of a polynucleotide molecule will base pair with the same number of contiguous nucleotides in a second polynucleotide molecule.
  • the “effective amount” or “therapeutically effective amount” is the amount of a composition that is required to ameliorate the symptoms of a disease relative to an untreated patient.
  • an effective amount is the amount of a composition sufficient to introduce an alteration in a gene of interest in a cell (e.g., a cell in vitro or in vivo).
  • the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 th Ed. Additionally, general principles of organic chemistry are described in “Organic Chemistry,” Thomas Sorrell, University Science Books, Sausalito: 1999, and “March's Advanced Organic Chemistry,” 5 th Ed., Ed.: Smith, M.B.
  • an “alkyl” group refers to a saturated aliphatic hydrocarbon group containing 1-15 (e.g., carbon atoms. An alkyl group can be straight or branched.
  • an alkyl and/or alkylene can be substituted with one or more substituents independently selected from halo, oxo, hydroxy, cyano, halo, alkoxy (e.g., C1-6 alkoxy), –C(O)-C1-6 alkoxy and –C(O)-C1-6 alkyl.
  • an alkyl and/or alkylene can be substituted with one or more substituents independently selected from halo, alkoxy, and hydroxy.
  • an alkyl and/or alkylene can be substituted with –OH (“hydroxy-substituted alkyl” or “hydroxy-substituted alkylene”).
  • a hydroxy- substituted alkyl/alkylene can be –(CH 2 ) n –CH(OH)–(CH 2 ) n –, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • an alkyl and/or alkylene can be substituted with –O– to form an alkoxy group.
  • an “alkoxy” group refers to an alkyl-O- group where “alkyl” has been defined previously.
  • the alkyl portion of the alkoxy group can be substituted (i.e., optionally substituted) as provided herein.
  • an alkoxy or an –O–substituted alkyl/alkylene can be –(CH 2 )n–O–(CH 2 )n–, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • an alkyl and/or alkylene can be substituted with both –OH and –O–. Examples of these instances include, but are not limited to, –(CH 2 )n–CH(OH)–(CH 2 )n– O–(CH 2 ) n – or –(CH 2 ) n –CH(OH)–O–(CH 2 ) n –, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • a heteroaryl group includes a benzofused ring system having 2 to 3 rings.
  • a benzofused group includes benzo fused with one or two 4 to 8 membered heterocycloaliphatic moieties (e.g., indolizyl, indolyl, isoindolyl, 3H-indolyl, indolinyl, benzo[b]furyl, benzo[b]thiophene-yl, quinolinyl, or isoquinolinyl).
  • heterocycloaliphatic moieties e.g., indolizyl, indolyl, isoindolyl, 3H-indolyl, indolinyl, benzo[b]furyl, benzo[b]thiophene-yl, quinolinyl, or isoquinolinyl.
  • bicyclic heteroaryls include indolizyl, indolyl, isoindolyl, 3H-indolyl, indolinyl, benzo[b]furyl, benzo[b]thiophenyl, quinolinyl, isoquinolinyl, indolizyl, isoindolyl, indolyl, benzo[b]furyl, bexo[b]thiophenyl, indazolyl, benzimidazyl, benzthiazolyl, purinyl, 4H-quinolizyl, quinolyl, isoquinolyl, cinnolyl, phthalazyl, quinazolyl, quinoxalyl, 1,8-naphthyridyl, or pteridyl.
  • a heteroaryl group has 1-3 heteroatoms independently selected from N, O, or S.
  • a heteroaryl group can be substituted (i.e., optionally substituted) with one or more substituents such as aliphatic [e.g., alkyl, alkenyl, or alkynyl]; cycloaliphatic; (cycloaliphatic)aliphatic; heterocycloaliphatic; (heterocycloaliphatic)aliphatic; aryl; heteroaryl; alkoxy; (cycloaliphatic)oxy; (heterocycloaliphatic)oxy; aryloxy; heteroaryloxy; (araliphatic)oxy; (heteroaraliphatic)oxy; aroyl; heteroaroyl; amino; oxo (on a non-aromatic carbocyclic or heterocyclic ring of a bicycl
  • substituents such as aliphatic [e.g., alkyl, alkenyl, or alkynyl]
  • a heteroaryl can be unsubstituted.
  • a “BS3” reagent refers to bis(sulfosuccinimidyl)suberate, a compound having a structure of: or [131]
  • a “BMH” reagent refers to bismaleimidohexane, a compound having a structure of: [132]
  • a “BM(PEG) 2 ” reagent refers to 1,8-bisamleimidodiethyleneglycol, a compound having a structure of: [133]
  • a “BMOE” reagent refers to bismaleimidoethane, a compound having a structure of: [134]
  • a “BM(PEG) 3 ” reagent refers to 1,11-bismaleimidotriethyleneglycol, a compound having a structure of:
  • RNA oligonucleotides 100 nucleotides or more in length
  • the coupling efficiency is not quantitative limiting the synthesis yield for long RNA oligonucleotides.
  • RNAs having 100-nucleotides or more Due to the similarity of the chemical and/or physical properties of these impurities and the FLP, it is difficult to remove said impurities during the purification process. This is particularly problematic for long RNAs having 100-nucleotides or more, because just several nucleotides shorter or longer becomes a trivial difference for long oligonucleotides (100 nucleotides or more in length) compared to much shorter oligonucleotides (i.e., 20 nucleotides in length). Hence, alternate synthesis, and purification methods are required for long-RNA synthesis.
  • the long RNAs, such as PEgRNAs, of this disclosure have a length of about 100 nucleotides, about 110 nucleotides, about 120 nucleotides, about 130 nucleotides, about 140 nucleotides, about 150 nucleotides, about 160 nucleotides, about 170 nucleotides, about 180 nucleotides, about 190 nucleotides, about 200 nucleotides, about 210 nucleotides, about 220 nucleotides, about 230 nucleotides, about 240 nucleotides, about 250 nucleotides, about 275 nucleotides, about 300 nucleotides, about 325 nucleotides, about 350 nucleotides, about 375 nucleotides, about 400 nucleotides, about 425 nucleotides, about 450 nucleotides, about 475 nucleotides, or about 500 nucleotides.
  • the long RNA has a length of about 240 nucleotides. In some embodiments, the long RNA has a length of about 250 nucleotides. In some embodiments, the long RNA has a length of about 300 nucleotides. In some embodiments, the long RNA has a length of about 400 nucleotides. In some embodiments, the long RNA has a length of about 500 nucleotides. [139] The present disclosure provides an approach termed “split synthesis.” In a split synthesis method, the long RNA is split into shorter fragments, sometimes containing chemical modifications on the 3’ and 5’ end, and the fragments are ligated (bound) through appropriate ligation methods to obtain a full-length product.
  • a chemical moiety in the form of a functional group is appended to the 3’ end and/or the 5’ end.
  • the chemical moiety is structurally different from a natural nucleotide, a natural sugar, and/or a natural internucleoside linkage.
  • the unreacted shorter fragments can be removed to obtain high purity full-length ligated product.
  • the purity of the full-length ligated product e.g., long RNA or PEgRNA
  • the purity of the full-length ligated product is over 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.
  • the long guide RNA has a length of about 140 nucleotides. In some embodiments, the long guide RNA has a length of about 150 nucleotides. In some embodiments, the long guide RNA has a length of about 170 nucleotides. In some embodiments, the long guide RNA has a length of about 190 nucleotides. In some embodiments, the long guide RNA has a length of about 200 nucleotides. In some embodiments, the long guide RNA has a length of about 210 nucleotides. In some embodiments, the long guide RNA has a length of about 220 nucleotides. In some embodiments, the long guide RNA has a length of about 230 nucleotides.
  • the long guide RNA has a length of about 240 nucleotides. In some embodiments, the long guide RNA has a length of about 250 nucleotides. In some embodiments, the long guide RNA has a length of about 300 nucleotides. In some embodiments, the long guide RNA has a length of about 400 nucleotides. In some embodiments, the long guide RNA has a length of about 500 nucleotides. [141] In certain aspects, the disclosure provides methods for synthesizing long RNAs. In some aspects, the disclosure provides methods for synthesizing prime editing guide RNA (PEgRNA).
  • PEgRNA prime editing guide RNA
  • the disclosure provides methods for synthesizing prime editing guide RNA (PEgRNA) comprising a guide RNA (gRNA) core. In some embodiments, the disclosure provides methods for synthesizing prime editing guide RNA (PEgRNA) comprising a chemically-modified guide RNA (gRNA) core. In some embodiments, the disclosure provides methods for synthesizing prime editing guide RNA (PEgRNA) comprising a non-chemically-modified guide RNA (gRNA) core. In some embodiments, the disclosure provides methods for synthesizing full-length prime editing guide RNA (PEgRNA) comprising a guide RNA (gRNA) core.
  • PEgRNA prime editing guide RNA
  • gRNA guide RNA
  • a method comprising, contacting a first RNA sequence with a second RNA sequence, wherein the first RNA sequence comprises a first chemical moiety at its 3’ end and the second RNA sequence comprises a second chemical moiety at its 5’ end; and ligating the first RNA sequence and the second RNA sequence to form a synthesized RNA by reacting the first chemical moiety of the first RNA sequence with the second chemical moiety of the second RNA sequence; wherein the ligation forms a linker in a stem loop structure, a tetraloop structure, and/or an extension arm of the synthesized RNA.
  • PEgRNA prime editing guide RNA
  • gRNA guide RNA
  • the method comprising, contacting a first RNA sequence with a second RNA sequence, wherein the first RNA sequence comprises a first chemical moiety at its 3’ end and the second RNA sequence comprises a second chemical moiety at its 5’ end; and ligating the first RNA sequence and the second RNA sequence to obtain the synthesized full-length PEgRNA, wherein the ligation forms a linker between the first chemical moiety of the first RNA sequence and the second chemical moiety of the second RNA sequence; and wherein the linker is present in the gRNA core of the synthesized full- length PEgRNA.
  • PEgRNA prime editing guide RNA
  • a spacer may be at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides.
  • the spacer is 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, or 25 nucleotides in length.
  • a PEgRNA may comprise a primer binding site (PBS) and an editing template (e.g., an RTT).
  • the extension arm of a PEgRNA may comprise a PBS and an editing template.
  • the length of the PBS sequence may vary depending on, e.g., the prime editor components, the search target sequence and other components of the PEgRNA.
  • the length of the primer binding site (PBS) varies from at least 2 nucleotides to 50 nucleotides.
  • the PBS is at least 6 nucleotides in length. In some embodiments, the PBS is about 4 to 16 nucleotides, about 6 to 16 nucleotides, about 6 to 18 nucleotides, about 6 to 20 nucleotides, about 8 to 20 nucleotides, about 10 to 20 nucleotides, about 12 to 20 nucleotides, about 14 to 20 nucleotides, about 16 to 20 nucleotides, or about 18 to 20 nucleotides in length. In some embodiments, the PBS is about 7 to 15 nucleotides in length. In some embodiments, the PBS is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length.
  • the editing template is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, or 350 nucleotides in length. In some embodiments, the editing template is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more nucleotides.
  • the editing template is about 5 to 10, 5 to 15, 5 to 20, 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, 5 to 50, 5 to 55, 5 to 60, 5 to 65, 5 to 70, 5 to 75, 5 to 80, 5 to 85, 5 to 90, 5 to 95, 5 to 100, 5 to 110, 5 to 120, 5 to 130, 5 to 140, 5 to 150, 15 to 20, 15 to 25, 15 to 30, 15 to 35, 15 to 40, 15 to 45, 15 to 50, 15 to 55, 15 to 60, 15 to 65, 15 to 70, 15 to 75, 15 to 80, 15 to 85, 15 to 90, 15 to 95, 15 to 100, 15 to 110, 15 to 120, 15 to 130, 15 to 140, 15 to 150, 25 to 30, 25 to 35, 25 to 40, 25 to 45, 25 to 50, 25 to 55, 25 to 60, 25 to 65, 25 to 70, 25 to 75, 25 to 80, 25 to 85, 25 to 90, 25 to 95, 25 to 100, 25 to 110, 25 to 120, 25 to 140, 25 to
  • the RTT is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the RTT is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the RTT is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length. [156] In some embodiments, a PEgRNA includes only RNA nucleotides and forms an RNA polynucleotide.
  • An editing template that comprises a DNA sequence may serve as a DNA synthesis template for a DNA polymerase in a prime editor, for example, a DNA-dependent DNA polymerase.
  • the PEgRNA may be a chimeric polynucleotide that comprises RNA in the spacer, gRNA core, and/or the PBS sequences and DNA in the editing template.
  • Components of a PEgRNA may be arranged in a modular fashion.
  • the spacer and the extension arm comprising a primer binding site sequence (PBS) and an editing template, e.g., a reverse transcriptase template (RTT), can be interchangeably located in the 5 ⁇ portion of the PEgRNA, the 3 ⁇ portion of the PEgRNA, or in the middle of the gRNA core.
  • a PEgRNA comprises, from 5’ to 3’: a spacer, a gRNA core, an editing template, and a PBS.
  • a PEgRNA comprises, from 5’ to 3’: an editing template, a PBS, a spacer, and a gRNA core.
  • the gRNA core may interact with a prime editor as described herein, for example, by association with a DNA binding domain, such as a DNA nickase of the prime editor.
  • a prime editor as described herein, for example, by association with a DNA binding domain, such as a DNA nickase of the prime editor.
  • a DNA binding domain such as a DNA nickase of the prime editor.
  • the gRNA core is capable of binding to a Cas9-based prime editor.
  • the gRNA core is capable of binding to a Cpf1-based prime editor.
  • the gRNA core is capable of binding to a Cas12b-based prime editor.
  • the gRNA core comprises regions and secondary structures involved in binding with specific CRISPR Cas proteins.
  • the gRNA core of a PEgRNA may comprise one or more regions of a base paired regions.
  • the gRNA core is a modified gRNA core.
  • the gRNA core is a chemically-modified gRNA core.
  • a gRNA core or a chemically-modified gRNA core comprises, from 5’ to 3’: a repeat sequence, a loop structure, an antirepeat sequence, a first stem loop, a second stem loop, and a third stem loop.
  • a gRNA core or a chemically-modified gRNA core comprises, from 5’ to 3’: a repeat sequence, a loop structure, an antirepeat sequence, a first stem loop, a second stem loop, and a third stem loop.
  • a repeat sequence and an antirepeat sequence refer to the nucleic acid secondary structure formed by the direct repeat region.
  • the repeat sequence and the antirepeat sequence may be connected by a loop structure, and the secondary structure formed by base pairing between the repeat and antirepeat sequence may be referred to as the direct repeat region.
  • the repeat, antirepeat, and the connecting loop structure may be referred to as a tetraloop.
  • a gRNA core or a chemically-modified gRNA core comprises, from 5’ to 3’: a tetraloop (TL), a first stem loop (SL1), a second stem loop (SL2), and a third stem loop (SL3).
  • a tetraloop TL
  • SL1 first stem loop
  • SL2 second stem loop
  • SL3 third stem loop
  • Exemplary structures of the gRNA core are shown in FIG.1 and FIG.2.
  • the tetraloop (TL) comprises a lower stem, a bulge, an upper stem, and a loop.
  • one or more nucleotides in the gRNA core is deleted, inserted, and/or substituted as compared to a wild type gRNA core.
  • An exemplary wild type gRNA core sequence is GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU GAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO.: 41).
  • the gRNA core comprises one or more nucleotide insertions, deletions, and/or substitutions in the lower stem or upper stem of the tetraloop.
  • the gRNA core comprises one or more nucleotide substitutions in the lower stem of the tetraloop.
  • the gRNA core comprises one or more nucleotide insertions in the upper stem of the tetraloop. In some embodiments, the gRNA core comprises one or more nucleotide insertions, deletions, and/or substitutions in the second stem loop as compared to a wild type gRNA core. In some embodiments, the gRNA core comprises one or more nucleotide insertions in the second stem loop. In some embodiments, the gRNA core comprises one or more nucleotide insertions, deletions, and/or substitutions in the third stem loop as compared to a wild type gRNA core.
  • the gRNA core comprises one or more nucleotide insertions in the lower stem of the tetraloop. In some embodiments, the gRNA core comprises one or more nucleotide insertions in the upper stem of the tetraloop. In some embodiments, the gRNA core comprises 1-30, 5-20, or 5-15 nucleotide insertions in the lower stem of the tetraloop. In some embodiments, the gRNA core comprises 3-30, 3-20, 3-16 or 3-12 nucleotide insertions in the lower stem of the tetraloop. In some embodiments, the gRNA core comprises 3-12 nucleotide insertions in the lower stem of the tetraloop.
  • the gRNA core has a 10 base pair extension in the upper stem of the tetraloop. In some embodiments, the gRNA core has a 11 base pair extension in the upper stem of the tetraloop. In some embodiments, the gRNA core has a 12 base pair extension in the upper stem of the tetraloop. [172] In some embodiments, the gRNA core comprises one or more nucleotide insertions in the SL2. In some embodiments, the gRNA core comprises 1-30, 5-20, or 5-15 nucleotide insertions in the SL2. In some embodiments, the gRNA core comprises 3-30, 3-20, 3-16 or 3- 12 nucleotide insertions in the SL2.
  • the gRNA core comprises 3-12 nucleotide insertions in the SL2. In some embodiments, the gRNA core comprises 4-30, 4-20, 4-16 or 4-12 nucleotide insertions in the SL2. In some embodiments, the gRNA core comprises 4-12 nucleotide insertions in the SL2. In some embodiments, the gRNA core comprises 8-30, 8-20, 8-16 or 8-12 nucleotide insertions in the SL2. In some embodiments, the gRNA core comprises 8-12 nucleotide insertions in the SL2.
  • the gRNA core comprises 1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotide insertions in the SL2. In some embodiments, the gRNA core comprises 10, 11 or 12 nucleotide insertions in the SL2. [173] In some embodiments, the gRNA core has a 1 base pair extension in the the SL2. In some embodiments, the gRNA core has a 2 base pair extension in the the SL2. In some embodiments, the gRNA core has a 3 base pair extension in the the SL2. In some embodiments, the gRNA core has a 1 base pair extension in the the SL2.
  • the gRNA core has a 4 base pair extension in the the SL2. In some embodiments, the gRNA core has a 5 base pair extension in the the SL2. In some embodiments, the gRNA core has a 6 base pair extension in the the SL2. In some embodiments, the gRNA core has a 7 base pair extension in the the SL2. In some embodiments, the gRNA core has a 8 base pair extension in the the SL2. In some embodiments, the gRNA core has a 9 base pair extension in the the SL2. In some embodiments, the gRNA core has a 10 base pair extension in the the SL2. In some embodiments, the gRNA core has a 11 base pair extension in the the SL2.
  • the gRNA core has a 12 base pair extension in the the SL2.
  • Split Synthesis [174] The split synthesis methods for preparing full-length PEgRNAs as described herein require a minimum of two RNA sequences (i.e., splitting the full-length PEgRNA into two or more parts), with each RNA sequence comprising at least one part of the full-length PEgRNA. [175] An important factor to consider in the split synthesis methods is choosing the appropriate split site for chemical ligations. Due to the additional chemical structure formed after chemical ligation, it is important to avoid the chemical structure interfering with the Cas9 nuclease interaction and activity by disturbing the gRNA/Cas9 complex.
  • the TL and SL2 are split sites for chemical ligations.
  • two RNA sequences are required, each sequence having at least one portion of the gRNA core.
  • a “portion” of the gRNA core corresponds to a section of the gRNA core that is smaller than the full-length gRNA core.
  • FIG.3A and FIG. 3B shows exemplary schemes of the split sites at the TL (FIG.3A) and SL2 (FIG.3B) of the gRNA core.
  • the first RNA sequence comprises a spacer and a first portion of a chemically-modified gRNA core.
  • the second RNA sequence comprises a second portion of a chemically-modified gRNA core, an extension arm, a chemically-modified extension arm, an RTT, and/or a PBS. In some embodiments, the second RNA sequence comprises a second portion of a chemically-modified gRNA core and an extension arm. In some embodiments, the second RNA sequence comprises a second portion of a chemically-modified gRNA core, an RTT, and a PBS. In some embodiments, the second RNA sequence comprises a second portion of a chemically-modified gRNA core and a chemically modified extension arm.
  • a “fragmented tetraloop” refer to a portion of a full-length tetraloop.
  • a fragmented tetraloop comprises 50% of the nucleotides of a full-length tetraloop.
  • a fragmented tetraloop comprises 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the nucleotides in length of a full-length tetraloop.
  • a fragmented tetraloop comprises 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 45% of the nucleotides in length of a full-length tetraloop.
  • a “fragmented SL2” refer to a portion of a full-length SL2.
  • a fragmented SL2 comprises 50% of the nucleotides of a full-length SL2.
  • the first portion of the chemically-modified gRNA core and the second portion of the chemically-modified gRNA core are different in length. In some embodiments, the first portion of the chemically-modified gRNA core and the second portion of the chemically-modified gRNA core are equal in length.
  • the first portion of the chemically-modified gRNA core is at least 5 nucleotides, at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least 35 nucleotides, at least 40 nucleotides, at least 45 nucleotides, at least 50 nucleotides, at least 55 nucleotides, at least 60 nucleotides, at least 65 nucleotides, at least 70 nucleotides, at least 75 nucleotides, at least 80 nucleotides, at least 85 nucleotides, at least 90 nucleotides, at least 95 nucleotides, at least 100 nucleotides, at least 105 nucleotides, or at least 110 nucleotides in length.
  • the first portion of the chemically-modified gRNA core is 5-120 or 10-110 nucleotides in length. In some embodiments, the first portion of the chemically-modified gRNA core is 5 nucleotides, 10 nucleotides, 15 nucleotides, 20 nucleotides, 25 nucleotides, 30 nucleotides, 35 nucleotides, 40 nucleotides, 45 nucleotides, 50 nucleotides, 55 nucleotides, 60 nucleotides, 65 nucleotides, 70 nucleotides, 75 nucleotides, 80 nucleotides, 85 nucleotides, 90 nucleotides, 95 nucleotides, 100 nucleotides, 105 nucleotides, or 110 nucleotides in length.
  • the second portion of the chemically-modified gRNA core is at least 5 nucleotides, at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least 35 nucleotides, at least 40 nucleotides, at least 45 nucleotides, at least 50 nucleotides, at least 55 nucleotides, at least 60 nucleotides, at least 65 nucleotides, at least 70 nucleotides, at least 75 nucleotides, at least 80 nucleotides, at least 85 nucleotides, at least 90 nucleotides, at least 95 nucleotides, at least 100 nucleotides, at least 105 nucleotides, or at least 110 nucleotides in length.
  • the fragmented tetraloop of the first portion of the chemically- modified gRNA core is 5 to 40, 10 to 30, or 10 to 20 nucleotides in length. In some embodiments, the fragmented tetraloop of the first portion of the chemically-modified gRNA core is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length. In some embodiments, the fragmented tetraloop of the second portion of the chemically-modified gRNA core is 5 to 40, 10 to 30, or 10 to 20 nucleotides in length.
  • the fragmented tetraloop of the second portion of the chemically-modified gRNA core is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length.
  • the fragmented SL2 of the first portion of the chemically- modified gRNA core is 5 to 30, 10 to 20 nucleotides in length.
  • the fragmented SL2 of the first portion of the chemically-modified gRNA core is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
  • the fragmented SL2 of the second portion of the chemically- modified gRNA core is 5 to 30, 10 to 20 nucleotides in length. In some embodiments, the fragmented SL2 of the second portion of the chemically-modified gRNA core is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. [185] In some embodiments, the chemically-modified gRNA core comprises a tetraloop, an extended tetraloop, an SL2 and/or an extended SL2. In some embodiments, the chemically- modified gRNA core comprises a tetraloop.
  • the chemically-modified gRNA core comprises an extended tetraloop.
  • the extended tetraloop comprises an additional 1-20 nucleotides in length compared to a native tetraloop.
  • the extended tetraloop comprises an additional 1-30, 5-20, or 5-15 nucleotides in length compared to a native tetraloop.
  • the extended tetraloop comprises an additional 3-30, 3-20, 3-16 or 3-12 nucleotides in length compared to a native tetraloop.
  • the extended tetraloop comprises an additional 3-12 nucleotides in length compared to a native tetraloop.
  • the extended tetraloop comprises an additional 6-12 nucleotides in length compared to a native tetraloopIn some embodiments, the extended tetraloop comprises an additional 1-20 nucleotides in the upper stem of the tetraloop compared to the upper step of a native tetraloop.
  • a “native tetraloop” is a tetraloop sequence that derives from a wild type gRNA core sequence. In some embodiments, a native tetraloop is a sequence of 5’-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAU-3’ (SEQ ID NO.: 22).
  • a native upper stem sequence of a tetraloop is the underlined nucleotides of of the following sequence 5’- GUUUUAGAGCUAGAAAUAGCAAGUUAAAAU-3’ (SEQ ID NO.: 22).
  • the chemically-modified gRNA core comprises an SL2.
  • the chemically-modified gRNA core comprises an extended SL2.
  • the extended SL2 comprises an additional 1-20 nucleotides in length compared to a native SL2.
  • the extended SL2 comprises an additional 1-30, 5-20, or 5-15 nucleotides in length compared to a native SL2.
  • the extended SL2 comprises an additional 3-30, 3-20, 3-16 or 3-12 nucleotides in length compared to a native SL2. In some embodiments the extended SL2 comprises an additional 3-12 nucleotides in length compared to a native SL2. In some embodiments the extended SL2 comprises an additional 4-30, 4-20, 4-16 or 4-12 nucleotides in length compared to a native SL2. In some embodiments the extended SL2 comprises an additional 4-12 nucleotides in length compared to a native SL2. In some embodiments the extended SL2 comprises an additional 9-30, 9-20, 9-16 or 9-12 nucleotides in length compared to a native SL2.
  • the extended SL2 comprises an additional 9-12 nucleotides in length compared to a native SL2.
  • a “native SL2” is a SL2 sequence that derives from a wild type gRNA core sequence.
  • a native SL2 is a sequence of 5’-ACUUGAAAAAGU-3’ (SEQ ID NO.: 23).
  • the number of additional nucleotides of the extension is determined by the chemical moieties and the linker therebetween present in the gRNA core of the PEgRNA. For example, larger and/or bulkier chemical moieties and/or linkers may necessitate a longer extension to ensure they are located sufficiently far away from the interactions between the PEgRNA and the Cas9 protein.
  • the extended TL or SL2 where the chemical moiety or linker is located comprises an additional 10, 11, 12, 13 or 14 nucleotides in length when the linker comprises a thioether linkage.
  • the extended TL or SL2 where the chemical moiety or linker is located comprises an additional 10, 11, 12, 13 or 14 nucleotides in length when the linker comprises a bisamide linkage. In some embodiments the extended TL or SL2 where the chemical moiety or linker is located comprises an additional 10, 11, 12, 13 or 14 nucleotides in length when the linker comprises a triazole linkage.
  • the split synthesis methods described herein form a full-length PEgRNA by joining two chemical moieties present at the 3’ end and/or 5’ ends of the RNA sequences.
  • the first RNA sequence comprises a first chemical moiety at its 3’ end.
  • the second RNA sequence comprises a second chemical moiety at its 5’ end. The first RNA sequence and the second RNA sequence are ligated via a chemical ligation (chemical reaction).
  • the first chemical moiety and the second chemical moiety react with each other or with a reagent thereby forming a linker between the two.
  • the first chemical moiety of the first RNA sequence comprises amino, –X1–NH2, –X1–OH, –X1–PO4-, –O(CO)X3, or a structure of any one of Formulas 1-6 or Formulas 9-11:
  • each X 1 and X 4 is independently absent or (O–CH 2 –CH 2 )n, (CH 2 –CH 2 –O)n, ((CH 2 )n-C(O)NH-CH 2 ), or an alkylene optionally substituted with one or more –OH or –O–, or –O–, “n” is an integer between 1 and 12; R 1 is halogen.
  • X 1 is absent.
  • X 1 is (O–CH 2 –CH 2 )n.
  • X1 is (CH 2 –CH 2 –O)n.
  • X1 is ((CH 2 )n-C(O)NH- CH 2 ).
  • X 1 is an alkylene. In some embodiments, X 1 is a C 1 -C 10 unsubstituted alkylene. In some embodiments, X1 is a C1-C6 alkylene. In some embodiments, X 1 is –CH 2 –. In some embodiments, X 1 is –CH 2 –CH 2 –. In some embodiments, X 1 is a C 6 unsubstituted alkylene. In some embodiments, X1 is a C1-C10 substituted alkylene with an – OH. In some embodiments, X 1 is a C 1 -C 10 substituted alkylene with an –O–.
  • X1 is a C1-C10 substituted alkylene with an –OH and an –O–. In some embodiments, X 1 is–O–. [192] In some embodiments, X4 is absent. In some embodiments, X4 is (O–CH 2 –CH 2 )n. In some embodiments, X 4 is (CH 2 –CH 2 –O)n. In some embodiments, X 4 is ((CH 2 )n-C(O)NH- CH 2 ). In some embodiments, X4 is an alkylene. In some embodiments, X4 is a C1-C10 unsubstituted alkylene. In some embodiments, X 4 is a C 1 -C 6 alkylene.
  • X4 is –CH 2 –. In some embodiments, X4 is –CH 2 –CH 2 –. In some embodiments, X4 is a C1-C10 substituted alkylene with an —OH. In some embodiments, X4 is a C1-C10 substituted alkylene with an –O–. In some embodiments, X4 is a C1-C10 substituted alkylene with an –OH and an –O–. In some embodiments, X4 is–O–. [193] In some embodiments “n” is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. [194] In some embodiments, X3 is 4-nitrophenoxy or imidazolyl.
  • X3 In some embodiments, X3 is a 4-nitrophenyl derivative.
  • R 1 is Cl. In some embodiments, R 1 is Br. In some embodiments, R 1 is I.
  • the first chemical moiety of the first RNA sequence comprises an amino. In some embodiments, the first chemical moiety of the first RNA sequence comprises –X 1 –NH 2 . In some embodiments, the first chemical moiety of the first RNA sequence comprises –X 1 –OH. In some embodiments, the first chemical moiety of the first RNA sequence comprises –X1–PO4. In some embodiments, the first chemical moiety of the first RNA sequence comprises –O(CO)X 3 .
  • the first chemical moiety of the first RNA sequence comprises a structure of Formula 3.
  • X 4 is an alkylene.
  • X4 is absent.
  • the first chemical moiety of the first RNA sequence comprises a structure of Formula 4.
  • X4 is an alkylene.
  • X 4 is absent.
  • the structure of Formula 4 is [202]
  • the first chemical moiety of the first RNA sequence comprises a structure of Formula 5.
  • X4 is an alkylene.
  • the first chemical moiety of the second RNA sequence comprises a structure of Formula 6.
  • X 5 is an alkylene.
  • X5 is –CH 2 –.
  • the second chemical moiety of the second RNA sequence is [204]
  • the first chemical moiety of the first RNA sequence comprises a structure of Formula 9.
  • X 4 is an alkylene.
  • X4 is absent.
  • the first chemical moiety of the first RNA sequence comprises a structure of Formula 10.
  • X4 is an alkylene. In some embodiments, in the structure of Formula 10, X 4 is absent.
  • the first chemical moiety of the first RNA sequence comprises a structure of Formula 11. In some embodiments, in the structure of Formula 11, X 4 is an alkylene. In some embodiments, in the structure of Formula 11, X4 is absent.
  • the second chemical moiety of the second RNA sequence comprises an amino, –X 2 –NH 2, –X 2 –OH, –X 2 –PO 4 -, –O(CO)X 3 , or a structure of any one of Formulas 6-8 or Formula 12:
  • each X2 and X5 is independently absent or (O-CH 2 -CH 2 )n, (CH 2 -CH 2 -O)n, ((CH 2 )n-C(O)NH-CH 2 ), or an alkylene optionally substituted with one or more –OH or –O–, or –O–, “n” is an integer between 1 and 12; R 2 is a heteroaryl.
  • X2 is absent.
  • X2 is (O–CH 2 –CH 2 )n.
  • X 2 is (CH 2 –CH 2 –O)n.
  • X 2 is ((CH 2 )n-C(O)NH- CH 2 ).
  • X2 is an alkylene. In some embodiments, X2 is a C1-C10 unsubstituted alkylene. In some embodiments, X 2 is a C 1 -C 6 alkylene. In some embodiments, X2 is –CH 2 –. In some embodiments, X2 is –CH 2 –CH 2 –. In some embodiments, X2 is a C1-C10 substituted alkylene with an —OH. In some embodiments, X 2 is a C 1 -C 10 substituted alkylene with an –O–. In some embodiments, X2 is a C1-C10 substituted alkylene with an –OH and an –O–.
  • X2 is –O–.
  • X5 is absent.
  • X5 is (O–CH 2 –CH 2 )n.
  • X5 is (CH 2 –CH 2 –O)n.
  • X5 is ((CH 2 )n-C(O)NH- CH 2 ).
  • X5 is an alkylene.
  • X5 is C1-C10 unsubstituted alkylene.
  • X5 is a C1-C6 alkylene.
  • X 5 is –CH 2 –.
  • X 5 is –CH 2 –CH 2 –. In some embodiments, X 5 is a C 1 -C 10 substituted alkylene with an –OH and an –O–. In some embodiments, X5 is –O–. [210] In some embodiments “n” is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. [211] In some embodiments, X3 is 4-nitrophenoxy or imidazolyl. In some embodiments, X3 In some embodiments, X3 is a 4-nitrophenyl derivative. [212] In some embodiments, R 2 is pyridine. In some embodiments, R 2 is pyrimidine.
  • the second chemical moiety of the second RNA sequence comprises an amino.
  • the second chemical moiety of the second RNA sequence comprises a structure of Formula 6.
  • X5 is an alkylene.
  • X 5 is –CH 2 –.
  • the second chemical moiety of the second RNA sequence is [215]
  • the second chemical moiety of the second RNA sequence comprises a structure of Formula 7.
  • X 5 is an alkylene.
  • X5 is –CH 2 –.
  • the first chemical moiety of the first RNA sequence comprises a structure of Formula 1, and the second chemical moiety of the second RNA sequence comprises an amino.
  • the first chemical moiety of the first RNA sequence comprises a structure of Formula 2
  • the second chemical moiety of the second RNA sequence comprises a structure of Formula 6.
  • the first chemical moiety of the first RNA sequence comprises a structure of Formula 3
  • the second chemical moiety of the second RNA sequence comprises an amino.
  • the first chemical moiety of the first RNA sequence comprises a structure of Formula 4
  • the second chemical moiety of the second RNA sequence comprises a structure of Formula 7.
  • the first chemical moiety of the first RNA sequence comprises a structure of Formula 5, and the second chemical moiety of the second RNA sequence comprises a structure of Formula 6.
  • the first chemical moiety of the first RNA sequence comprises a structure of Formula 2
  • the second chemical moiety of the second RNA sequence comprises a structure of Formula 8.
  • the first chemical moiety of the first RNA sequence comprises a structure of Formula 6, and the second chemical moiety of the second RNA sequence comprises a structure of Formula 6.
  • the first chemical moiety of the first RNA sequence comprises a structure of Formula 9, and the second chemical moiety of the second RNA sequence comprises a structure of Formula 7.
  • the first chemical moiety of the first RNA sequence comprises a structure of Formula 2
  • the second chemical moiety of the second RNA sequence comprises a structure of Formula 8.
  • the first chemical moiety of the first RNA sequence comprises a structure of Formula 10
  • the second chemical moiety of the second RNA sequence comprises a structure of Formula 12.
  • the first chemical moiety of the first RNA sequence comprises a structure of Formula 10
  • the second chemical moiety of the second RNA sequence comprises a structure of Formula 6.
  • the first chemical moiety of the first RNA sequence comprises –X1–NH2
  • the second chemical moiety of the second RNA sequence comprises –X2–NH2.
  • the first chemical moiety of the first RNA sequence comprises –X1–OH
  • the second chemical moiety of the second RNA sequence comprises –X2– PO4-.
  • the first chemical moiety of the first RNA sequence comprises an amino
  • the second chemical moiety of the second RNA sequence comprises – O(CO)X3.
  • the first chemical moiety of the first RNA sequence comprises –O(CO)X 3
  • the second chemical moiety of the second RNA sequence comprises an amino
  • the first chemical moiety of the first RNA sequence is located on the 3’ end of the US4, US5, US6, US7, or US8 nucleotide of the tetraloop.
  • the first chemical moiety of the first RNA sequence is located on the 3’ end of the US4 nucleotide of the tetraloop. In some embodiments, the first chemical moiety of the first RNA sequence is located on the 3’ end of the US5 nucleotide of the tetraloop. In some embodiments, the first chemical moiety of the first RNA sequence is located on the 3’ end of the US6 nucleotide of the tetraloop. In some embodiments, the first chemical moiety of the first RNA sequence is located on the 3’ end of the US7 nucleotide of the tetraloop.
  • the first chemical moiety of the first RNA sequence is located on the 3’ end of the US8 nucleotide of the tetraloop. [234] In some embodiments, the first chemical moiety of the first RNA sequence is located on the 3’ end of the SL2-4, SL2-5, SL2-6, SL2-7, or SL2-8 nucleotide of the SL2. In some embodiments, the first chemical moiety of the first RNA sequence is located on the 3’ end of the SL2-4 nucleotide of the SL2. In some embodiments, the first chemical moiety of the first RNA sequence is located on the 3’ end of the SL2-5 nucleotide of the SL2.
  • the first chemical moiety of the first RNA sequence is located on the 3’ end of the SL2-6 nucleotide of the SL2. In some embodiments, the first chemical moiety of the first RNA sequence is located on the 3’ end of the SL2-7 nucleotide of the SL2. In some embodiments, the first chemical moiety of the first RNA sequence is located on the 3’ end of the SL2-8 nucleotide of the SL2. [235] In some embodiments, the second chemical moiety of the second RNA sequence is located on the 5’ end of the US5, US6, US7, US8, or US9 nucleotide of the tetraloop.
  • the second chemical moiety of the second RNA sequence is located on the 5’ end of the US5 nucleotide of the tetraloop. In some embodiments, the second chemical moiety of the second RNA sequence is located on the 5’ end of the US6 nucleotide of the tetraloop. In some embodiments, the second chemical moiety of the second RNA sequence is located on the 5’ end of the US7 nucleotide of the tetraloop. In some embodiments, the second chemical moiety of the second RNA sequence is located on the 5’ end of the US8 nucleotide of the tetraloop.
  • the second chemical moiety of the second RNA sequence is located on the 5’ end of the SL2-7 nucleotide of the SL2. In some embodiments, the second chemical moiety of the second RNA sequence is located on the 5’ end of the SL2-8 nucleotide of the SL2. In some embodiments, the second chemical moiety of the second RNA sequence is located on the 5’ end of the SL2-9 nucleotide of the SL2. [237] In some embodiments, the method further comprises a third RNA sequence comprising a third chemical moiety at its 5’ end and a fourth chemical moiety at its 3’ end.
  • the third chemical moiety of the third RNA sequence comprises an amino, –X2–NH2, –X2–OH, –X2–PO4-, –O(CO)X3, or a structure of any one of Formulas 6-8 or Formula 12: wherein: each X 2 and X 5 is independently absent or (O-CH 2 -CH 2 )n, (CH 2 -CH 2 -O)n, ((CH 2 )n-C(O)NH-CH 2 ), or an alkylene optionally substituted with one or more –OH or –O–, or-O-, “n” is an integer between 1 and 12; R 2 is a heteroaryl.
  • the second linker is present in the chemically-modified gRNA core of the synthesized full-length PEgRNA. [243] In some embodiments, the second linker is present in the junction of the synthesized full-length PEgRNA. [244] In some embodiments, the synthesized full-length PEgRNA comprises, in 5’ to 3’ order, a spacer, a chemically-modified gRNA core, a junction, and an extension arm. [245] In some embodiments, the extension arm comprises a reverse transcription template (RTT) and a primer binding site (PBS). In some embodiments, the extension arm is a chemically-modified extension arm.
  • RTT reverse transcription template
  • PBS primer binding site
  • the first portion of the chemically-modified gRNA core comprises a fragmented tetraloop.
  • each chemical moiety is a functional group appended to the 3’ end and/or the 5’ end of a polynucleotide.
  • the chemical moiety is structurally different from a natural nucleotide, a natural sugar, and/or a natural internucleoside linkage.
  • the second RNA sequence comprises a second portion of the chemically-modified gRNA core, an extension arm, a chemically-modified extension arm, an RTT, and/or a PBS.
  • the second RNA sequence comprises a second portion of the chemically-modified gRNA core and an extension arm. In some embodiments, the second portion of the chemically-modified gRNA core comprises a fragmented SL2. In some embodiments, the second RNA sequence comprises a chemically-modified extension arm. [250] In some embodiments, the third RNA sequence comprises a third portion of the chemically-modified gRNA core, a fourth portion of the chemically-modified gRNA core, and/or a junction. In some embodiments, the third RNA sequence comprises a third portion of the chemically-modified gRNA core and a fourth portion of the chemically-modified gRNA core.
  • the first chemical moiety of the first RNA sequence is located on the 3’ end of the US4 nucleotide of the tetraloop. In some embodiments, the first chemical moiety of the first RNA sequence is located on the 3’ end of the US5 nucleotide of the tetraloop. In some embodiments, the first chemical moiety of the first RNA sequence is located on the 3’ end of the US6 nucleotide of the tetraloop. In some embodiments, the first chemical moiety of the first RNA sequence is located on the 3’ end of the US7 nucleotide of the tetraloop.
  • the third chemical moiety of the third RNA sequence is located on the 5’ end of the US7 nucleotide of the tetraloop. In some embodiments, the v chemical moiety of the third RNA sequence is located on the 5’ end of the US8 nucleotide of the tetraloop. In some embodiments, the third chemical moiety of the third RNA sequence is located on the 5’ end of the US9 nucleotide of the tetraloop. [253] In some embodiments, the second chemical moiety of the second RNA sequence is located on the 5’ end of the SL2-5, SL2-6, SL2-7, SL2-8, or SL2-9 nucleotide of the SL2.
  • the second chemical moiety of the second RNA sequence is located on the 5’ end of the SL2-5 nucleotide of the SL2. In some embodiments, the second chemical moiety of the second RNA sequence is located on the 5’ end of the SL2-6 nucleotide of the SL2. In some embodiments, the second chemical moiety of the second RNA sequence is located on the 5’ end of the SL2-7 nucleotide of the SL2. In some embodiments, the second chemical moiety of the second RNA sequence is located on the 5’ end of the SL2-8 nucleotide of the SL2.
  • the first chemical moiety of the first RNA sequence and the fourth chemical moiety of the third RNA sequence each comprise: an amino, –X1–NH2, –X1–OH, –X1–PO4-, –O(CO)X3, or a structure of any one of Formulas 1-6 or Formulas 9-11: wherein: each X 1 and X 4 is independently absent or (O–CH 2 –CH 2 )n, (CH 2 –CH 2 –O)n, ((CH 2 )n-C(O)NH-CH 2 ), or an alkylene optionally substituted with one or more –OH or –O–, or –O–, “n” is an integer between 1 and 12; R 1 is halogen.
  • the first chemical moiety of the first RNA sequence and/or the fourth chemical moiety of the third RNA sequence comprises —O(CO)X3.
  • the first chemical moiety of the first RNA sequence and/or the fourth chemical moiety of the third RNA sequence comprises a structure of Formula 1.
  • X4 is an alkylene.
  • X 4 is–CH 2 –CH 2 –.
  • the first chemical moiety of the first RNA sequence and/or the fourth chemical moiety of the third RNA sequence comprises a structure of Formula 2.
  • R 1 is Cl, Br, or I. In some embodiments, R 1 is Br.
  • R 1 is I.
  • the structure of Formula 2 is [262]
  • the second chemical moiety of the second RNA sequence and/or the third chemical moiety of the third RNA sequence comprises an amino.
  • the second chemical moiety of the second RNA sequence and/or the third chemical moiety of the third RNA sequence comprises a structure of Formula 6.
  • X5 is an alkylene.
  • X5 is –CH 2 –.
  • the second chemical moiety of the second RNA sequence and/or the third chemical moiety of the third RNA sequence is [264] In some embodiments, the second chemical moiety of the second RNA sequence and/or the third chemical moiety of the third RNA sequence comprises a structure of Formula 7. In some embodiments, in the structure of Formula 7, X 5 is an alkylene. In some embodiments, in the structure of Formula 7, X5 is –CH 2 –. [265] In some embodiments, the second chemical moiety of the second RNA sequence and/or the third chemical moiety of the third RNA sequence comprises a structure of Formula 8. In some embodiments, in the structure of Formula 8, X5 is an alkylene.
  • X5 is –CH 2 –.
  • the second chemical moiety of the second RNA sequence is and/or the third chemical moiety of the third RNA sequence .
  • the first chemical moiety of the first RNA sequence and the fourth chemical moiety of the third RNA sequence comprises an amino.
  • the first chemical moiety of the first RNA sequence and the fourth chemical moiety of the third RNA sequence comprises –X1–NH2.
  • the second chemical moiety of the second RNA sequence and the third chemical moiety of the third RNA sequence comprises a structure of Formula 6.
  • X5 is an alkylene.
  • X5 is – CH 2 –.
  • the second chemical moiety of the second RNA sequence and the third chemical moiety of the third RNA sequence is Ligation [267]
  • Enzymatic ligation or chemical ligation can be utilized to ligate the chemical moieties and produce the long RNAs described herein.
  • enzymatic ligation is used to ligate the chemical moieties.
  • chemical ligation is used to ligate the chemical moieties.
  • both enzymatic ligation and chemical ligation are used to ligate the chemical moieties.
  • Chemical Ligation is used to ligate the chemical moieties.
  • appropriate ligation chemistries can be designed.
  • the chemical moieties described herein can incorporate functional groups that are specific to particular ligation chemistries.
  • Scheme 1 Some examples of chemistries suitable for chemical ligations in the split synthesis methods described herein are shown in Scheme 1 below:
  • Another exemplary chemistry for use in the split synthesis methods described herein includes the use of a thioether ligation as shown in Schemes 2 and 3 below.
  • the thioether ligation strategy uses a thiol modified RNA sequence to react with the bromine containing RNA sequence to form a thioether linker. This strategy does not require the two RNA sequences to be pre-annealed.
  • the split site can be in a native TL or SL2 without any extension. In some embodiments, this strategy is referred to as a non- templated strategy.
  • Another exemplary chemistry for use in the split synthesis methods described herein includes the use of a bisamide ligation as shown in Scheme 4 below.
  • the bisamide strategy uses one molecule of BS3 (bis(sulfosuccinimidyl)suberate) with two NHS ester groups to link two amino modified RNA sequences through two amide bond formations on a single BS3 molecule (Scheme 4). This requires the two RNA sequences to be pre-annealed as the two amino groups need to be positioned in proximity before adding the BS3 reagent for the ligation.
  • the fragmented gRNA core comprises an extended TL or SL2.
  • Another exemplary chemistry includes the cycloaddition reaction between two RNA sequences carrying an azide and alkyne functional groups at the respective ends. The reaction is driven by the ring strain of alkyne functional group in the cyclic ring instead of traditional alkyne activation by copper.
  • an azide is either partnered with A) DBCO-alkyne or B) BCN-alkyne (Scheme 4.1).
  • RNAs can be treated with the activated NHS-esters of azide or alkyne partners (Scheme 4.1.1). Additionally, azide functional groups can be introduced on the RNAs on the nucleotides directly at the 5’-end of the last nucleotide in the position of 5’- OH or as a modification on the purine or pyrimidine bases without requiring using the amine handle and NHS-esters.
  • Another exemplary chemistry includes bismaleimide ligation with native stem loops using a splint.
  • two thiol containing RNAs are ligated together using BMX reagent, where X is a variable in length and nature of the linker itself as shown in Scheme 4.2.
  • the amine functionalized RNAs can be treated with succinimidyl 3-(2- pyridyldithio)propionate, SPDP, reagent followed by TCEP-mediated reduction to produce the thiolated RNA (Scheme 4.2.1).
  • the splint ligation occurs between and within the gRNA core and the RTT of the PEgRNA. In some embodiments, the splint ligation occurs between and within the RTT and the PBS of the PEgRNA. [296] In some embodiments, splint ligation requires a minimum of two fragments (2-part ligation) and can be increased further to three fragments (3-part ligation), four fragments (4- part ligation), five fragments (5-part ligation), six fragments (6-part ligation), seven fragments (7-part ligation), eight fragments (8-part ligation), nine fragments (9-part ligation), or ten fragments (10-part ligation).
  • Another aspect of the disclosure provides a method for synthesizing a full-length prime editing guide RNA (PEgRNA) comprising a guide RNA (gRNA) core, the method comprising, providing a first RNA sequence, wherein the first RNA sequence comprises a first chemical moiety at its 3’ end; providing a second RNA sequence, wherein the second RNA sequence comprises a second chemical moiety at its 5’ end and a free 3’ OH; providing a third RNA sequence, wherein the third RNA sequence comprises a 5’- monophosphate; ligating the first, second, and third RNA sequences to obtain the synthesized full- length PEgRNA, wherein the ligation forms (i) a first linker between the first chemical moiety of the first RNA sequence and the second chemical moiety of the second RNA sequence, and (ii) a second linker between the 3’
  • Linker [307] The split synthesis methods described herein form a full-length PEgRNA that comprises a linker.
  • the linker derived from the joining of the first chemical moiety of the first RNA sequence and the second chemical moiety of the second RNA sequence.
  • the linker is present in the tetraloop or SL2 of the gRNA core. In some embodiments, the linker is present in the tetraloop or SL2 of the modified gRNA core. In some embodiments, the linker is present in the tetraloop or SL2 of the chemically- modified gRNA core. In some embodiments, the linker is present in the tetraloop of the chemically-modified gRNA core.
  • the linker is present in the SL2 of the chemically-modified gRNA core. In some embodiments, the linker is a chemical linker. [309] In some embodiments, the linker is present between the US4 and US9 nucleotides of the tetraloop. In some embodiments, the linker is present between the US4 and US5 nucleotides of the tetraloop. In some embodiments, the linker is present between the US5 and US6 nucleotides of the tetraloop. In some embodiments, the linker is present between the US6 and US7 nucleotides of the tetraloop.
  • the linker is present between the US7 and US8 nucleotides of the tetraloop. In some embodiments, the linker is present between the US8 and US9 nucleotides of the tetraloop. [310] In some embodiments, the linker is present between the SL2-4 and SL2-9 nucleotides of the SL2. In some embodiments, the linker is present between the SL2-4 and SL2-5 nucleotides of the SL2. In some embodiments, the linker is present between the SL2-5 and SL2-6 nucleotides of the SL2. In some embodiments, the linker is present between the SL2-6 and SL2-7 nucleotides of the SL2.
  • Another aspect of the disclosure includes a method for synthesizing a full-length prime editing guide RNA (PEgRNA) comprising a chemically-modified guide RNA (gRNA) core, the method comprising, providing a first RNA sequence, wherein the first RNA sequence comprises an amino modified phosphodiester backbone at its 3’end; providing a second RNA sequence; wherein the second RNA sequence comprises an amino modified phosphodiester backbone at its 5’end; annealing the first and second RNA sequences to form a pre-annealed duplex; providing a reagent; contacting the pre-annealed duplex with the reagent to form a linker, wherein forming the linker results in the formation of the synthesized full-length PEgRNA; and wherein the linker is present in the chemically-modified gRNA core of the synthesized full-length PEgRNA.
  • PEgRNA prime editing guide RNA
  • gRNA chemically-modified guide RNA
  • Another aspect of the disclosure includes an RNA sequence of Formula R: wherein X 1 is absent or (O-CH 2 -CH 2 )n, (CH 2 -CH 2 -O)n, ((CH 2 )n-C(O)NH-CH 2 ), or an alkylene optionally substituted with one or more –OH or –O–, or –O–; and “n” is an integer between 1 and 12.
  • Another aspect of the disclosure includes an RNA sequence of Formula U: wherein X 2 is absent or (O-CH 2 -CH 2 )n, (CH 2 -CH 2 -O)n, ((CH 2 )n-C(O)NH-CH 2 ), or an alkylene optionally substituted with one or more –OH or –O–, or –O–; and “n” is an integer between 1 and 12.
  • Another aspect of the disclosure includes an RNA sequence of Formula Y: ; wherein X 2 is absent or (O-CH 2 -CH 2 )n, (CH 2 -CH 2 -O)n, ((CH 2 )n-C(O)NH-CH 2 ), or an alkylene optionally substituted with one or more –OH or –O–, or –O–; and “n” is an integer between 1 and 12.
  • the synthetic RNA or synthesized full-length PEgRNA is at least 85 nucleotides, at least 90 nucleotides, at least 95 nucleotides, at least 100 nucleotides, at least 110 nucleotides, at least 120 nucleotides, at least 130 nucleotides, at least 140 nucleotides, at least 150 nucleotides, at least 160 nucleotides, at least 170 nucleotides, at least 180 nucleotides, at least 190 nucleotides, at least 200 nucleotides, at least 210 nucleotides, at least 220 nucleotides, at least 230 nucleotides, at least 240 nucleotides, at least 250 nucleotides, at least 275 nucleotides, at least 300 nucleotides, at least 325 nucleotides, at least 350 nucleotides, at least 375 nucleotides
  • the first RNA sequence is SEQ ID NO.: 1, 3, 5, 7, 9, 19, 24, 30, 60, 62, 66, 69, 74, 89, 91, 93, or 112.
  • the second RNA sequence is SEQ ID NO.: 2, 4, 6, 8, 10, 20, 25, 31, 61, 63, 67, 70, 73, 75, 90, 108, or 121.
  • the synthesized full-length PEgRNA is SEQ ID NO.: 17, 18, 21, 26, 32, 64, 65, 68, 72, 77-88, 97-102, 104-106, 109-111, 114-116, 118-120, or 122-123.
  • the synthesized full-length PEgRNA is SEQ ID NO.: 17, 18, 21, 64, 65, 68, 72, 77-88, 97-102, 104-106, 109-111, 114- 116, 118-120, or 122-123.
  • the first RNA sequence is SEQ ID NO.: 89, 91, or 93
  • the second RNA sequence is SEQ ID NO.: 90
  • the third RNA sequence is SEQ ID NO.: 92 or 94.
  • the first RNA sequence is SEQ ID NO.: 69
  • the second RNA sequence is SEQ ID NO.: 75
  • the third RNA sequence is SEQ ID NO.: 103.
  • the first RNA sequence is SEQ ID NO.: 112
  • the second RNA sequence is SEQ ID NO.: 108
  • the third RNA sequence is SEQ ID NO.: 117.
  • the first RNA sequence is SEQ ID NO.: 24 or 30.
  • the second RNA sequence is SEQ ID NO.: 25 or 31.
  • the synthesized full-length PEgRNA is SEQ ID NO.: 26 or 32.
  • the linker of the full-length PEgRNA does not affect CRISPR associated (Cas) protein binding and/or prime editor binding. In some embodiments, the linker of the chemically-modified PEgRNA does not affect CRISPR associated (Cas) protein binding and/or prime editor.
  • long RNAs such as a full-length PEgRNA, are prepared using the chemical ligation methods described herein. In some embodiments, long RNAs, such as a full-length PEgRNA, are prepared using the enzymatic ligation methods described herein.
  • long RNAs such as a full-length PEgRNA
  • long RNAs are prepared using a combination of the chemical ligation and enzymatic ligation methods described herein.
  • the gRNA core can be prepared with any of the chemical ligation methods described herein.
  • the rest of the PEgRNA elements e.g., spacer, RTT, and PBS
  • the chemically-ligated gRNA core can then be incorporated to the other elements via the enzymatic ligation methods described herein.
  • the chemically-ligated gRNA core can also be incorporated to the other elements via other methods known to a person of ordinary skill in the art.
  • the gRNA core when preparing a full-length PEgRNA, can be prepared with a 3-part chemical ligation, as shown in FIG.4A and FIG.4B, and as described above.
  • the rest of the PEgRNA elements e.g., spacer, RTT, and PBS
  • the chemically-ligated gRNA core can then be incorporated to the other elements via the enzymatic ligation methods described herein.
  • the chemically-ligated gRNA core can also be incorporated to the other elements via other methods known to a person of ordinary skill in the art.
  • any combination of chemical and enzymatic ligation can be utilized to prepare the full-length PEgRNA and it is not limited to the examples provided above.
  • a combination of one chemical ligation and one enzymatic ligation are utilized to prepare the full-length PEgRNA.
  • a combination of two chemical ligations and two enzymatic ligations are utilized to prepare the full-length PEgRNA.
  • a combination of three chemical ligations and three enzymatic ligations are utilized to prepare the full-length PEgRNA.
  • a combination of one chemical ligation and two enzymatic ligations are utilized to prepare the full-length PEgRNA.
  • a combination of one chemical ligation and three enzymatic ligations are utilized to prepare the full-length PEgRNA.
  • a combination of two chemical ligations and one enzymatic ligation are utilized to prepare the full-length PEgRNA.
  • a combination of three chemical ligations and one enzymatic ligation are utilized to prepare the full-length PEgRNA.
  • a combination of three chemical ligations and two enzymatic ligations are utilized to prepare the full-length PEgRNA.
  • the PEgRNA synthesized by the methods described herein comprises a tag sequence in addition to the other elements, e.g., spacer, gRNA core (e.g., chemically-modified gRNA core), primer binding site, and editing template.
  • the tag sequence comprises a region of complementarity to the editing template.
  • the tag sequence comprises a region of complementarity to the PBS.
  • the tag sequence comprises a region of complementarity to the editing template and/or the PBS.
  • the tag sequence comprises a region of complementarity to the editing template and does not have substantial complementarity to the PBS.
  • the tag sequence comprises a region of complementarity to the editing template and does not have complementarity to the PBS. In some embodiments, the tag sequence does not have perfect complementarity with the PBS. In some embodiments, the tag sequence does not have perfect complimentarity with the gRNA core. In some embodiments, the tag sequence and the editing template each comprises a region of complementarity to each other, wherein the 3’ end of the region of complementarity in the editing template is at a position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more bases 5' of the 3' half of the editing template. In some embodiments, the region of complementarity in the tag sequence is at a 5’ portion of the tag sequence.
  • the tag sequence does not have substantial complementarity to the spacer. In some embodiments, the tag does not have complementarity to the spacer. In some embodiments, the tag sequence does not have perfect complementarity to the spacer. [390] In some embodiments, the tag sequence is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 nucleotides in length. In some embodiments, the tag sequence is at least 4, at least 6, at least 8 nucleotides in length. In some embodiments, the tag sequence is from 4 to 22, 4 to 10, or 6 to 8 nucleotides in length.
  • the tag sequence comprises a nucleic acid sequence selected from any nucleic acid sequence disclosed in SEQ ID NOs 62-1960 of PCT Publication No.: WO 2023/096977.
  • the PEgRNA synthesized by the methods described herein comprises a motif at the 3’ end of the PBS in addition to the other elements, e.g., spacer, gRNA core (e.g., chemically-modified gRNA core), primer binding site, and editing template.
  • pseudoknot is derived form a potato roll leaf virus (PLRV)), or any combination thereof.
  • the one or more nucleic acid moieties comprises a hairpin.
  • the hairpin comprises a sequence of any one of SEQ ID NOs: 42-44 or 46-48.
  • the one or more nucleic acid moieties comprises a pseudoknot.
  • the pseudoknot is derived from potato roll-leaf virus.
  • the pseudoknot comprises the sequence of SEQ ID NO.: 45.
  • the one or more nucleic acid moieties comprises a MS2 hairpin.
  • the quadruplex comprises the sequence of SEQ ID NO.: 51 or 52.
  • the one or more nucleic acid moieties comprise a structure derived form a replication recognition sequence of a retrovirus.
  • the nucleic acid moiety comprises a sequence derived from a replication recognition sequence of a Moloney Murine leukemia virus (MMLV).
  • the one or more nucleic acid moieties comprise a nucleic acid sequence selected from SEQ ID Nos: 53-56. [398] Table A.
  • the prime editor comprises additional polypeptides or polypeptide domains involved in prime editing, for example, a polypeptide domain having 5’ endonuclease activity, e.g., a 5' endogenous DNA flap endonucleases (e.g., FEN1), for helping to drive the prime editing process towards the edited product formation.
  • the prime editor further comprises an RNA-protein recruitment polypeptide, for example, a MS2 coat protein.
  • a prime editor may be engineered.
  • the polypeptide components of a prime editor do not naturally occur in the same organism or cellular environment.
  • the polypeptide components of a prime editor may be of different origins or from different organisms.
  • a prime editor comprises a DNA binding domain and a DNA polymerase domain that are derived from different species.
  • a prime editor comprises a Cas polypeptide and a reverse transcriptase polypeptide that are derived from different species.
  • a prime editor may comprise a S. pyogenes Cas9 polypeptide and a Moloney murine leukemia virus (M- MLV) reverse transcriptase polypeptide.
  • M- MLV Moloney murine leukemia virus
  • polypeptide domains of a prime editor may be fused or linked by a peptide linker to form a fusion protein.
  • a prime editor comprises one or more polypeptide domains provided in trans as separate proteins, which are capable of being associated to each other through non-peptide linkages or through aptamers or recruitment sequences.
  • a prime editor may comprise a DNA binding domain and a reverse transcriptase domain associated with each other by an RNA-protein recruitment aptamer, e.g., an MS2 aptamer, which may be linked to a PEgRNA.
  • Prime editor polypeptide components may be encoded by one or more polynucleotides in whole or in part.
  • a single polynucleotide, construct, or vector encodes the prime editor fusion protein.
  • multiple polynucleotides, constructs, or vectors each encode a polypeptide domain or portion of a domain of a prime editor, or a portion of a prime editor fusion protein.
  • a prime editor fusion protein may comprise an N-terminal portion fused to an intein-N and a C-terminal portion fused to an intein-C, each of which is individually encoded by an AAV vector.
  • Flap Endonuclease [404]
  • a prime editor further comprises additional polypeptide components, for example, a flap endonuclease (FEN, e.g.,FEN1).
  • FEN flap endonuclease
  • the flap endonuclease excises the 5’ single stranded DNA of the edit strand of the target gene and assists incorporation of the intended nucleotide edit into the target gene.
  • the FEN is linked or fused to another component.
  • the FEN is provided in trans, for example, as a separate polypeptide or polynucleotide encoding the FEN.
  • a prime editor or prime editing composition comprises a flap nuclease.
  • the flap nuclease is a FEN1, or any FEN1 functional variant, functional mutant, or functional fragment thereof.
  • the flap nuclease is a TREX2, EXO1, or any other flap nuclease known in the art, or any functional variant, functional mutant, or functional fragment thereof.
  • the flap nuclease has an amino acid sequence that is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to any of the flap nucleases described herein or known in the art.
  • PEgRNA modular arrangement [407] Components of a PEgRNA may be arranged in a modular fashion.
  • the spacer, the primer binding site sequence (PBS) and the editing template can be interchangeably located in the 5’ portion of the PEgRNA, the 3’ portion of the PEgRNA, or in the middle of the gRNA core.
  • a PEgRNA comprises a PBS and an editing template sequence in 5’ to 3’ order.
  • the gRNA core of a PEgRNA of this disclosure may be located in between a spacer and an extension arm (i.e., the PBS and editing template) of the PEgRNA.
  • the gRNA core of a PEgRNA may be located at the 3’ end of a spacer.
  • a PEgRNA comprises, from 5’ to 3’: an extension arm, a spacer, and a gRNA core. In some embodiments, the PEgRNA comprises, from 5’ to 3’: an editing template a PBS, a spacer, and a gRNA core. In some embodiments, a first PEgRNA comprises a structure: 5 ⁇ -[first spacer]-[first gRNA core]-[first editing template]-[first primer binding site sequence]-3’. In some embodiments, a first PEgRNA comprises a structure: 5 ⁇ - [first editing template]-[first primer binding site sequence]- [first spacer]-[first gRNA core]-3’.
  • a second PEgRNA comprises a structure: 5 ⁇ -[second spacer]-[second gRNA core]-[second editing template]-[second primer binding site sequence]-3’. In some embodiments, a second PEgRNA comprises a structure: 5 ⁇ - [second editing template]-[second primer binding site sequence]- [second spacer]-[second gRNA core]-3’. [408] In some embodiments, a spacer sequence may further comprise additional nucleotides beside a region of complementarity to genomic search target sequence.
  • a spacer sequence (as well as the full PEgRNA sequence) may comprise an additional G at the 5’ end, for example, wherein the 5’ most nucleotide of the spacer (or the PEgRNA) is not a G.
  • the extension arm of a first PEgRNA may be partially complementary to the spacer of the first PEgRNA.
  • the editing template (e.g., RTT) of a first PEgRNA is partially complementary to the spacer of the first PEgRNA.
  • the editing template (e.g., RTT) and the primer binding site (PBS) of the first PEgRNA are each partially complementary to the spacer of the first PEgRNA.
  • the extension arm of a PEgRNA may comprise a primer binding site (PBS) and an editing template (e.g., an RTT).
  • the extension arm of a second PEgRNA may be partially complementary to the spacer of the second PEgRNA.
  • the editing template (e.g., RTT) of a second PEgRNA is partially complementary to the spacer of the second PEgRNA.
  • the editing template (e.g., RTT) and the primer binding site (PBS) of the second PEgRNA are each partially complementary to the spacer of the second PEgRNA.
  • the PBS is at least 4 nucleotides in length. In some embodiments, the PBS is at least 6 nucleotides in length. In some embodiments, the PBS is about 4 to 12 nucleotides, about 6 to 12 nucleotides, about 8 to 12 nucleotides, about 10 to 12 nucleotides, 4 to 14 nucleotides, about 6 to 14 nucleotides, about 8 to 14 nucleotides, about 10 to 14 nucleotides, about 12 to 14 nucleotides, 4 to 16 nucleotides, about 6 to 16 nucleotides, about 8 to 16 nucleotides, about 10 to 16 nucleotides, about 6 to 18 nucleotides, about 6 to 20 nucleotides, about 8 to 20 nucleotides, about 10 to 20 nucleotides, about 12 to 20 nucleotides, about 14 to 20 nucleotides, about 16 to 20 nucleotides, or about 18 to 20 nucleo
  • a PBS may initiate synthesis of a new single stranded DNA encoded by the editing template at the nick site.
  • a PBS is at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to a region of the edit strand of the target gene.
  • a PBS is perfectly complementary, or 100% complementary, to a region of the edit strand of the target gene.
  • Prime Editing Another aspect of the present disclosure includes a prime editing complex comprising: (i) the full-length PEgRNA synthesized by the methods described herein; and (ii) a prime editor comprising a DNA binding domain and a DNA polymerase domain.
  • the DNA binding domain is a CRISPR associated (Cas) protein domain.
  • the term “prime editing” refers to programmable editing of a target DNA using a prime editor complexed with a PEgRNA to incorporate an intended nucleotide edit into the target DNA through target-primed DNA synthesis.
  • a target polynucleotide e.g., a target gene of prime editing may comprise a double stranded DNA molecule having two complementary strands: a first strand that may be referred to as a “target strand” or a “non-edit strand”, and a second strand that may be referred to as a “non-target strand,” or an “edit strand.”
  • a spacer sequence is complementary or substantially complementary to a specific sequence on the target strand, which may be referred to as a “search target sequence”.
  • the spacer sequence anneals with the target strand at the search target sequence.
  • the target strand may also be referred to as the “non-Protospacer Adjacent Motif (non-PAM strand).”
  • the non- target strand may also be referred to as the “PAM strand”.
  • the PAM strand comprises a protospacer sequence and optionally a protospacer adjacent motif (PAM) sequence.
  • PAM sequence refers to a short DNA sequence immediately adjacent to the protospacer sequence on the PAM strand of the target gene.
  • a PAM sequence may be specifically recognized by a programmable DNA binding protein, e.g., a Cas nickase or a Cas nuclease
  • a specific PAM is characteristic of a specific programmable DNA binding protein, e.g., a Cas nickase or a Cas nuclease.
  • a protospacer sequence refers to a specific sequence in the PAM strand of the target gene that is complementary to the search target sequence.
  • a spacer sequence may have a substantially identical sequence as the protospacer sequence on the edit strand of a target gene, except that the spacer sequence may comprise Uracil (U) and the protospacer sequence may comprise Thymine (T).
  • the nick site is upstream of a specific PAM sequence on the PAM strand of the double stranded target DNA. In some embodiments, the nick site is downstream of a specific PAM sequence on the PAM strand of the double stranded target DNA. In some embodiments, the nick site is 3 base pairs upstream of the PAM sequence, and the PAM sequence is recognized by a Streptococcus pyogenes Cas9 nickase, a P. lavamentivorans Cas9 nickase, a C. diphtheriae Cas9 nickase, a N. cinerea Cas9, a S. aureus Cas9, or a N.
  • the nick site is 3 base pairs upstream of the PAM sequence, and the PAM sequence is recognized by a Cas9 nickase, wherein the Cas9 nickase comprises a nuclease active HNH domain and a nuclease inactive RuvC domain.
  • the nick site is 2 base pairs upstream of the PAM sequence, and the PAM sequence is recognized by a S. thermophilus Cas9 nickase.
  • a PEgRNA complexes with and directs a prime editor to bind to the search target sequence of the target gene.
  • the bound prime editor generates a nick on the edit strand (PAM strand) of the target gene at the nick site.
  • a primer binding site (PBS) of the PEgRNA anneals with a free 3 ⁇ end formed at the nick site, and the prime editor initiates DNA synthesis from the nick site, using the free 3 ⁇ end as a primer.
  • a single-stranded DNA encoded by the editing template of the PEgRNA is synthesized.
  • the newly synthesized single- stranded DNA comprises one or more intended nucleotide edits compared to the endogenous target gene sequence.
  • the editing template of a PEgRNA is complementary to a sequence in the edit strand except for one or more mismatches at the intended nucleotide edit positions in the editing template partially complementary to the editing template may be referred to as an “editing target sequence”.
  • the newly synthesized single stranded DNA has identity or substantial identity to a sequence in the editing target sequence, except for one or more insertions, deletions, or substitutions at the intended nucleotide edit positions.
  • the newly synthesized single-stranded DNA equilibrates with the editing target on the edit strand of the target gene for pairing with the target strand of the target gene.
  • the editing target sequence of the target gene is excised by a flap endonuclease (FEN), for example, FEN1.
  • FEN flap endonuclease
  • the FEN is an endogenous FEN, for example, in a cell comprising the target gene.
  • the FEN is provided as part of the prime editor, either linked to other components of the prime editor or provided in trans.
  • the newly synthesized single stranded DNA which comprises the intended nucleotide edit, replaces the endogenous single stranded editing target sequence on the edit strand of the target gene.
  • the newly synthesized single stranded DNA and the endogenous DNA on the target strand form a heteroduplex DNA structure at the region corresponding to the editing target sequence of the target gene.
  • the newly synthesized single-stranded DNA comprising the nucleotide edit is paired in the heteroduplex with the target strand of the target DNA that does not comprise the nucleotide edit, thereby creating a mismatch between the two otherwise complementary strands.
  • the mismatch is recognized by DNA repair machinery, e.g., an endogenous DNA repair machinery.
  • the intended nucleotide edit is incorporated into the target gene.
  • the Cas protein domain has nickase activity.
  • the Cas protein domain is a Cas9.
  • the Cas9 comprises a mutation in an HNH domain.
  • the Cas9 comprises a H840A mutation in the HNH domain.
  • the Cas protein domain is a Cas12b.
  • the Cas protein domain is a Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas14a, Cas14b, Cas14c, Cas14d, Cas14e, Cas14f, Cas14g, Cas14h, Cas14u, or a Cas ⁇ .
  • the DNA polymerase domain is a reverse transcriptase.
  • the reverse transcriptase is a retrovirus reverse transcriptase.
  • the reverse transcriptase is a Moloney murine leukemia virus (M-MLV) reverse transcriptase.
  • M-MLV Moloney murine leukemia virus
  • the DNA polymerase and the DNA binding domain are fused or linked to form a fusion protein.
  • Delivery [419] Prime editing compositions described herein can be delivered to a cellular environment with any approach known in the art. Components of a prime editing composition can be delivered to a cell by the same mode or different modes.
  • a prime editor can be delivered as a polypeptide or a polynucleotide (DNA or RNA) encoding the polypeptide.
  • a PEgRNA can be delivered directly as an RNA or as a DNA encoding the PEgRNA.
  • the polynucleotide encoding one or more prime editing composition components is a part of, or is encoded by, a vector.
  • the vector is a viral vector.
  • the vector is a non-viral vector. Exemplary delivery methods are shown in Table 1 below. [421] Table 1: Exemplary delivery methods
  • a prime editing composition for example, prime editor polypeptide components and PEgRNA/ngRNA are introduced to a target cell by nanoparticles.
  • the prime editor polypeptide components and the PEgRNA and/or ngRNA form a complex in the nanoparticle.
  • Any suitable nanoparticle design can be used to deliver genome editing system components or nucleic acids encoding such components.
  • the nanoparticle is inorganic.
  • the nanoparticle is organic.
  • a prime editing composition is delivered to a target cell, e.g., a hepatocyte, in an organic nanoparticle, e.g., a lipid nanoparticle (LNP) or polymer nanoparticle.
  • LNP lipid nanoparticle
  • a pharmaceutically-acceptable carrier comprises any vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the compound from one site (e.g., the delivery site) of the body, to another site (e.g., organ, tissue or portion of the body).
  • manufacturing aid e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid
  • solvent encapsulating material involved in carrying or transporting the compound from one site (e.g., the delivery site) of the body, to another site (e.g., organ, tissue or portion of the body).
  • compositions can additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
  • a pharmaceutically acceptable excipient includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
  • rOLI051 in presence of sodium chloride (150 mM) and a bridging oligonucleotide, OLI230, (1:2:1 equiv. for rOLI050:rOLI051:OLI230). rOLI050 and rOLI051 were then reduced using TCEP to generate thiol moieties. BMH (250 equiv.) was then added to the desalted, reduced rOLI050 and rOLI051 (at a concentration of 10 ⁇ M per RNA, 1 equiv.) and the reaction proceeded for 2 h at room temperature at pH 6.5 under aqueous conditions (50 mM phosphate buffer pH 6.5, 2.5% DMF).
  • BMH 250 equiv.
  • rOLI053 and rOLI051 at a concentration of 10 ⁇ M per RNA, 1 equiv.
  • This reaction was then desalted to remove excess reagents, concentrated, and buffer exchanged into 50 mM CHES pH 9.0.
  • the annealed RNA complex (at a concentration of 10 ⁇ M per RNA, 1 equiv.) was reacted with BS3 (500 equiv.) for 1 h at room temperature.
  • rOLI052, rOLI055 and rOLI051 were annealed (at a concentration of 20-50 ⁇ M per RNA, 1 equiv.) with sodium chloride (150 mM) and a bridging oligonucleotide, OLI399, (1 equiv. for rOLI052:rOLI055:rOLI051:OLI399). rOLI055 and rOLI051 were then reduced using TCEP to generate thiol moieties.
  • BMH 250 equiv.
  • rOLI055 and rOLI051 at a concentration of 10 ⁇ M per RNA, 1 equiv.
  • This reaction was then desalted using Cytiva NAP TM columns to remove residual small molecule from RNA.
  • the solution was then concentrated, and buffer exchanged into 50 mM CHES pH 9.0.
  • the annealed RNA (at a concentration of 10 ⁇ M per RNA, 1 equiv.) were reacted with BS3 (500 equiv.) for 1 h at room temperature.
  • rOLI017_BMH_ rOLI007 was next annealed with rOLI001 (at a concentration of 20-50 ⁇ M, 1 equiv.) in presence of sodium chloride (150 mM) and a bridging splint, OLI231 (1 equiv. for rOLI001: rOLI017_BMH_ rOLI007:OLI231).
  • the annealed RNA mixture (at a concentration of 10 ⁇ M per RNA, 1 equiv.) was reacted with BS3 (2000 equiv.) for 1 h at room temperature at pH 9.0 under aqueous conditions (50 mM CHES pH 9.0).
  • rOLI001 and rOLI027 were then reduced using TCEP to generate thiol moieties.
  • BMH 250 equiv.
  • This reaction was then desalted using Cytiva NAP TM columns to remove residual small molecule from RNA.
  • the solution was then concentrated, and buffer exchanged into UltraPure TM water using centrifugal filtration devices.
  • RNA was analyzed by RP-HPLC to confirm ligation, and then frozen until purification.
  • rOLI001_BMH_ rOLI027 and rOLI028 were annealed (at a concentration of 20-50 ⁇ M per RNA, 1 equiv.) with sodium chloride (150 mM).
  • the annealed RNA mixture (at a concentration of 10 ⁇ M per RNA, 1 equiv.) was reacted with BS3 (500 equiv.) for 1 h at room temperature at pH 9.0 under aqueous conditions (50 mM CHES pH 9.0).
  • Short RNA sequences carrying functional groups (at the ends or internally through a side chain on the ribose sugar or nucleobase) such as aniline derivatives can be selectively coupled under oxidation conditions to phenol derivatives to produce a ligated RNA as shown in Scheme 24.
  • Step-2 Reduction of O-nitrophenol-RNA to O-aminophenol-RNA was performed at 10-100 ⁇ M of RNA with 100 mM sodium dithionite (Na 2 S 2 O 3 ) at pH 6.5 in 100 mM phosphate buffer for 20 minutes at room temperature. This reaction was then desalted using Cytiva NAP TM columns to remove the unreacted small molecule reagents from RNAs and used in the next coupling reaction, step-3.
  • the PEgRNA can be generated from one 5’ fragment, and one or more 3’ fragments which are 5’ terminally modified with a free monophosphate to facilitate the ligation by way of a nucleotide ligase enzyme. Free 3’ ends and phosphoroylated 5’ ends are brought into proximity by way of sequence complementarity resulting in base pairing hybridization either between the RNA fragments directly (self-templated) or via an additional complementary DNA splint (splinted).
  • a nucleic acid ligase such as, but not limited to, T4 RNA Ligase I, T4 RNA Ligase II, or T4 DNA Ligase can generate a natural phosphodiester linkage between fragments, yielding the full-length PEgRNA.
  • Other ligase variants can also be used to ligate the fragments.
  • Scheme 26 below shows a potential fragment functionalization for enzymatic split synthesis, where “Upstream” or 5’ end RNA fragments have a free 3’ OH available for ligation, while “Downstream” or 3’ end RNA fragments are terminally functionalized with a 5’-monophosphate.
  • the RNA duplex can form a native secondary structure mimic to the full length PEgRNA, resulting in a net increase in the effective local concentration of the terminal 3’- OH and 5’-monophosphate functional ends required for ligase activity.
  • Subsequent ligase catalysis forms a phosphodiester bond between the RNA fragments which covalently seals the nick and generates a full-length PEgRNA. Accordingly, during self-templated ligation two RNA fragments with significant complementarity are enzymatically conjugated. The first step is to anneal the fragments, bringing the reactive functional groups together and creating a nicked double-stranded substrate for ligase catalysis.
  • Step 2 Enzymatic conjugation
  • Freshly prepared enzyme master mix (13 ⁇ L) was added to the annealed RNA mixture (7 ⁇ L).
  • the master mix contained molecular biology grade water (5.5 ⁇ L), T4 RNA Ligase 2 buffer (10X, 2 ⁇ L), PEG8000 (50% w/v, 4 ⁇ L), RNase inhibitor (40U/ ⁇ L, 0.5 ⁇ L), and T4 RNA Ligase 2 (10U/ ⁇ L, 1 ⁇ L).
  • the reaction proceeded in a thermocycler at 25°C for 120 minutes.
  • the synthesis products were then analyzed by 10% TBE-Urea denaturing PAGE and fluorescence gel imaging.
  • RNA fragments and the conjugation product [583] EXAMPLE 11.2 – Templated Enzymatic Ligation [584] Templated ligation is another strategy used for enzymatic split synthesis of PEgRNAs. In here, two functionalized, single-stranded RNA fragments are conjugated with ligase, similarly to self-templated ligation.
  • a short, complementary nucleic acid splint facilitates a trimeric secondary structure formation during templated ligation.
  • the nucleic acid splint is designed such that it bears proximal sequence complementarity of both the 3’ end of the upstream RNA fragment and 5’ end of the downstream RNA fragment to facilitate a splinted trimeric structure as shown in Scheme 27 below. Consequently, the RNA fragments do not require complementarity at the ligation site, and so the advantage of templated ligation for PEgRNA synthesis is that the splinted approach enables ligation at PEgRNA locations which lack secondary structure and anywhere in the sequence without extensions.
  • Schematic representation of templated ligation Black and light solid lines indicate upstream and downstream fragments, respectively.
  • Dotted line indicates splint that is complementary to the 3’-end of upstream fragment and 5’-end of downstream fragment. Splint facilitates bringing the functional ends of the two fragments to proximity for ligation.
  • the first step is to anneal the DNA-RNA hybrid complex, bringing the RNA fragments and their functional groups together.
  • the DNA splint is designed such that DNA-RNA hybridization forms a double-stranded structure with a nick between the RNA fragments.
  • a phosphodiester bond is formed between the 5’monophosphate and native 3’-hydroxyl of the RNA fragments, yielding a full-length pegRNA.
  • Step 1 Hybridization
  • 588 Equimolar ratio of RNA X, 5’-phosphorylated RNA Y, and DNA splint (0.01nmol, 1 ⁇ L each) were combined in molecular biology grade water (4 ⁇ L) and annealed in a thermocycler. The thermocycling program included heating the DNA-RNA mixture to 65°C for 3 minutes, followed by snap cooling on ice for 2 minutes. The DNA-RNA hybrid was maintained on ice for no longer than 5 minutes before enzyme addition.
  • Step 2 Enzymatic conjugation
  • DNA splints L, M, and N were designed to anneal to RNA’s X and Y, facilitating the formation of a nicked, double-stranded substrate for ligase activity. Furthermore, RNA Y was functionalized with a 5’-monophosphate to enable ligation to the native 3’-hydroxyl of RNA X. During the reaction, RNA’s X and Y were first annealed to a single DNA splint. Subsequently, the DNA-RNA hybrid was treated with ligase to catalyze the formation of a phosphodiester bond between the splinted RNA fragments.
  • RNA fragments and the conjugation product [595] EXAMPLE 11.3 –Enzymatic Ligation [596] Methods [597] All control/comparative RNA sequences prepared through linear solid-phase synthesis are synthesized and purified by Axo Labs. [598] Enzymatic ligation protocol [599] A self-splinted guide RNA ligation approach was utilized for enzymatic ligation. Both the acceptor and donor split fragments (A1+D1 or A2+D2) were used in 1:1 molar ratio. The solutions of both oligos were mixed along with 2X annealing solution (0.2 mM EDTA + 200 mM KCl).
  • the resulting solution was heated to 75 °C and cooled slowly to 25 °C at 0.1 °C/sec in a thermocycler.
  • the annealed oligo mixture was used for the ligation reaction.
  • the ligation reaction was setup by mixing the annealing mixture, RNase-free T4 RNA Ligase II (NEB, Cat. No. M0239L), T4 Rnl2 Reaction Buffer, SUPERase•InTM RNase Inhibitor (Invitrogen, Cat. No. AM2694), 50% aq. PEG8000, and RNase-free water to a final oligo concentration of 0.5 mM.
  • the reaction mixture was incubated at 37 °C for 1 h.
  • RNA product was isolated by using Monarch® RNA Cleanup Kit (NEB, Cat. No. T2030S/T2040S/T2050S) using manufacturer’s protocols. The resultat full-length RNA was obtained in good purity and could be directly used for downstream applications without further LC-based purification.
  • Analytical UPLC method [601] The purity of the ligation products was analyzed by Agilent 1290 Infinity II UPLC system.
  • a buffer system consisting of mobile phase A: 0.1M Hexylamine acetate (HAA) buffer pH 6.9 – 7.0 and mobile phase B: 0.1M HAA in H2O/ACN/MeOH (5/16/4 v/v/v) was used at a flow rate of 0.2 ml/min.
  • An ACQUITY, BEH C18, 130 ⁇ , 1.7 ⁇ m, 2.1 x 150 mm column at 80 °C was used using 0.00-40.00 min – 54-72% B, 40-42.60 min – 72-90% B, 42.60-45.60 min – 90% B, 45.20-45.30 min, 90-54% B, 45.30-52.00 min – 54% B gradient.
  • Acceptor and donor fragments are listed in Table 23. Acceptor A1 and donor D1 fragments were synthesized with a split in the tetraloop (TL) and acceptor A2 and donor D2 were synthesized with a split in the stem-loop 2 (SL2). [606] The ligation reactions were conducted as described above. At the end of the ligation reactions, all samples were analyzed by UPLC utilizing the buffer gradient described in the analytical UPLC method. FIG.18 shows the UPLC profiles of A1, D1 fragments and A1-D1 ligated product (FLP1-T4).
  • the UPLC profile of A1-D1 product (FLP1-T4) shown in the bottom panel of FIG.18 suggests that the ligated product is clean with ⁇ 1% of unreacted A1 and D1 fragments remaining.
  • ligation reactions were conducted between A2 and D2.
  • samples were analyzed by UPLC utilizing the buffer gradient described in the analytical UPLC method.
  • FIG.19 shows the UPLC profiles of A1, D1 fragments and A1- D1 ligated product (FLP2-T4).
  • the UPLC profile of A1-D1 product (FLP2-T4) shown in the bottom panel of FIG.19 suggests that the ligated product is clean with ⁇ 3% of unreacted A2 and D2 fragments remaining.
  • Table 23 Sequence information for acceptor and donor oligos along with full- length products and corresponding molecular weights.
  • RNAs equivalent to A1, D1 and A2, D2 ligation products were synthesized on an automated DNA/RNA synthesizer. Both the enzymatically ligated and synthetic full-length products were analyzed on UPLC for comparison.
  • FIG.20 shows the comparison of enzymatically ligated A1-D1, and synthetic products
  • FIG.21 shows the comparison of enzymatically ligated A2-D2 and synthetic products. Analysis of the results suggest that in both cases, the enzymatically ligated products show higher purity than the fully synthesized products (Table 24).
  • Table 24 Table 24.

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