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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
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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
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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]
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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
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  • 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
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/35—Nature of the modification
  • C12N2310/351—Conjugate
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  • 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.
• Prime editing can be used to correct disease associated gene mutations, and can be used for treating disease with a genetic component.
• technical challenges remain, limiting the rapid growth of gene editing technology in therapeutic areas.
• One of the major challenges is the production of high-quality long RNA oligonucleotides.
• [6] In general, the synthesis of RNA/DNA oligonucleotides is achieved by solid phase synthesis on automated synthesizers. However, the solid phase synthesis of long RNA oligonucleotides suffers from low yield and low purity of the final product. The coupling efficiency is not quantitative, limiting the yield for long-RNA oligonucleotides. These non- quantitative couplings generate shorter and longer impurities which are difficult to remove, especially in long RNAs.
• PEgRNAs full-length prime editing guide RNAs
• the disclosure provides 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 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.
• 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, 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
• 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 C-1: providing a second RNA sequence of Formula D-1: 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-1: 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, BM(PEG) 2 , BMOE, BM(PEG) 3 , or BMB reagent of the formulas: contacting the pre-annealed duplex with the BMH, BM(PEG) 2 , BMOE, BM(PEG) 3 , or BMB reagent to form a linker, wherein forming the linker results in the formation of the synthesized full-length PEgRNA of Formulas K-O:
• 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
• 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.
• 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 U: annealing the first and second RNA sequences to form a pre-annealed duplex; 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 W: 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
• 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 X: providing a second RNA sequence of Formula Y: annealing the first and second RNA sequences to form a pre-annealed duplex; 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 Z:
• 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.
• the disclosure provides a full-length PEgRNA comprising a chemically-modified gRNA core, wherein the full-length PEgRNA is produced by: 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 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 chemically-modified gRNA core of the full-length PEgRNA.
• the disclosure provides a full-length PEgRNA comprising a chemically-modified gRNA core, wherein the chemically-modified gRNA core comprises one or more linkers of Formula AA: 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; M is a linker, wherein the linker comprises an alkylene optionally substituted with one or more of –OH, -O-, -S-, or (O-CH 2 -CH 2 )n; or a structure of any one of Formulas AA-1 to AA-10: [27] In certain aspects, the disclosure provides a prime editing complex comprising: (i) the full-length PEgRNA prepared by
• the disclosure provides a lipid nanoparticle (LNP) or ribonucleoprotein (RNP) comprising the prime editing complex disclosed herein, or a component thereof.
• LNP lipid nanoparticle
• RNP ribonucleoprotein
• the disclosure provides a cell comprising the full-length PEgRNA prepared by the methods described herein, the prime editing complex disclosed herein, or the LNP or RNP disclosed herein.
• the disclosure provides a pharmaceutical composition comprising (i) the full-length PEgRNA prepared by the methods described herein, the prime editing complex disclosed herein, or the LNP or RNP disclosed herein; and (ii) a pharmaceutically acceptable carrier.
• 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 C: 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 D: 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 C-1: 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 J: 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 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.
• the disclosure provides an RNA sequence of Formula S: 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 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 X: 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.
• 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, wherein the first RNA sequence comprises a free 3’ OH and the second RNA sequence comprises a 5’-monophosphate; 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, reverse transcription editing template (RTT), or primer binding site (PBS) of the synthesized full-length PEgRNA.
• RTT reverse transcription editing template
• PBS primer binding site
• 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; 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 first linker
• 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.7 shows a RP-HPLC analysis of the thioether ligation.
• the bottom chromatogram is the RNA010 functionalized by SBAP.
• the middle chromatogram is the RNA011 functionalized by SPDP.
• the top chromatogram is the thioether ligation reaction.
• FIG.8 shows a LC-MS analysis of the thioether ligation.
• FIG.9A shows split synthesis of PEGRNAL1001 using thioether ligation.
• the figure is a denaturing PAGE (15%) analysis of the purified ligation product with different loading amounts (Lane 1: oligonucleotide length ladder; Lane 2: 1.8 pmol loading; Lane 3: 0.9 pmol loading; Lane 4: 0.5 pmol loading; Lane 5: 0.2 pmol loading).
• FIG.9B shows split synthesis of PEGRNAL1001 using thioether ligation.
• FIG.10A shows split synthesis of PEGRNAL1005 using thioether ligation.
• the figure shows a denaturing PAGE (15%) analysis of purified ligation product with different loading amounts (Lane 1: oligonucleotide length ladder; Lane 2: 1.6 pmol loading; Lane 3: 0.8 pmol loading; Lane 4: 0.5 pmol loading; Lane 5: 0.2 pmol loading).
• FIG.10B shows split synthesis of PEGRNAL1005 using thioether ligation.
• the figure shows an RP-HPLC analysis of purified ligation product.
• 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.
• FIG.16 shows a denaturing PAGE (10%) result of a self-templated ligation reaction.
• FIG.17 shows a denaturing PAGE (10%) result of a templated ligation reaction.
• Lane 1 ssRNA size standard (300-50mer); Lane 2) RNA’s X & Y and Splint L without ligase; Lane 3) RNA’s X & Y and splint M without ligase; Lane 4) RNA’s X & Y and Splint N without ligase; Lane 5) RNA’s X & Y and splint L with ligase; Lane 6) RNA’s X & Y and splint M with ligase; Lane 7) RNA’s X & Y and splint N with ligase; Lane 8) RNA’s X & Y with ligase; Lane 9) pegRNA Z full-length control.
• FIG.18 shows analytical UPLC traces of LC-purified acceptor A1 (Upper panel) and donor D1 (Middle panel) RNA oligonucleotides along with the enzymatically ligated full- length product FLP1-T4 with T4 RNA Ligase II (Bottom panel).
• FIG.19 shows analytical UPLC traces of LC-purified acceptor A2 (Upper panel) and donor D2 (Middle panel) RNA oligonucleotides along with the enzymatically ligated full- length product FLP2-T4 with T4 RNA Ligase II (Bottom panel).
• the enzymatically ligated RNA FLP2-T4 shows good purity in absence of LC-based purification with ⁇ 3% of A2+D2 remaining.
• FIG.20 shows analytical UPLC traces of the full-length product synthesized through linear solid-phase synthesis FLP1-LS (after LC purification) (top panel) and the enzymatically, ligated full-length product FLP1-T4 with T4 RNA Ligase II (in absence of LC purification) (Bottom panel).
• FIG.21 shows analytical UPLC traces of the full-length product synthesized through linear solid-phase synthesis FLP1-LS (after LC purification) (top panel) and the enzymatically, ligated full-length product FLP1-T4 with T4 RNA Ligase II (in absence of LC purification) (Bottom panel).
• FIG.22 shows dose-dependent editing of enzymatically ligated, linearlly synthesized and control PEgRNAs in primary human hepatocytes. ngRNA001 was used.
• FIG.23 shows a polyacrylamide gel analysis of the reaction products of SPAAC chemical ligation.
• FIG.24 shows a polyacrylamide gel analysis of 3-part ligation products.
• FIG.25 shows a RP-HPLC analysis of 3-part ligation products.
• FIG.26 shows a PAGE-urea analysis of oxidative ligations of aminophenol-RNAs with aniline-RNAs.
• FIG. 27 shows a PAGE-urea analysis of oxidative ligations of of thiol-RNA to catechol-RNA.
• RNAs such as full-length prime editing guide RNAs (PEgRNAs).
• the methods provided herein for synthesizing long RNAs, such as full-length PEgRNAs involve 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.
• the first and second RNA sequences are ligated to obtain the long RNA, such as a full-length PEgRNA.
• the ligation forms a linker between the first and second RNA and, in some embodiments, can be present in the gRNA core of the long RNAs, such as full-length PEgRNA.
• 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.
• the term “about” meaning within an acceptable error range for the particular value should be assumed.
• the term “substantially” as used herein may refer to a value approaching 100% of a given value. In some embodiments, the term may refer to an amount that may be at least about 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% of a total amount. In some embodiments, the term may refer to an amount that may be about 100% of a total amount.
• protein and “polypeptide” can be used interchangeably to refer to a polymer of two or more amino acids joined by covalent bonds (e.g., an amide bond) that can adopt a three-dimensional conformation.
• a protein or polypeptide comprises at least 10 amino acids, 15 amino acids, 20 amino acids, 30 amino acids or 50 amino acids joined by covalent bonds (e.g., amide bonds).
• a protein comprises at least two amide bonds.
• a protein comprises multiple amide bonds.
• a protein comprises an enzyme, enzyme precursor proteins, regulatory protein, structural protein, receptor, nucleic acid binding protein, a biomarker, a member of a specific binding pair (e.g., a ligand or aptamer), or an antibody.
• a protein may be a full-length protein (e.g., a fully processed protein having certain biological function).
• a protein may be a variant or a fragment of a full-length protein.
• a Cas9 protein domain comprises an H840A amino acid substitution compared to a naturally occurring S. pyogenes Cas9 protein.
• a variant of a protein or enzyme for example a variant reverse transcriptase, comprises a polypeptide having an amino acid sequence that is about 60% identical, about 70% identical, about 80% identical, about 90% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, about 99% identical, about 99.5% identical, or about 99.9% identical to the amino acid sequence of a reference protein.
• a protein comprises one or more protein domains or subdomains.
• polypeptide domain when used in the context of a protein or polypeptide, refers to a polypeptide chain that has one or more biological functions, e.g., a catalytic function, a protein-protein binding function, or a protein-DNA function.
• a protein comprises multiple protein domains.
• a protein comprises multiple protein domains that are naturally occurring.
• a protein comprises multiple protein domains from different naturally occurring proteins.
• a prime editor may be a fusion protein comprising a Cas9 protein domain of S. pyogenes and a reverse transcriptase protein domain of Moloney murine leukemia virus.
• 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 polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA.
• the polynucleotide may comprise one or more other nucleotide bases, such as inosine (I), which is read by the translation machinery as guanine (G).
• a polynucleotide e.g., PEgRNA
• PEgRNA may be modified.
• the terms “modified” or “modification” refers to chemical modification with respect to the A, C, G, T and U nucleotides.
• modifications may be on the nucleoside base and/or sugar portion of the nucleosides that comprise the polynucleotide.
• the modification may be on the internucleoside linkage (e.g., phosphate backbone).
• multiple modifications are included in the modified nucleic acid molecule.
• a single modification is included in the modified nucleic acid molecule.
• a polynucleotide e.g., PEgRNA
• PEgRNA may be chemically modified.
• 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 gRNA core is a chemically-modified gRNA core.
• a “chemically-modified gRNA core” refers to a gRNA core containing a non-nucleotidic chemical moiety, a modified nucleobase, a modified sugar, and/or a modified phosphate backbone.
• the term “moiety” refers to a specific segment or functional group of a molecule.
• a “chemical moiety” can be a chemical entity embedded in or appended to a molecule.
• a “chemical moiety” can also be a part or functional group of a molecule that can react with another chemical moiety.
• a chemical moiety is a functional group appended to the 3’ end and/or the 5’ end of a polynucleotide.
• a chemical moiety is a hydroxyl (-OH) group.
• a chemical moiety is a phosphate (-PO4) group.
• the chemical moiety is not a nucleic acid moiety, or polynucleotide moiety, connected to a sugar moiety of the rest of a polynucleotide as it would do if it is part of a polynucleotide.
• 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.
• Complement, “complementary,” or “complementarity” as used herein, refers to the ability of two polynucleotide molecules to base pair with each other.
• Complementary polynucleotides may base pair via hydrogen bonding, which may be Watson Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding.
• an adenine on one polynucleotide molecule will base pair to a thymine or uracil on a second polynucleotide molecule and a cytosine on one polynucleotide molecule will base pair to a guanine on a second polynucleotide molecule.
• Two polynucleotide molecules are complementary to each other when a first polynucleotide molecule comprising a first nucleotide sequence can base pair with a second polynucleotide molecule comprising a second nucleotide sequence.
• the two DNA molecules 5 ⁇ -ATGC-3 ⁇ and 5 ⁇ -GCAT-3 ⁇ are complementary, and the complement of the DNA molecule 5 ⁇ -ATGC-3 ⁇ is 5 ⁇ -GCAT-3 ⁇ .
• 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.
• “Substantially complementary” as used herein refers to a degree of complementarity that can be 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% over all or a portion of two polynucleotide molecules.
• the portion of complementarity may be a region of 10, 15, 20, 25, 30, 35, 40, 45, 50, or more nucleotides.
• “Substantial complementary” can also refer to a 100% complementarity over a portion of two polynucleotide molecules.
• the portion of complementarity between the two polynucleotide molecules is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% of the length of at least one of the two polynucleotide molecules or a functional or defined portion thereof.
• the term “subject” and its grammatical equivalents as used herein may refer to a human or a non-human.
• a subject may be a mammal.
• a human subject may be male or female.
• a human subject may be of any age.
• a subject may be a human embryo.
• a human subject may be a newborn, an infant, a child, an adolescent, or an adult.
• a human subject may be up to about 100 years of age.
• a human subject may be in need of treatment for a genetic disease or disorder.
• treatment or “treating” and their grammatical equivalents may refer to the medical management of a subject with an intent to cure, ameliorate, or ameliorate a symptom of, a disease, condition, or disorder.
• Treatment may include active treatment, that is, treatment directed specifically toward the improvement of a disease, condition, or disorder.
• Treatment may include causal treatment, that is, treatment directed toward removal of the cause of the associated disease, condition, or disorder.
• this treatment may include palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, condition, or disorder.
• Treatment may include supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the disease, condition, or disorder.
• a condition may be pathological.
• a treatment may not completely cure or prevent a disease, condition, or disorder.
• a treatment ameliorates, but does not completely cure or prevent a disease, condition, or disorder.
• a subject may be treated for 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, indefinitely, or life of the subject.
• the term “ameliorate” and its grammatical equivalents means to decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.
• the terms “prevent” or “preventing” means delaying, forestalling, or avoiding the onset or development of a disease, condition, or disorder for a period of time. Prevent also means reducing risk of developing a disease, disorder, or condition. Prevention includes minimizing or partially or completely inhibiting the development of a disease, condition, or disorder.
• a composition prevents a disorder by delaying the onset of the disorder for 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, indefinitely, or life of a subject.
• effective amount or “therapeutically effective amount” may refer to a quantity of a composition, for example a composition comprising a construct, that can be sufficient to result in a desired activity upon introduction into a subject as disclosed herein.
• An effective amount of the prime editing compositions can be provided to the target gene or cell, whether the cell is ex vivo or in vivo.
• An effective amount can be the amount to induce, for example, at least about a 2- fold change (increase or decrease) or more in the amount of target nucleic acid modulation (e.g., expression of a gene to produce functional a protein) observed relative to a negative control.
• An effective amount or dose can induce, for example, about 2-fold increase, about 3- fold increase, about 4-fold increase, about 5-fold increase, about 6-fold increase, about 7-fold increase, about 8-fold increase, about 9-fold increase, about 10-fold increase, about 25-fold increase, about 50-fold increase, about 100-fold increase, about 200-fold increase, about 500- fold increase, about 700-fold increase, about 1000-fold increase, about 5000-fold increase, or about 10,000-fold increase in target gene modulation (e.g., expression of a target gene to produce a functional protein).
• target gene modulation e.g., expression of a target gene to produce a functional protein.
• the amount of target gene modulation may be measured by any suitable method known in the art.
• 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.
• alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-dodecanyl, n- tridecanyl, n-tetradecanyl, n-pentadecanyl, or 2-ethylhexyl.
• An “alkylene” refers to an alkyl group, as defined above, linking at least two other groups, i.e., a divalent hydrocarbon radical.
• the two moieties linked to the alkylene can be linked to the same atom or different atoms of the alkylene.
• a straight chain alkylene can be the bivalent radical of –(CH 2 )n–, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
• Alkylene groups include, but are not limited to, methylene, ethylene, propylene, isopropylene, butylene, isobutylene, sec-butylene, pentylene and hexylene.
• an alkyl and/or alkylene can be substituted (i.e., optionally substituted) with one or more substituents independently selected from halo, cycloaliphatic [e.g., cycloalkyl or cycloalkenyl], heterocycloaliphatic [e.g., heterocycloalkyl or heterocycloalkenyl], aryl, heteroaryl, alkoxy, aroyl, heteroaroyl, acyl [e.g., (aliphatic)carbonyl, (cycloaliphatic)carbonyl, or (heterocycloaliphatic)carbonyl], nitro, cyano, amido [e.g., (cycloalkylalkyl)carbonylamino, arylcarbonylamino, aralkylcarbonylamino, (heterocycloalkyl)carbonylamino, (heterocycloalkylalkyl)carbonylamino, heteroary
• an alkyl and/or alkylene can be substituted with one or more substituents independently selected from carboxy (such as HOOC-alkyl, alkoxycarbonyl, and alkylcarbonyloxy), cyano, hydroxy, alkoxy, acyl, aralkyl, aryl, heteroaryl, sulfonylaminoalkyl (such as alkyl-SO2-amino), amino, amido, cycloaliphatic, or halo.
• carboxy such as HOOC-alkyl, alkoxycarbonyl, and alkylcarbonyloxy
• cyano cyano
• hydroxy alkoxy
• acyl aralkyl
• aryl aryl
• heteroaryl sulfonylaminoalkyl
• 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 “carbonyl” refers to –C(O)–.
• a “halogen” or “halo” group refers to fluorine, chlorine, bromine or iodine.
• a “heteroaryl” group refers to a monocyclic, bicyclic, or tricyclic ring system having 4 to 15 ring atoms wherein one or more of the ring atoms is a heteroatom (e.g., N, O, S, or combinations thereof) and in which the monocyclic ring system is aromatic or at least one of the rings in the bicyclic or tricyclic ring systems is aromatic.
• 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.
• heteroaryl examples include azetidinyl, pyridyl, 1H- indazolyl, furyl, pyrrolyl, thienyl, thiazolyl, oxazolyl, imidazolyl, tetrazolyl, benzofuryl, isoquinolinyl, benzthiazolyl, xanthene, thioxanthene, phenothiazine, dihydroindole, benzo[1,3]dioxole, benzo[b]furyl, benzo[b]thiophenyl, indazolyl, benzimidazolyl, benzthiazolyl, puryl, cinnolyl, quinolyl, quinazolyl,cinnolyl, phthalazyl, quinazolyl, quinoxalyl, isoquinolyl, 4H-quinolizyl, benzo-1,2,5-thiadiazolyl, or 1,
• monocyclic heteroaryls include furyl, thiophene-yl, 2H-pyrrolyl, pyrrolyl, oxazolyl, thazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, 1,3,4-thiadiazolyl, 2H-pyranyl, 4-H-pranyl, pyridyl, pyridazyl, pyrimidyl, pyrazolyl, pyrazyl, or 1,3,5-triazyl.
• Monocyclic heteroaryls are numbered according to standard chemical nomenclature.
• 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:
• an optionally substituted group can have a substituent at each substitutable position of the group, and when more than one position in any given structure can be substituted with more than one substituent selected from a specified group, the substituent can be either the same or different at every position.
• a ring substituent such as a heterocycloalkyl, can be bound to another ring, such as a cycloalkyl, to form a spiro-bicyclic ring system, e.g., both rings share one common atom.
• RNA/DNA oligonucleotides are within the scope of the disclosure.
• structures depicted herein also are meant to include compounds that differ only in the presence of one or more isotopically enriched atoms.
• compounds having the present structures except for the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a 13 C- or 14 C-enriched carbon are within the scope of this disclosure.
• Such compounds are useful, for example, as analytical tools or probes in biological assays, or as therapeutic agents.
• Split Synthesis [138] In general, the synthesis of RNA/DNA oligonucleotides is achieved by solid phase synthesis on automated synthesizers.
• 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 140 nucleotides. In some embodiments, the long RNA has a length of about 150 nucleotides. In some embodiments, the long RNA has a length of about 170 nucleotides. In some embodiments, the long RNA has a length of about 190 nucleotides. In some embodiments, the long RNA has a length of about 200 nucleotides. In some embodiments, the long RNA has a length of about 210 nucleotides. In some embodiments, the long RNA has a length of about 220 nucleotides. In some embodiments, the long RNA has a length of about 230 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 purity of the full-length ligated product is over 55%. In some embodiments, the purity of the full-length ligated product (e.g., long RNA or PEgRNA) is over 60%. In some embodiments, the purity of the full-length ligated product (e.g., long RNA or PEgRNA) is over 70%. In some embodiments, the purity of the full-length ligated product (e.g., long RNA or PEgRNA) is over 75%. In some embodiments, the purity of the full-length ligated product (e.g., long RNA or PEgRNA) is over 80%.
• the purity of the full-length ligated product is over 90%. In some embodiments, the purity of the full-length ligated product (e.g., long RNA or PEgRNA) is over 95%. In some embodiments, the full-length ligated product (e.g., long RNA or PEgRNA) contains less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of unreacted starting materials (e.g., RNA fragments such as donor D1 and acceptor A1 or RNA sequences containing chemical moieties as described herein).
• unreacted starting materials e.g., RNA fragments such as donor D1 and acceptor A1 or RNA sequences containing chemical moieties as described herein.
• the full-length ligated product (e.g., long RNA or PEgRNA) contains less than 10% of unreacted starting materials (e.g., RNA fragments such as donor D1 and acceptor A1 or RNA sequences containing chemical moieties as described herein). In some embodiments, the full-length ligated product (e.g., long RNA or PEgRNA) contains less than 9% of unreacted starting materials. In some embodiments, the full-length ligated product (e.g., long RNA or PEgRNA) contains less than 8% of unreacted starting materials. In some embodiments, the full-length ligated product (e.g., long RNA or PEgRNA) contains less than 7% of unreacted starting materials.
• unreacted starting materials e.g., RNA fragments such as donor D1 and acceptor A1 or RNA sequences containing chemical moieties as described herein.
• the full-length ligated product e.g., long RNA or PEgRNA
• the full-length ligated product (e.g., long RNA or PEgRNA) contains less than 6% of unreacted starting materials. In some embodiments, the full-length ligated product (e.g., long RNA or PEgRNA) contains less than 5% of unreacted starting materials. In some embodiments, the full-length ligated product (e.g., long RNA or PEgRNA) contains less than 4% of unreacted starting materials. In some embodiments, the full-length ligated product (e.g., long RNA or PEgRNA) contains less than 3% of unreacted starting materials.
• the full-length ligated product (e.g., long RNA or PEgRNA) contains less than 2% of unreacted starting materials. In some embodiments, the full-length ligated product (e.g., long RNA or PEgRNA) contains less than 1% of unreacted starting materials. In some embodiments, the full-length ligated product (e.g., long RNA or PEgRNA) is produced at a yield of about 50%, 55%, 60%, 65%, 70%, 75% or more. In some embodiments, full-length ligated product (e.g., long RNA or PEgRNA) produced at a yield of about 60%.
• the full-length ligated product (e.g., long RNA or PEgRNA) is produced at a yield of about 70%. In some embodiments, the full-length ligated product (e.g., long RNA or PEgRNA) is produced at a yield of about 80%. In some embodiments, the full-length ligated product (e.g., long RNA or PEgRNA) is produced at a yield of about 90%.
• the methods according to all aspects of this disclosure can equally be methods for synthesizing long guide RNAs other than PEgRNA. For example, methods described herein are suitable for synthesizing guide RNA for other Cas9 or Cas12 gene editing systems.
• the long guide RNAs of this disclosure have a length of over 100 nucleotides. In some embodiments, the long guide RNAs 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,
• 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
• PEgRNA refers to a guide polynucleotide that comprises one or more intended nucleotide edits for incorporation into the target DNA.
• the PEgRNA associates with and directs a prime editor to incorporate the one or more (e.g., two or more, three or more, four or more, or five or more) intended nucleotide edits into the target gene via prime editing.
• the gRNA core is a modified gRNA core.
• the synthesized full-length PEgRNA comprises, in 5’ to 3’ order, a spacer, a gRNA core, and an extension arm.
• the synthesized full-length PEgRNA comprises, in 5’ to 3’ order, a spacer, a chemically-modified gRNA core, and an extension arm.
• the extension arm comprises a reverse transcription template (RTT) and a primer binding site (PBS).
• RTT reverse transcription template
• PBS primer binding site
• Spacers [147] A spacer may guide a prime editing complex to a genomic locus with identical or substantially identical sequence during prime editing.
• the PEgRNA comprises a spacer.
• the length of the spacer varies from at least 10 nucleotides to 100 nucleotides.
• 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.
• the spacer is from 15 nucleotides to 30 nucleotides in length, 15 to 25 nucleotides in length, 18 to 22 nucleotides in length, 10 to 20 nucleotides in length, 20 to 30 nucleotides in length, 30 to 40 nucleotides in length, 40 to 50 nucleotides in length, 50 to 60 nucleotides in length, 60 to 70 nucleotides in length, 70 to 80 nucleotides in length, or 90 nucleotides to 100 nucleotides in length.
• the spacer is 20 nucleotides in length. In some embodiments, the spacer is 17 to 18 nucleotides in length.
• the length of the spacer varies from at least 10 nucleotides to 100 nucleotides.
• 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.
• the spacer is from 15 nucleotides to 30 nucleotides in length, 15 to 25 nucleotides in length, 18 to 22 nucleotides in length, 10 to 20 nucleotides in length, 20 to 30 nucleotides in length, 30 to 40 nucleotides in length, 40 to 50 nucleotides in length, 50 to 60 nucleotides in length, 60 to 70 nucleotides in length, 70 to 80 nucleotides in length, or 90 nucleotides to 100 nucleotides in length.
• the spacer is 20 nucleotides in length. In some embodiments, the spacer is 17 to 18 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.
• a primer binding site may be at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, 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, or at least 50 nucleotides in length.
• 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 PBS is 8, 9, 10, 11, 12, 13, or 14 nucleotides in length.
• An extension arm of a PEgRNA may comprise an editing template that serves as a DNA synthesis template for the DNA polymerase in a prime editor during prime editing.
• the length of an editing template may vary depending on, e.g., the prime editor components, the search target sequence and other components of the PEgRNA.
• the editing template serves as a DNA synthesis template for a reverse transcriptase, and the editing template is referred to as a reverse transcription editing template (RTT).
• RTT reverse transcription editing template
• the length of the editing template varies from at least 2 nucleotides to 350 nucleotides.
• 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.
• a PEgRNA is a chimeric polynucleotide that includes both RNA and DNA nucleotides.
• a PEgRNA can include DNA in the spacer sequence, the gRNA core, or the extension arm.
• a PEgRNA comprises DNA in the spacer sequence.
• the entire spacer sequence of a PEgRNA is a DNA sequence.
• the PEgRNA comprises DNA in the gRNA core, for example, in a stem region of the gRNA core.
• the PEgRNA comprises DNA in the extension arm, for example, in the editing template.
• 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.
• a gRNA core of a PEgRNA associates with a programmable DNA binding domain in a prime editor.
• the gRNA core comprises a direct repeat, a first stem loop, and a second stem loop.
• the gRNA core further comprises a third stem loop.
• a guide RNA core (also referred to herein as the gRNA core, gRNA scaffold, or gRNA backbone sequence) of a PEgRNA may contain a polynucleotide sequence that binds to a DNA binding domain (e.g., Cas9) of a prime editor.
• 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.
• the tetraloop comprises one or more base paired regions: a base paired “lower stem” adjacent to the spacer sequence and a base paired “upper stem” following the lower stem, where the lower stem and upper stem may be connected by a “bulge” comprising unpaired RNAs.
• a stem loop (or a hairpin loop) is base pairing pattern that can occur in single-stranded nucleic acids.
• a stem loop may be formed when two regions of the same nucleic acid strand are at least partially complementary in nucleotide sequence when read in opposite directions, therefore, the base-pairs can form a double helix that comprises an unpaired loop.
• Stem loops within a gRNA core described herein may be numbered starting from the 5’ to the 3’ end of the gRNA core.
• the “first stem loop” would be the first stem loop (not including any direct repeats) at the 5’ end proximal to the direct repeat of the gRNA core sequence.
• a “second stem loop” would be the second stem loop (not including any direct repeats) following the first stem loop in a 5’ to 3’ direction
• the “third stem loop” would be the third stem loop following the second stem loop in a 5’ to 3’ direction, and so on.
• the gRNA core comprises nucleotide alterations as compared to a wild type gRNA core.
• 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 comprises 4-30, 4-20, 4-16 or 4-12 nucleotide insertions in the lower stem of the tetraloop. In some embodiments, the gRNA core comprises 4-12 nucleotide insertions in the lower stem of the tetraloop. In some embodiments, the gRNA core comprises 6-30, 6-20, 6- 16 or 6-12 nucleotide insertions in the lower stem of the tetraloop. In some embodiments, the gRNA core comprises 6-12 nucleotide insertions in the lower stem of the tetraloop.
• 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 lower stem of the tetraloop. In some embodiments, the gRNA core comprises 10, 11 or 12 nucleotide insertions in the lower stem of the tetraloop. In some embodiments, the gRNA core comprises 1-30, 5-20, or 5-15 nucleotide insertions in the upper stem of the tetraloop.
• 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 upper stem of the tetraloop.
• the gRNA core has extensions in the TL or SL2 as shown in FIG.5A and FIG.5B.
• the gRNA core has a 1 base pair extension in the upper stem of the tetraloop.
• the gRNA core has a 2 base pair extension in the upper stem of the tetraloop.
• the gRNA core has a 3 base pair extension in the upper stem of the tetraloop.
• the gRNA core has a 4 base pair extension in the upper stem of the tetraloop. In some embodiments, the gRNA core has a 5 base pair extension in the upper stem of the tetraloop. In some embodiments, the gRNA core has a 6 base pair extension in the upper stem of the tetraloop. In some embodiments, the gRNA core has a 7 base pair extension in the upper stem of the tetraloop. In some embodiments, the gRNA core has a 8 base pair extension in the upper stem of the tetraloop. In some embodiments, the gRNA core has a 9 base pair extension in the upper 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 second portion of the chemically-modified gRNA core is 5-120 or 10-110 nucleotides in length. In some embodiments, the second 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 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 4-30, 4-20, 4-16 or 4-12 nucleotides in length compared to a native tetraloop. In some embodiments the extended tetraloop comprises an additional 4-12 nucleotides in length compared to a native tetraloop. In some embodiments the extended tetraloop comprises an additional 6-30, 6-20, 6-16 or 6-12 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 1.
• X 4 is an alkylene.
• X4 is–CH 2 –CH 2 –.
• the first chemical moiety of the first RNA sequence comprises a structure of Formula 2.
• R 1 is Cl, Br, or I.
• R 1 is Br.
• R 1 is I.
• the structure of Formula 2 is [199]
• the first chemical moiety of the first RNA sequence comprises a structure of Formula 2.
• X 4 is an alkylene.
• 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 second chemical moiety of the second RNA sequence comprises a structure of Formula 8.
• X5 is an alkylene.
• X5 is –CH 2 –.
• the second chemical moiety of the second RNA sequence is [217]
• the second chemical moiety of the second RNA sequence comprises a structure of Formula 12.
• X5 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 US9 nucleotide of the tetraloop. [236] 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. In some embodiments, 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.
• 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 RNA sequence comprises a third portion of the chemically-modified gRNA core.
• the third portion of the chemically- modified gRNA core comprises a fragmented tetraloop, a first stem loop, and a fragmented SL2.
• 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 fourth chemical moiety of the third RNA sequence comprises 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:
• each X1 and X4 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.
• Three-part Split Synthesis [241] Another method to synthesize long RNAs involves a three-part split synthesis.
• one aspect of the disclosure described herein includes 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 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; where
• 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 chemically-modified gRNA core comprises, in 5’ to 3’ order, a tetraloop, a first stem loop (SL1), a second stem loop (SL2) and a third stem loop (SL3).
• a tetraloop comprises a lower stem, a bulge, an upper stem, and a loop.
• the junction is present, in 5’ to 3’ order, between the chemically-modified gRNA core and the extension arm.
• the first RNA sequence comprises a spacer and a first portion of the chemically-modified gRNA core.
• 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 third portion of the chemically-modified gRNA core comprises a fragmented tetraloop and the fourth portion of the chemically-modified gRNA core comprises a fragmented SL2.
• the third RNA sequence comprises a third portion of the chemically-modified gRNA core and a junction.
• the third portion of the chemically-modified gRNA core comprises a fragmented tetraloop.
• the junction comprises the fourth chemical moiety.
• 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.
• the third chemical moiety of the third RNA sequence is located on the 5’ end of the US5, US6, US7, US8, or US9 nucleotide of the tetraloop.
• the third chemical moiety of the third RNA sequence is located on the 5’ end of the US5 nucleotide of the tetraloop.
• the third chemical moiety of the third RNA sequence is located on the 5’ end of the US6 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 second chemical moiety of the second RNA sequence is located on the 5’ end of the SL2-9 nucleotide of the SL2.
• the fourth chemical moiety of the third 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.
• the fourth chemical moiety of the third RNA sequence is located on the 3’ end of the SL2-4 nucleotide of the SL2.
• the fourth chemical moiety of the third RNA sequence is located on the 3’ end of the SL2-5 nucleotide of the SL2.
• the fourth chemical moiety of the third RNA sequence is located on the 3’ end of the SL2-6 nucleotide of the SL2. In some embodiments, the fourth chemical moiety of the third RNA sequence is located on the 3’ end of the SL2-7 nucleotide of the SL2. In some embodiments, the fourth chemical moiety of the third RNA sequence is located on the 3’ end of the SL2-8 nucleotide of the SL2. [255] In some embodiments, the second chemical moiety of the second RNA sequence is located on the 5’ end of the chemically-modified extension arm. [256] In some embodiments, the fourth chemical moiety of the third RNA sequence is located on the 3’ end of the junction.
• 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 second chemical moiety of the second RNA sequence and the third chemical moiety of the third RNA sequence each comprise: 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: wherein: 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.
• the first chemical moiety of the first RNA sequence and/or the fourth chemical moiety of the third RNA sequence comprises an amino. In some embodiments, the first chemical moiety of the first RNA sequence and/or the fourth chemical moiety of the third RNA sequence comprises —X 1 –NH 2 . In some embodiments, the first chemical moiety of the first RNA sequence and/or the fourth chemical moiety of the third RNA sequence comprises –X 1 –OH. In some embodiments, the first chemical moiety of the first RNA sequence and/or the fourth chemical moiety of the third RNA sequence comprises –X 1 –PO 4 .
• 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).
• RNAs with two thiol groups at respective ends can be ligated by heat annealing of the two RNA strands in the presence of a splint, TCEP reduction, followed by the addition of bismaleimide linker, as exemplified below with BMOE (Scheme 4.2.2).
• BMOE bismaleimide linker
• the bismaleimide ligation and BS3 ligation chemistries can be combined in a 3-part ligation method, as shown in Scheme 4.3.
• Another exemplary chemistry includes oxidative ligation of aniline-RNA and aminophenol-RNA.
• the selective reactivity of phenols with anilines can be used as a ligation chemistry for synthesizing longer RNAs.
• 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, Scheme 4.4.
• Another exemplary chemistry includes oxidative ligation of thiol-RNA and Catechol- RNA.
• the selective reactivity of thiols with catechols can be used as a ligation chemistry for synthesizing longer RNAs.
• Short RNA sequences carrying functional groups (at the ends or internally through a side chain on the ribose sugar or nucleobase) such as thiol derivatives or catechols derivatives can be selectively coupled under oxidation conditions to produce a ligated RNA, Scheme 4.5.
• Another exemplary chemistry includes the ligation of phosphorylated (5’ or 3’) RNA sequence with a free OH (5’ or 3’) RNA sequence in the presence of an N-cyanoimidazole activated phosphate of another RNA producing longer RNAs with native phosphodiester linkage as well as phosphodiester linkages with additional spacers, Scheme 5.
• Another exemplary chemistry includes chemical thiolation of 3’-maleimide functionalized RNA sequence with an appropriate 5’-thiol (SH) of a second RNA sequence, where the resultant succinimide derivative may undergo hydrolysis to produce an open-chain linker. Thiolation of maleimide is highly chemo selective and may need no template or splints, Scheme 6.
• Another exemplary chemistry includes producing a carbamate linkage, which is a mimic of phosphate that contains the same number of atoms as to native phosphate. This mimic allows maintaining similar internucleotide distance and exert nuclease resistance compared to phosphodiester linkage.
• a 3’-C6- NH 2 of a RNA sequence could react with an activated ester 5’-X(CO)O of a second RNA sequence resulting in a synthetic carbamate bridge.
• Enzymatic Ligation [285] In some embodiments, enzymatic ligation is used to ligate the chemical moieties.
• a long RNA such as a PEgRNA
• a long RNA is split into two or more short fragments. Then, the fragments are functionalized and ligated by enzymatic ligation to generate full-length PEgRNA.
• 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, wherein the first RNA sequence comprises a free 3’ OH and the second RNA sequence comprises a 5’-monophosphate; 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 PEg
• 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 chemical moieties described herein can incorporate functional groups that are specific for enzymatic ligation.
• the two or more fragments may contain chemically modifications of the sugar, phosphodiester backbone, or nucleobase canonical structures.
• 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 is T4 RNA Ligase I, T4 RNA Ligase II, or T4 DNA Ligase.
• Scheme 8 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. Schematic of upstream and downstream fragments are shown.
• the light shaded segments are complementary and facilitate self-templated ligation.
• Self-templated Ligation In some embodiments, two functionalized, single-stranded RNA fragments with complementary base pairs are hybridized for enzymatic ligation (e.g., ligase-mediated conjugation). In some embodiments, the fragments take natural advantage of the sequence complementarity driven structure of any region within the PEgRNA to help facilitate a very specific ligation reaction between the fragments. Once these fragments are annealed under controlled buffered conditions, the RNA duplex forms 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.
• the ligation occurs at any region within the PEgRNA. In some embodiments, the ligation occurs between and within the spacer of the PEgRNA. In some embodiments, the ligation occurs between and within the gRNA core of the PEgRNA. In some embodiments, the ligation occurs between and within the RTT of the PEgRNA. In some embodiments, the ligation occurs between and within the PBS of the PEgRNA. In some embodiments, the ligation occurs between and within the spacer and the gRNA core of the PEgRNA.
• the ligation occurs between and within the TL, SL1, SL2, or SL3 of the gRNA core of the PEgRNA. In some embodiments, the ligation occurs between and within the TL of the gRNA core of the PEgRNA. In some embodiments, the ligation occurs between and within the SL1 of the gRNA core of the PEgRNA. In some embodiments, the ligation occurs between and within the SL2 of the gRNA core of the PEgRNA. In some embodiments, the ligation occurs between and within the SL3 of the gRNA core of the PEgRNA. In some embodiments, the ligation occurs between and within the gRNA core and the RTT of the PEgRNA.
• the ligation occurs between and within the RTT and the PBS of the PEgRNA.
• self-templated 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).
• the location of the fragments in the two-part to ten-part ligations is similar to that described in the sections above.
• the efficiency of self-templated ligation reactions can be improved by modifying the conjugation sites.
• stem-loop modifications in the form of stem extensions and sequence changes help facilitate secondary structure formation that strengthen favorable structures that promote efficient ligations to form. By increasing the number of complementary base pairs between the fragments and the melt temperature of the RNA duplex they form, the conjugation site is more stable, and the ultimate effect we hypothesize would be an increase in ligation efficiency.
• designing modified stem-loops it is critical to avoid disrupting the formation of the PEgRNA-Cas9 complex.
• the Tetraloop and Stem-loop 2 are amenable to sequence modifications and secondary structure extensions ranging from 1 to 8 base pairs.
• Template Ligation In some embodmients, a template ligation is used for enzymatic split synthesis of PEgRNA. In here, two functionalized, single-stranded RNA fragments are conjugated with ligase, similarly to self-templated ligation. In addition, 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 9 below.
• the RNA fragments may or may 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.
• the splint is a DNA strand.
• the splint is a modified nucleic acid strand. Schematic representation of a splint 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 ligations are possible at non-structured regions of the PEgRNA.
• the regions are between and within SL1 and SL2.
• the regions are within the 3’ extension region to further elongate the RTT portion of PEgRNA.
• ligations in SL1 and SL3 are also possible without the need to extend the SL3 sequence. In some embodiments, this ligation may require a stepwise annealing approach to ensure proper triplex hybridization to occur prior to enzymatic ligation.
• the splint base pairs with each RNA fragment, bridging them together to form a nicked, double-stranded splint-RNA hybrid.
• Subsequent ligase catalysis forms a phosphodiester bond between the RNA fragments of the splint-RNA hybrid, covalently sealing the nick and generating a full-length PEgRNA annealed to a splint.
• the splint will be an extra oligonucleotide that must be removed during purification steps to yield the desired PEgRNA product, free from impurities.
• the splint ligation occurs at any region within the PEgRNA. In some embodiments, the splint ligation occurs between and within the spacer of the PEgRNA. In some embodiments, the splint ligation occurs between and within the gRNA core of the PEgRNA. In some embodiments, the splint ligation occurs between and within the RTT of the PEgRNA. In some embodiments, the splint ligation occurs between and within the PBS of the PEgRNA. In some embodiments, the splint ligation occurs between and within the spacer and the gRNA core of the PEgRNA.
• the splint ligation occurs between and within the TL, SL1, SL2, or SL3 of the gRNA core of the PEgRNA. In some embodiments, the splint ligation occurs between and within the TL of the gRNA core of the PEgRNA. In some embodiments, the splint ligation occurs between and within the SL1 of the gRNA core of the PEgRNA. In some embodiments, the splint ligation occurs between and within the SL2 of the gRNA core of the PEgRNA. In some embodiments, the splint ligation occurs between and within the SL3 of the gRNA core of the PEgRNA.
• 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’
• 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.
• the method further comprises providing at least one additional RNA sequence comprising a chemical moiety at its 3’ end, and at least one additional RNA sequence comprising a chemical moiety at its 5’ end.
• the method further comprises providing at least one additional RNA sequence comprising a free 3’ OH, and at least one additional RNA sequence comprising 5’-monophosphate.
• ligating the additional RNA sequences results in the formation of a third linker.
• ligating the additional RNA sequences results in the formation of a third and a fourth linker.
• ligating the additional RNA sequences results in the formation of a third, fourth, and fifth linker.
• ligating the additional RNA sequences results in the formation of a third, fourth, fifth, and sixth linker.
• 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 chemically and enzymatically ligating RNA fragments.
• PEgRNA prime editing guide RNA
• gRNA guide RNA
• 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 at least two RNA sequences comprising chemical moieties at their 3’ and/or 5’ ends, at least two RNA sequences comprising a free 3’ OH, and at least two RNA sequences comprising a 5’-monophosphate; and chemically and enzymatically ligating the RNA sequences, wherein ligating the RNA sequences produce one to three linkers formed with the chemical ligation and one to three linkers formed with the enzymatic ligation.
• PEgRNA prime editing guide RNA
• gRNA guide RNA
• 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.
• the linker is present between the SL2-7 and SL2-8 nucleotides of the SL2. In some embodiments, the linker is present between the SL2-8 and SL2-9 nucleotides of the SL2.
• Another aspect of the disclosure described herein is 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; 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 link
• 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 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 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 X1, X2, X4, 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; M1 is an amino, a halogen, a hydroxyl, a phosphate ion, a maleimide, an
• X 4 is an alkylene and M 1 is [315] In some embodiments, in Formula A, X4 is an alkylene and M1 is a halogen. [316] In some embodiments, in Formula A, X4 is ((CH 2 )n-C(O)NH-CH 2 ), and M1 is a halogen. [317] In some embodiments, in Formula A, X4 is absent and M1 is an aldehyde-substituted phenyl.
• X4 is an alkylene and M1 is [319] In some embodiments, in Formula A, X 4 is an alkylene and M 1 is [320] In some embodiments, in Formula A, X 4 is an alkylene and M 1 is [321] In some embodiments, in Formula A, X 4 is an alkylene and M 1 is a maleimide. [322] In some embodiments, in Formula B, “a” is 0, X5 is absent, and M2 is an amino. [323] In some embodiments, in Formula B, “a” is 1, X 5 is an alkylene, and M 2 is a thiol.
• each X 1 and X 2 is independently a C 1 -C 10 unsubstituted alkylene.
• each X 1 and X 2 is independently a C 6 unsubstituted alkylene.
• X1 is a C1-C10 substituted alkylene with an –OH and an –O– group.
• X 1 is a C 1 -C 10 substituted alkylene with an –OH.
• X1 is a C1-C10 substituted alkylene with an –O– group.
• X1 is an –O– group.
• X 2 is (O-CH 2 -CH 2 )n.
• 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 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
• 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 of Formula C-1: providing a second RNA sequence of Formula D-1: 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-1: wherein each X 1 and X 2 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.
• each X1 and X2 is independently a C1-C10 unsubstituted alkylene. In some embodiments, each X1 and X2 is independently a C6 unsubstituted alkylene. In some embodiments, X 1 is a C 1 -C 10 substituted alkylene with an – OH and an –O–. In some embodiments, X1 is an –O–. In some embodiments, X2 is (-O-CH 2 - CH 2 )n.
• Another aspect of the present 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 of Formula F: o ua ; 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
• Another aspect of the present 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 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, BM(PEG) 2 , BMOE, BM(PEG) 3 , or BMB reagent of the formulas: contacting the pre-annealed duplex with the BMH, BM(PEG) 2 , BMOE, BM(PEG) 3 , or BMB reagent to form a linker, wherein forming the linker results in the formation of the synthesized full-length PEgRNA of Formulas K-O:
• Another aspect of the present 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 of Formula I-1: 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 for a second pre-annealed duplex; providing a BS3 reagent; contacting the second pre-annealed duplex with the
• Another aspect of the present 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 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: 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
• Another aspect of the present 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 of Formula I: providing a second RNA sequence of Formula U: annealing the first and second RNA sequences to form a pre-annealed duplex; 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 W: 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
• each X 1 and X 2 is independently a C 1 -C 10 unsubstituted alkylene. In some embodiments, each X1 and X2 is independently a C6 unsubstituted alkylene. In some embodiments, X 1 is a C 1 -C 10 substituted alkylene with an –OH and an –O–. In some embodiments, X1 is an –O–. In some embodiments, X2 is (-O-CH 2 -CH 2 )n. [341] In embodiments of certain aspects, the linker is present in the tetraloop or SL2 of the chemically-modified gRNA core.
• the linker is present in the tetraloop of the chemically-modified gRNA core. In some embodiments, the linker is present in the upper stem of the tetraloop. In some embodiments, the linker is present in the SL2 of the chemically-modified gRNA core. [342] In some embodiments, the linker is located between the US4 and US5 nucleotides, between the US5 and US6 nucleotides, between the US6 and US7 nucleotides, between the US7 and US8 nucleotides, or between the US8 and US9 nucleotides of the tetraloop.
• Another aspect of the present disclosure includes a full-length PEgRNA comprising a chemically-modified gRNA core, wherein the synthetic full-length PEgRNA is produced by: 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 synthetic 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 chemically-modified gRNA core of the synthetic full-length PEgRNA.
• Another aspect of the present disclosure includes a full-length PEgRNA comprising a chemically-modified gRNA core, wherein the chemically-modified gRNA core comprises one or more linkers of Formula AA: 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; M is a linker, wherein the linker comprises an alkylene optionally substituted with one or more of –OH, -O-, -S-, or (O-CH 2 -CH 2 )n,; or a structure of any one of Formulas AA-1 to AA-10:
• the linker is a C6 unsubstituted alkylene. In some embodiments, the linker is a C 1 -C 10 substituted alkylene with a –S–. In some embodiments, the linker is a structure of any one of of Formulas AA-1 to AA-10. In some embodiments, the linker is a structure of Formula AA-1. In some embodiments, the linker is a structure of Formula AA-2. In some embodiments, the linker is a structure of Formula AA-3. In some embodiments, the linker is a structure of Formula AA-4. In some embodiments, the linker is a structure of Formula AA-5.
• the linker is a structure of Formula AA-6. In some embodiments, the linker is a structure of Formula AA-7. In some embodiments, the linker is a structure of Formula AA-8. In some embodiments, the linker is a structure of Formula AA-9. In some embodiments, the linker is a structure of Formula AA-10. [347] In some embodiments, X1 is a C1-C10 unsubstituted alkylene. In some embodiments, X1 is a C6 unsubstituted alkylene. In some embodiments, X1 is a C1-C10 substituted alkylene with an –OH and an –O–.
• X1 is a C1-C10 substituted alkylene with an –OH. In some embodiments, X1 is a C1-C10 substituted alkylene with an –O–. In some embodiments, X 1 is an –O–. In some embodiments, X 1 is (-O-CH 2 -CH 2 )n. [348] 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. In some embodiments, the linker is present in the upper stem of the tetraloop.
• the linker is present in the SL2 of the chemically-modified gRNA core. [349] In some embodiments, the linker is located between the US4 and US5 nucleotides, between the US5 and US6 nucleotides, between the US6 and US7 nucleotides, between the US7 and US8 nucleotides, or between the US8 and US9 nucleotides of the tetraloop.
• the linker is located between the SL2-4 and SL2-5 nucleotides, between the SL2-5 and SL2-6 nucleotides, between the SL2-6 and SL2-7 nucleotides, between the SL2-7 and SL2-8 nucleotides, or between the SL2-8 and SL2-9 nucleotides of the SL2.
• Another aspect of the disclosure includes an RNA sequence of Formula C: 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.
• Another aspect of the disclosure includes an RNA sequence of Formula D: 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.
• Another aspect of the disclosure includes an RNA sequence of Formula C-1: 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 D-1: 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.
• Another aspect of the disclosure includes an RNA sequence of Formula F: 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 G: 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.
• Another aspect of the disclosure includes an RNA sequence of Formula I: 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.
• Another aspect of the disclosure includes an RNA sequence of Formula J: 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 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 S: 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 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 X: 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.
• 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 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, or at least 190 nucleotides in length at least 200 nucleotides, at least 210 nucleotides, at least 220 nucleotides, at least 230 nucleotides, at least 240 nucleotides, at least 250 nucleotides in length, at least 275 nucleotides, at least 300 nucleotides, at least 325 nucleotides, at least 350 nucleotides, at least 375 nucleotides, at least 400 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 first RNA sequence when using chemical ligation, is SEQ ID NO.: 1, 3, 5, 7, 9, 19, 60, 62, 66, 69, 74, 89, 91, 93, or 112.
• the second RNA sequence when using chemical ligation, is SEQ ID NO.: 2, 4, 6, 8, 10, 20, 61, 63, 67, 70, 73, 75, 90, 108, or 121.
• 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.: 1, 3, 5, 7, 9, 69, 75, 89, 91, 93, or 112; the second RNA sequence is SEQ ID NO.: 2, 4, 6, 8, 10, 75, 90, or 108, and the third RNA sequence is SEQ ID NO.: 12, 13, 14, 15, 92, 94, 103, 107, 113, or 117.
• the synthesized full-length PEgRNA is SEQ ID NO.: 97-102, 104-106, 109-111, 114-116, or 118-120.
• 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.: 69
• the second RNA sequence is SEQ ID NO.: 107
• the third RNA sequence is SEQ ID NO.: 108.
• the first RNA sequence is SEQ ID NO.: 112
• the second RNA sequence is SEQ ID NO.: 75
• the third RNA sequence is SEQ ID NO.: 113.
• 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.
• Additional PEgRNA elements may comprise, or further comprise, additional polynucleotide components such as a structural motif at the 3’ end of the extension arm, tag sequences, and/or flap endonucleases as described in PCT publication No.: WO 2023/096977 and WO 2023/086558, the contents of each are herein incorporated by reference in their entireties.
• 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.
• the one or more nucleic acid moieties comprise a hairpin (e.g., hairpin comprising a region of self-complementarity, optionally wherein the region of self- complementary comprises 2, 3, 4, 5, 6, 7, 8 , 9, 10 or more contiguous complementary basepairs), a quadruplex (e.g., a G-quadruplex or a C-quadruplex, optionally wherein the G- quadruplex or the C-quadruplex is derived from a VEGF gene promoter), a tRNA sequence (e.g., a tRNA sequence, optionally wherein the tRNA sequence is a tRNA (Proline) sequence), an aptamer (e.g., an aptamer derived from a viral protein-binding sequence, optionally wherein the aptamer comprises a viral reverse transcriptase recruitment sequence, optionally wherein the aptamer comprises a MS2 protein binding sequence or a Moloney Murine leukemia
• a hairpin e
• 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 nucleotide sequence of the MS2 hairpin (or also referred to as the “MS2 aptamer”) is: GCCAACATGAGGATCACCCATGTCTGCAGGGCC (SEQ ID NO.: 58). In some embodiments, the nucleotide sequence of the MS2 aptamer comprises the sequence of SEQ ID NO.: 50. In some embodiments, a MS2 coat protein (MCP) recognizes the MS2 hairpin.
• MCP MS2 coat protein
• the amino acid sequence of the MCP is: GSASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQ NR KYTIKVEVPKVATQTVGGEELPVAGWRSYLNMELTIPIFATNSDCELIVKAMQGLLK DG NPIPSAIA ANSGIY (SEQ ID NO.: 59).
• the one or more nucleic acid moieties comprises a G- quadruplex or a C-quadruplex.
• the one or more nucleic acid moieties comprises a quadruplex from a VEGF gene promoter.
• 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.
• the gRNA core of a PEgRNA may be located at the 5’ end of a spacer. In some embodiments, the gRNA core of a PEgRNA may be located at the 3’ end of an extension arm. In some embodiments, the gRNA core of a PEgRNA may be located at the 5’ end of an extension arm. In some embodiments, a PEgRNA comprises, from 5’ to 3’: a spacer, a gRNA core, and an extension arm. In some embodiments, 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 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.
• An extension arm of a PEgRNA may comprise a primer binding site sequence (also referred to as a primer binding site, PBS, or PBS sequence) that hybridizes with a free 3’ end of a single stranded DNA in the target gene generated by nicking with a prime editor.
• 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. In some embodiments, the length of the primer binding site (PBS) varies from at least 2 nucleotides to 50 nucleotides.
• a primer binding site may be at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, 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, or at least 50 nucleotides in length.
• 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
• 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. In some embodiments, the PBS is 8, 9, 10, 11, 12, 13, or 14 nucleotides in length. [411]
• the PBS of a first PEgRNA may be complementary or substantially complementary to a DNA sequence in the second strand of the target gene.
• the PBS of a second PEgRNA may be complementary or substantially complementary to a DNA sequence in the first strand of the target gene.
• 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 double stranded target DNA comprises a nick site on the PAM strand (or non-target strand).
• a “nick site” refers to a specific position in between two nucleotides or two base pairs of the double stranded target DNA.
• the position of a nick site is determined relative to the position of a specific PAM sequence.
• the nick site is the particular position where a nick will occur when the double stranded target DNA is contacted with a nickase, for example, a Cas nickase, that recognizes a specific PAM sequence.
• 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
• LNPs are formulated from cationic, anionic, neutral lipids, or combinations thereof.
• neutral lipids such as the fusogenic phospholipid DOPE or the membrane component cholesterol, are included to enhance transfection activity and nanoparticle stability.
• LNPs are formulated with hydrophobic lipids, hydrophilic lipids, or combinations thereof. Lipids may be formulated in a wide range of molar ratios to produce an LNP. Any lipid or combination of lipids that are known in the art can be used to produce an LNP. Exemplary lipids used to produce LNPs are provided in Table 2 below.
• components of a prime editing composition form a complex prior to delivery to a target cell.
• a PEgRNA can form a complex prior to delivery to the target cell.
• a prime editing polypeptide e.g., a prime editor fusion protein
• a guide polynucleotide e.g., a PEgRNA or ngRNA
• RNP ribonucleoprotein
• the RNP comprises a prime editor fusion protein in complex with a PEgRNA.
• RNPs may be delivered to cells using known methods, such as electroporation, nucleofection, or cationic lipid- mediated methods, or any other approaches known in the art.
• delivery of a prime editing composition or complex to the target cell does not require the delivery of foreign DNA into the cell.
• the RNP comprising the prime editing complex is degraded over time in the target cell.
• Exemplary lipids for use in nanoparticle formulations and/or gene transfer are shown in Table 2 below. [425] Table 2: Exemplary lipids for nanoparticle formulation or gene transfer
• compositions comprising the full-length PEgRNA, the prime editing complex, or the LNP or RNP described herein.
• pharmaceutical composition refers to a composition formulated for pharmaceutical use.
• the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
• the pharmaceutical composition comprises additional agents, e.g., for specific delivery, increasing half-life, or other therapeutic compounds.
• 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).
• a pharmaceutically acceptable carrier is “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the tissue of the subject (e.g., physiologically compatible, sterile, physiologic pH, etc.)
• Formulations of the pharmaceutical compositions described herein can be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient(s) into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping and/or packaging the product into a desired single- or multi-dose unit.
• 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.
• the prime editing method comprises contacting a target gene, with a PEgRNA prepare by the methods described herein and a prime editor (PE) 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.
• a method for editing a gene comprises contacting the gene with the prime editing complex described herein, wherein the PEgRNA directs the prime editor to incorporate the intended nucleotide edit in the gene, thereby editing the gene.
• the editing efficiency is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% equivalent as compared to editing with an unmodified control PEgRNA.
• the prime editor synthesizes a single stranded DNA encoded by the editing template, wherein the single stranded DNA replaces the editing target sequence and results in incorporation of the intended nucleotide edit into a region corresponding to the editing target sequence.
• the subject is a human.
• the method further comprising administering the cell to the subject after incorporation of the intended nucleotide edit.
• RNA/DNA oligonucleotides are produced by solid phase synthesis on automated synthesizers.
• solid phase synthesis of long RNA oligonucleotides suffers from low yield and purity of the final product.
• the coupling efficiency is not quantitative, thus limiting the synthesis yield for long-RNA oligonucleotides.
• RNAs > 100-nt because just several nucleotides shorter or longer becomes a trivial difference for long oligonucleotides (> 100-nt length) compared to much shorter oligonucleotides (20-nt length).
• alternate synthesis, and purification methods are required for long-RNA synthesis.
• synthesizing long RNAs, such as PEgRNAs via split synthesis using functionalized fragments.
• the functionalized fragments can be joined together via enzymatic ligation or chemical conjugation.
• PEgRNAs have four major components: a spacer, a gRNA core, a Reverse Transcription Template (RTT), and a Primer Binding Site (PBS), in 5’ to 3’ order.
• the gRNA core has four major components: a tetraloop (TL), a Stem Loop 1 (SL1), a Stem Loop 2 (SL2), and a Stem Loop 3 (SL3). An illustration of these major components is shown in FIGs.1 and 2.
• TERT Reverse Transcription Template
• PBS Primer Binding Site
• TL and SL2 are considered as the potential split sites for chemical ligations.
• Other potential split sites include the junction between the gRNA core and the RTT and the end of the PBS and extensions of the 3’ end.
• EXAMPLE 2 Split Synthesis of Long RNAs via Thioether Ligation
• the thioether ligation uses a thiol modified fragment to react with the bromine containing fragment to form a thioether linkage (Schemes 10 and 11).
• One amino-modified fragment is functionalized by bromine containing NHS ester SBAP (succinimidyl 3- (bromoacetamido)propionate), and the other amino-modified fragment is functionalized by pyridyl disulfide NHS ester SPDP (succinimidyl 3-(2-pyridyldithio)propionate).
• TCEP tris(2- carboxyethyl)phosphine
• SBAP Succinimidyl 3-(bromoacetamido)propionate
• Step 2 SPDP modification
• SPDP Succinimidyl 3-(2-pyridyldithio)propionate
• RNA-002 Amino-functionalized RNA-002, Table 4 (60 nmol in water is treated with freshly prepared solution of SPDP in DMF/phosphate buffer at pH 8.0 under denaturing conditions. After 2 h at room temperature, the reaction mixture is quenched with 1 M Tris.HCland the mixture is desalted using a Sephadex TM G25 DNA grade Nap-25 column as per the manufacture’s recommendations. The desalted RNA product is then concentrated. The concentrated disulfide product is analyzed via RP-HPLC and observed a quantitative conversion in general. The product without purification is then either used immediately in a next step or stored at -20 °C until the next use.
• Step 3 Thioether conjugation of SBAP and SPDP modified RNA fragments
• disulfide-RNA-002 product is reduced to reveal the reactive thiol-RNA-002 product using tris-(2-carboxyethyl)phosphine solution (TCEP, neutral pH) and conjugated to bromo-RNA-001 product under denaturing conditions.
• TCEP tris-(2-carboxyethyl)phosphine solution
• disulfide- RNA-002 product is reduced with 50 eq. of TCEP solution at room temperature for 30 minutes.
• the reaction mixture is desalted using a Sephadex TM G25 DNA grade Nap-5 column as per the manufacture’s recommendations.
• the resultant thiol-RNA- 002 product (28.4 nmol) is mixed with bromo-RNA-001 product (31.24 nmol) and concentrated.
• the above concentrate of bromo-RNA-001 product and thiol-RNA-002 product mixture (26.75 nmol) is added followed by the addition of DMF and allowed to react at room temperature for 2 h.
• the mixture is desalted using a Sephadex TM G25 DNA grade Nap-25 column as per the manufacture’s recommendations.
• the crude products are analyzed on analytical RP-HPLC and denaturing TBE-urea PAGE analysis.
• RNA010 and RNA011 were tested using model fragment sequences RNA010 and RNA011 (Table 4).
• the RNA010 was treated with SBAP and the RNA011 was treated with SPDP to functionalize amino groups on each fragment.
• the ligation conditions were optimized for parameters like pH (6.0 – 8.0), DMF solvent volume percentage (0-50%), without pre-annealing.
• the ligation reaction products were analyzed by denaturing PAGE (Polyacrylamide Gel Electrophoresis), FIG.6.
• the ligated (FLP) yield is quantified as 65% based on the band intensity on the gel image.
• the ligation reaction was also verified by RP-HPLC (FIG.7) and LC-MS (FIG.8).
• PEGRNAL1001 was synthesized with a thioether linkage in the TL using SBAP functionalized RNA001 and SPDP functionalized RNA002.
• PEGRNAL1005 was synthesized with a thioether linkage in the SL2 using SPDP functionalized RNA003 and SBAP functionalized RNA004.
• Prime editing systems including PE3 and PE3b, are described in Anzalone, A.V., Randolph, P.B., Davis, J.R. et al., “Search-and-replace genome editing without double-strand breaks or donor DNA,” Nature 576, 149–157 (2019) which is incorporated herein by reference.
• EXAMPLE 4 – Split Synthesis of Long RNAs via Bisamide Conjugation uses one molecule of BS3 (bis(sulfosuccinimidyl)suberate) with two NHS ester groups to link two amino modified fragments through two amide bond formations on a single BS3 molecule (Scheme 12).
• This conjugation requires the two fragments to be pre-annealed and the two amino groups need to be positioned in proximity before adding the BS3 reagent.
• the split site has an extended stem loop so that a stable duplex forms, which in turn facilitates the reaction between the amino groups of the two fragments and the BS3 reagent.
• Step 1 Pre-annealing
• RNA011 1.3 nmol, 3.03 ⁇ L
• RNA010 1.3 nmol, 3.03 ⁇ L
• ultrapure water 40.34 ⁇ L
• NaCl 5.2 ⁇ L, 1.5 M
• Step 2 BS3-mediated conjugation
• BS3 reagent 13 ⁇ L, variable equivalents from 500 to 3000
• CHES N-Cyclohexyl-2-aminoethanesulfonic acid
• the reaction is quenched after 10 min with 1M tris buffer (pH 7.5) to a final concentration of 50 mM.
• the above mixture is desalted using Nap-5 column followed by concentration with Amicon centrifugal device 0.5-mL (10 KDa cutoff).
• the products are analyzed by RP-HPLC and denaturing PAGE analysis. [473] Table 7. Model RNA fragments and the conjugation [474] Results [475]
• the conjugation was analyzed by PAGE (FIG.13).
• the yield of the conjugated product (RNAL1) was up to 70% based on the band intensity on the gel image.
• the ligation reaction was also verified by RP-HPLC (FIG. 14) and LC-MS (FIG.15).
• EXAMPLE 4.1 – Split Synthesis of Long RNAs via Bisamide Conjugation [477] Using similar conjugation to the one described in Example 4, the following 153-nt long PEgRNAs were synthesized (Table 7.1). The ligation was performed at either the tetraloop (TL) or SL2, where each had an 11-bp extension at their respective stem structures. [478] Table 7.1. RNA fragments and the conjugation [479] EXAMPLE 4.2 – Split Synthesis of Long RNAs via Bisamide Conjugation [480] To further demonstrate the value of ligation chemistries for building long RNAs, we synthesized a longer 193-nt long PEgRNA.
• SL2 ligation was appropriate to synthesize the target with almost similar length 5’- and 3’-split fragments.
• the SL2 had an 11-bp extension at the stem structure.
• the bisamide ligated PEgRNA and the corresponding fragments for SL2 ligation are shown in Table 7.2. [481] Table 7.2. RNA fragments and the conjugation g /5 [482] EXAMPLE 5 – Strain-promoted copper-free azide and alkyne cycloaddition reaction (SPAAC) [483] This strategy involves the cycloaddition reaction between two RNA sequences carrying an azide and alkyne functional groups at the respective ends.
• SPAAC Strain-promoted copper-free azide and alkyne cycloaddition reaction
• the reaction is driven by the ring strain of alkyne functional group in the cyclic ring instead of traditional alkyne activation by copper.
• a splint was used to bring the both the reactive functional groups in the proximity for a successful ligation reaction.
• RNAs were treated with the activated NHS-esters of azide or alkyne partners (Scheme 14). 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.
• DBCO modification of amino-functionalized RNA oligonucleotides [490]
• the input for this process is azide-functionalized rOLI001 and alkyne-functionalized rOLI019 in UltraPure TM water.
• rOLI019 with a 5’amino-end (typically at a concentration of 10 ⁇ M – 50 ⁇ M, 1 equiv.) was modified with DBCO-C6-NHS ester (200 equiv.) for 2 h at room temperature at pH 8.0 under denaturing conditions (40/60 v/v% DMF and 50 mM phosphate buffer pH 8.0), as shown in FIG.23.
• RNA oligonucleotides at the native TL site using SPAAC [492] The input for this process is azido-rOLI001 (containing a 3’ azido), alkyne-rOLI019 (containing 5’ DBCO), and OLI231 (splint oligonucleotide with complementarity to TL ligation site).
• azido-rOLI001 and alkyne-rOLI019 were annealed (at a concentration of 20-50 ⁇ M per RNA, 1 equiv. azido-rOLI001 and 1 equiv.
• alkyne-rOLI019 with sodium chloride (150 mM) and a bridging oligonucleotide, OLI231, (1:1:1 equiv. for azido-rOLI001: alkyne- rOLI019:OLI231), as shown in Table 8.
• azido-rOLI001 and alkyne-rOLI019 were then combined (at a concentration of 10 ⁇ M per RNA, 1 equiv.) and the reaction proceeded for 2 h at room temperature at pH 7.5 under aqueous conditions (20 mM HEPES buffer pH 7.5, 5 mM EDTA, 158 mM NaCl).
• RNA modification of amino-functionalized RNA oligonucleotides and BMX ligations [502] Amino-functionalized RNAs (rOLI001/rOLI003/rOLI005/rOLI007, at a concentration of 10 ⁇ M – 50 ⁇ M, 1 equiv.) were modified with SPDP (160 equiv.) for 2 h at room temperature at pH 8.0 under denaturing conditions (40/60 v/v% DMF and 50 mM phosphate buffer pH 8.0). After 2 h, the reaction was quenched with tris ( ⁇ 1,000 equiv., as a solution of 1 M Tris HCl pH 7.5).
• This reaction was then desalted using Cytiva NAP TM columns to remove residual SPDP from RNAs. Following desalting, the crude was analyzed by RP- HPLC to confirm modification followed by concentration.
• This SPDP-modified RNA was annealed to another complimentary SPDP-modified RNA in presence of a splint oligonucleotide (OLI231 for TL ligation and OLI232 for SL2 ligation), reduced, and then ligated to form a longer RNAs using BMX reagents.
• a splint oligonucleotide OLI231 for TL ligation and OLI232 for SL2 ligation
• rOLI001 containing a 3’ protected thiol
• rOLI003 containing 5’ protected thiol
• OLI231 complementarity to TL ligation site
• rOLI001 and rOLI003 were annealed (at a concentration of 20-50 ⁇ M per RNA, 1 equiv. rOLI001 and 1 equiv. rOLI003) in presence of sodium chloride (150 mM) and a bridging oligonucleotide, OLI231, (1:1:1 equiv.
• rOLI001 and rOLI003 were then reduced using TCEP to generate thiol moieties.
• BMH 250 equiv.
• 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).
• rOLI054 and rOLI051 were annealed (at a concentration of 20-50 ⁇ M per RNA, 1 equiv. rOLI054 and 2 equiv. rOLI051) with sodium chloride (150 mM) and a bridging oligonucleotide, OLI399, (1:2:1 equiv. for rOLI054:rOLI051:OLI399). rOLI054 and rOLI051 were then reduced using TCEP to generate thiol moieties.
• rOLI052, rOLI053 and rOLI051 were annealed (at a concentration of 20-50 ⁇ M per RNA, 1 equiv.) with sodium chloride (150 mM) and a bridging oligonucleotide, OLI231, (1 equiv. for rOLI052:rOLI053:rOLI051:OLI231). rOLI053 and rOLI051 were then reduced using TCEP to generate thiol moieties.
• 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.
• the input for this process is rOLI052 (containing a 3’ amino and 3’ 11-mer extension at the TL site), rOLI055 (containing 5’ amino and 3’ protected thiol, a 5’ 11-mer extension at the TL site, and a 3’ 4-mer extension at the end of the SL3), and rOLI051 (containing 5’ protected thiol).
• 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 and rOLI007 were reduced using TCEP to generate thiol moieties.
• BMH 250 equiv.
• This reaction was then desalted using Cytiva NAP TM followed by concentration, crude analysis and RP-HPLC purification.
• 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).
• RNA-2 and RNA-3 were then reduced using TCEP to generate thiol moieties.
• BMH 250 equiv.
• rOLI026_BMH_rOLI007 and rOLI009 were annealed (at a concentration of 20-50 ⁇ M per RNA, 1 equiv.) with sodium chloride (150 mM).
• 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 at pH 9.0 under aqueous conditions (50 mM CHES pH 9.0). After 1 h, the reaction was quenched with tris (as a solution of 1 M Tris HCl pH 7.5, ⁇ 1,000 equiv.).
• rOLI009, rOLI029, and rOLI028 were annealed (at a concentration of 20-50 ⁇ M per RNA, 1:1:1 equiv.) with sodium chloride (150 mM) and splint sequence (2 equiv.) to promote the maximum desired product formation.
• a solution of BS3 (500 equiv.) was then added to annealed rOLI009, rOLI029, and rOLI028, and splint OLI300 in the reaction buffer (50 mM CHES pH 9.0). The reaction proceeded for 1 h at room temperature. After 1 h, the reaction is quenched with tris ( ⁇ 1000 equiv., as a solution of 1 M Tris pH 7.5).
• 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-1 Amino-functionalized RNAs (rOLI001/rOLI019, at a concentration of 10 ⁇ M - 50 ⁇ M, 1 equiv.) were modified with corresponding NHS-esters (160 equiv.) for 2 h at room temperature at pH 8.0 under denaturing conditions (40/60 v/v% DMF and 50 mM phosphate buffer pH 8.0), Scheme 24. After 2 h, the reaction was quenched with tris ( ⁇ 1,000 equiv., as a solution of 1 M Tris HCl pH 7.5).
• 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.
• Step-3 Aniline-RNA, rOLI001 (1 eq.) was coupled with O-aminophenol-RNA, rOLI019 (5-10 eq.) at pH 6.0 under oxidative conditions of 5 mM K3FeCN6 (100 eq.) at room temperature for 30 minutes. The reaction was quenched with TCEP followed by the desalting using Cytiva NAP TM columns resulted in a ligated RNA which is analyzed by the PAGE-urea (FIG.26) and LCMS. Table 18 shows the sequences used in this example. [563] Table 18.
• EXAMPLE 10 Oxidative ligation of thiol-RNA and Catechol-RNA [565]
• the selective reactivity of thiols with catechols can be used as a ligation chemistry for synthesizing longer RNAs.
• Short RNA sequences carrying functional groups (at the ends or internally through a side chain on the ribose sugar or nucleobase) such as thiol derivatives or catechols derivatives can be selectively coupled under oxidation conditions to produce a ligated RNA, Scheme 25.
• Step-1 Amino-functionalized RNAs (rOLI001/rOLI019, at a concentration of 10 ⁇ M - 50 ⁇ M, 1 equiv.) were modified with corresponding NHS-esters (160 equiv.) for 2 h at room temperature at pH 8.0 under denaturing conditions (40/60 v/v% DMF and 50 mM phosphate buffer pH 8.0), Scheme 25. After 2 h, the reaction was quenched with tris ( ⁇ 1,000 equiv., as a solution of 1 M Tris HCl pH 7.5). This reaction was then desalted using Cytiva NAP TM columns to remove the unreacted small molecule reagents from RNAs.
• Step-2 and step-3 Oxidation of phenol-RNA obtained in the above step to catechol- RNA was performed using abTYR (Tyrosinase isolated from Agaricus bisporus). The oxidation was typically performed at 10-50 ⁇ M of RNA concentration (10 eq.) with abTYR (14 U/L, 0.2 eq.) in 20 mM phosphate buffer pH 6.5 for 20 min at room temperature. To the above mixture was added the freshly TCEP-reduced thiol-RNA (1 eq.) and allowed the ligation for 30 minutes.
• abTYR Tyrosinase isolated from Agaricus bisporus
• oligonucleotides Synthesis of oligonucleotides (RNA or DNA) is commonly achieved on automated DNA/RNA synthesizers using a solid support. Synthetic methods have been well established for producing several therapeutic oligonucleotides used for antisense (20-30-nt), siRNA (19-21- nt), aptamers (30-90-nt), and sgRNAs up to 100-nt. However, making >120-nt long nucleic acids faces technical challenges of synthesis and their purification. These challenges demand finding alternate methods to build >120-nt long prime editors at the quantities and purities required.
• 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.
• EXAMPLE 11.1 – Self-templated Enzymatic Ligation [574] In this approach, two functionalized, single-stranded RNA fragments with complementary base pairs are hybridized for ligase-mediated conjugation (Scheme 26). In here, the full-length PEgRNA is split into two or more fragments that take natural advantage of the sequence complementarity driven structure of either the Tetraloop or Stem-loop 2 features within the scaffold of a PEgRNA to help facilitate a very specific ligation reaction between the fragments.
• 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.
• RNA A and 5’-phosphorylated RNA B (0.01nmol, 1 ⁇ L each) were combined in molecular biology grade water (5 ⁇ L) and annealed in a thermocycler.
• the thermocycling program included heating the RNA mixture to 65°C for 3 minutes, followed by snap cooling on ice for 2 minutes. The RNA was maintained on ice for no longer than 5 minutes before enzyme addition.
• 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’s A and B were designed to anneal and form SL2, and thus, the nicked, double-stranded substrate necessary for ligase activity. Furthermore, RNA B was functionalized with a 5’-monophosphate to enable ligation to the native 3’-hydroxyl of RNA A. RNA’s A and B were first pre-annealed and then treated with ligase to catalyze the formation of a phosphodiester bond.
• 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
• 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.
• the templated ligation approach was tested using DNA splints L, M, and N (Table 21) to bridge fragments RNA X and RNA Y (Table 22) and split synthesize pegRNA Z (Table 22).
• 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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