Methods for synthesizing RNA molecules

Abstract

The present disclosure relates to methods of synthesizing medium length RNAs by splint-mediated ligation of RNA fragments.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 62 / 941,174, filed November 27, 2019, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure generally relates to the fields of molecular biology and biotechnology, including the synthesis of nucleic acids and methods for synthesizing RNA molecules (also known as guide RNAs) associated with endonucleases. Background Technology

[0004] The RNA-guided, DNA-targeting principle of CRISPR (Crisis-Repetitive Clusters)-Cas (CRISPR-related) systems has been widely adopted in the field. CRISPR-Cas systems can be divided into two main categories: Class I systems utilize multiple Cas protein complexes (such as Class I, III, and IV CRISPR-Cas systems), and Class II systems utilize a single Cas protein (such as Class II, V, and VI CRISPR-Cas systems). Class II CRISPR-Cas-based systems have been used for genome editing and require Cas peptides or variants guided by customizable guide RNA (gRNA) for programmable DNA targeting. Guide RNAs in Class II CRISPR-Cas-based systems are typically 30–130 nucleotides in length (Chylinski et al. (2013) RNABiology, 10(5):726–737).

[0005] Methods for synthesizing gRNA include, for example, intracellular transcription of exogenous plasmids or solid-phase synthesis using phosphoramide chemistry. Direct chemical synthesis of gRNA allows for the incorporation of chemical modifications, thereby increasing the chemical stability of the RNA, reducing its immunogenicity, and minimizing potential off-target effects (i.e., cleavage of genomic DNA at undesirable sites). One limitation of the chemical synthesis of certain sequences (such as gRNA) is the desired length of the single-stranded RNA, which is typically around 60 to 100 nucleotides (nts) for gRNA. For example, if the phosphoramide chemistry method used has a length of approximately 0.99... X Given the coupling efficiency (where X is the number of nucleotides), when synthesizing gRNAs approximately 100 nucleotides in length, the entire synthesis process is expected to produce about 30-40% full-length product (FLP). For RNA molecules approximately 100 nucleotides in length, it is currently impossible to completely separate the FLP from the remaining byproducts formed by incomplete coupling (truncated products) and deprotection using standard purification methods (e.g., chromatography). Due to these limitations, there is a need to design more efficient methods for synthesizing gRNAs. Summary of the Invention

[0006] The applicant has discovered improved methods for synthesizing RNA, particularly medium-length RNA (mlRNA), such as guide RNA for gene editing. Therefore, this disclosure provides a method for synthesizing mlRNA using a splint-mediated ligation of two or more RNA fragments. In some aspects, this disclosure provides a method for synthesizing mlRNA using a splint-mediated ligation of two or three RNA fragments. Such a method may include, for example: providing a first RNA fragment including a terminal region containing a 5' phosphate moiety, and a second RNA fragment including a terminal region containing a 3' hydroxyl group, wherein the first RNA fragment, the second RNA fragment, or both, at least includes a portion of a sequence capable of, for example, binding to an RNA-guided endonuclease; providing a splint oligonucleotide including a first portion of a terminal region complementary to the first RNA fragment, the terminal region including a 5' phosphate moiety, and a second portion of a terminal region complementary to the second RNA fragment, the terminal region including a 3' hydroxyl group; hybridizing the first RNA fragment, the second RNA fragment, and the splint oligonucleotide together to form a complex; and using a ligase to ligate the first and second RNA fragments at a ligation site present in the complex, thereby synthesizing mlRNA or a portion of mlRNA.

[0007] In some aspects, the method includes providing: (a) a first RNA fragment comprising a terminal region containing a 3' hydroxyl group; (b) a second RNA fragment comprising: (i) a terminal region containing a 5' phosphate moiety, and (ii) a terminal region containing a 3' hydroxyl group; and (c) a third RNA fragment comprising a terminal region containing a 5' phosphate moiety; (d) a first splice oligonucleotide comprising (i) a first portion complementary to the terminal region containing the 3' hydroxyl group of the first RNA fragment; (ii) a second portion complementary to the first terminal region containing the 5' phosphate moiety of the second RNA fragment; and (e) a second splice oligonucleotide comprising (i) a first portion complementary to the second terminal region containing the 3' hydroxyl group of the second RNA fragment; and (i) a second portion complementary to the first terminal region containing the 5' phosphate moiety of the second RNA fragment; and (i) a third RNA fragment comprising a first portion complementary to the second terminal region containing the 3' hydroxyl group of the second RNA fragment; and (i) a third RNA fragment comprising ... i) a second portion complementary to the terminal region of the 5' phosphate portion containing the third RNA fragment; and (f) a ligase, wherein the first RNA fragment, the second RNA fragment, the third RNA fragment, the first splice oligonucleotide, and the second splice oligonucleotide are hybridized together to form a complex, the complex comprising a first linker site between the 3' hydroxyl group of the first RNA fragment and the 5' phosphate group of the second RNA fragment, and a second linker site between the 3' hydroxyl group of the second RNA fragment and the 5' phosphate group of the third RNA fragment, wherein the ligase causes the first and second RNA fragments to be joined at the first linker site, and the second and third RNA fragments to be joined at the second linker site, thereby synthesizing mlRNA, or a portion of mlRNA. In some aspects, the first RNA fragment, the second RNA fragment, the third RNA fragment, or a combination thereof comprises at least, for example, a portion of a sequence that binds to an RNA-guided endonuclease (e.g., Cas9). In some aspects, the first RNA fragment, the second RNA fragment, the third RNA fragment, or a combination thereof comprises a spacer region sequence that targets a target sequence in a target DNA (e.g., a genomic DNA molecule).

[0008] In one aspect, this document provides a method for synthesizing guide RNA (gRNA), the method comprising: providing a first RNA fragment comprising a terminal region containing a 5' phosphate moiety, and a second RNA fragment comprising a terminal region containing a 3' hydroxyl group, wherein the first RNA fragment, the second RNA fragment, or both contain at least a portion of a sequence capable of binding to an RNA-guided endonuclease; providing a splice oligonucleotide comprising a first portion of a terminal region complementary to the first RNA fragment, the terminal region comprising a 5' phosphate moiety, and a second portion of a terminal region complementary to the second RNA fragment, the terminal region comprising a 3' hydroxyl group; and hybridizing the first RNA fragment, the second RNA fragment, and the splice oligonucleotide together to form a complex; and using a ligase to ligate the first and second RNA fragments at a linker site between the RNA complexes, thereby synthesizing gRNA. In some embodiments, the length of each of the first and second RNA fragments is 10 to 90 nucleotides. In some embodiments, the length of the second RNA fragment is 40 nucleotides or less. In some embodiments, the 5' phosphate moiety is a 5'-phosphate or a 5'-thiophosphate. In some embodiments, the ligase is T4 DNA ligase, T4 RNA ligase I, or T4 RNA ligase II. In some embodiments, the splice oligonucleotide is a DNA or RNA oligonucleotide. In some embodiments, the splice oligonucleotide is 20 to 100 nucleotides in length. In some embodiments, the splice oligonucleotide is attached to a solid carrier. In some embodiments, the gRNA is 30 to 160 nucleotides in length. In some embodiments, the gRNA contains a sequence complementary to a sequence in the target DNA. In some embodiments, the target DNA is mammalian DNA. In some embodiments, the target DNA is human DNA. In some embodiments, the ligation site corresponds to a site in the tetraloop portion of the stem-loop structure in the synthesized gRNA. In some embodiments, the ligation site corresponds to a site in the helical portion of the stem-loop structure in the synthesized gRNA. In some embodiments, the first RNA fragment, the second RNA fragment, or both contain at least one secondary structure, and hybridizing the first RNA fragment, the second RNA fragment, and the splice oligonucleotide produces a free energy lower than that of the secondary structure with the lowest free energy. In some embodiments, the method includes ligating three or more RNA fragments. In some embodiments, providing the first and second RNA fragments includes synthesizing the first and second RNA fragments by enzymatic synthesis or phosphoramide chemistry. In some embodiments, the second RNA fragment is synthesized in a 5' to 3' or 3' to 5' orientation. In some embodiments, providing the first and second RNA fragments includes purifying the first and second fragments after synthesis. In some embodiments, providing the splint oligonucleotide includes synthesizing the splint oligonucleotide by enzymatic synthesis or phosphoramide chemistry.In some embodiments, providing the splice oligonucleotide includes purifying the splice oligonucleotide after synthesis. In some embodiments, purification includes purification by chromatographic methods. In some embodiments, the chromatographic methods are reversed-phase HPLC, ion-exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, affinity chromatography, or polyacrylamide gel purification, or any combination thereof. In some embodiments, the first RNA fragment, the second RNA fragment, or both contain at least one modification in the RNA backbone. In some embodiments, the modification is selected from the group consisting of: 2'-methoxy (2'OMe), 2'-fluoro (2'fluoro), 2'-O-methoxy-ethyl (MOE), locked nucleic acid (LNA), unlocked nucleic acid (UNA), bridging nucleic acid, 2'-deoxynucleic acid (DNA), and peptide nucleic acid (PNA). In some embodiments, the first RNA fragment, the second RNA fragment, or both contain at least one base modification. In some embodiments, the base modification is selected from the group consisting of: 2-aminopurine, inosine, thymine, 2,6-diaminopurine, 2-pyrimidinone, and 5-methylcytosine. In some embodiments, the first RNA fragment, the second RNA fragment, or both contain at least one phosphate-thioester bond. In some embodiments, hybridization includes hybridization in solution. In some embodiments, the concentrations of the splice oligonucleotide, the first RNA fragment, and the second RNA fragment in solution are approximately equal. In some embodiments, the ligation of the first and second RNA fragments is performed at 15°C–45°C. In some embodiments, the ligation of the first and second RNA fragments is performed at about 37°C. In some embodiments, the ligation of the first and second RNA fragments is carried out for about 0.1 to about 48 hours. In some embodiments, the ligation of the first and second RNA fragments further includes the use of a protease or a chelating agent. In some embodiments, the chelating agent is EDTA, EGTA, or a combination of both. In some embodiments, the ligation of the first and second RNA fragments further includes the use of one or more crowding agents. In some embodiments, one or more crowding agents include polyethylene glycol (PEG). Ethylene glycol, dextran, or any combination thereof. In some embodiments, the ligation of the first and second RNA fragments is performed to at least 10% completion. In some embodiments, the ligation of the first and second RNA fragments is performed to at least 90% completion. In some embodiments, the method further includes purifying the gRNA after synthesis. In some embodiments, the purification of the gRNA includes purification using chromatographic methods. In some embodiments, the chromatographic methods are reversed-phase HPLC, ion-exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, affinity chromatography, or polyacrylamide gel purification, or any combination thereof. In some embodiments, the RNA-guided endonuclease is a small Cas nuclease or a small RNA-guided endonuclease. In some embodiments, the RNA-guided endonuclease is selected from the group consisting of Cas9, Cas12, aCas13, and their variants. In some embodiments, the RNA-guided endonuclease is Streptococcus pyogenes Cas9 (SpyCas9) or Staphylococcus aureus (SaCas9). In some implementations, the RNA-guided endonuclease is a variant of Cas9, and the variant of Cas9 is selected from the group consisting of: small Cas9, dead Cas9 (dCas9), and Cas9 nickase.

[0009] In another aspect, this disclosure provides a method for synthesizing gRNA, the method comprising providing (a) a first RNA fragment comprising a terminal region containing a 3' hydroxyl group; (b) a second RNA fragment comprising a first terminal region containing a 5' phosphate moiety and a second terminal region containing a 3' hydroxyl group; (c) a third RNA fragment comprising a terminal region containing a 5' phosphate moiety; (d) a first splice oligonucleotide comprising (i) a first portion complementary to the terminal region containing the 3' hydroxyl group of the first RNA fragment; and (ii) a second portion complementary to the first terminal region containing the 5' phosphate moiety of the second RNA fragment; and (e) a second splice oligonucleotide comprising (i) a second terminal region containing the 3' hydroxyl group of the second RNA fragment. The gRNA comprises (i) a first region complementary to the terminal region of the 5' phosphate portion of the third RNA fragment; and (ii) a second region complementary to the terminal region of the third RNA fragment containing the 5' phosphate portion; and (f) a ligase, wherein hybridization of the first, second, and third RNA fragments with the first and second splice oligonucleotides results in the formation of a complex having a first linker site between the 3' hydroxyl group of the first RNA fragment and the 5' phosphate group of the second RNA fragment, and a second linker site between the 3' hydroxyl group of the second RNA fragment and the 5' phosphate group of the third RNA fragment; and wherein the ligase causes the ligation of the first and second RNA fragments at the first linker site, and the ligation of the second and third RNA fragments at the second linker site, thereby synthesizing gRNA. In some embodiments, the gRNA comprises a first RNA fragment 5' to 3' linked to the second RNA fragment via a first phosphodiester bond and a second RNA fragment linked to the third RNA fragment via a second phosphodiester bond. In some embodiments, the first phosphodiester bond is formed between the 3' hydroxyl group of the first RNA fragment and the 5' phosphate group of the second RNA fragment, and wherein the second phosphodiester bond is formed between the 3' hydroxyl group of the second RNA fragment and the 5' phosphate group of the third RNA fragment. In some embodiments, the gRNA is a single-molecule gRNA (sgRNA). In some embodiments, the sgRNA is about 30 to about 160 nucleotides in length, or about 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or 160 nucleotides in length. In some embodiments, the first linker site corresponds to a site in a first stem-loop structure, wherein the first stem-loop structure is formed by hybridization of the smallest CRISPR repeat sequence and the smallest tracrRNA sequence in the synthesized gRNA. In some embodiments, the site in the first stem-loop structure is in a tetraloop portion or a helical portion. In some embodiments, the second linker site corresponds to a site in a second stem-loop structure. In some embodiments, the second stem-loop is present in the tracrRNA sequence of the gRNA.In some embodiments, the sites in the second stem-ring structure are in the four-ring portion or the spiral portion.

[0010] In some aspects, this disclosure provides a method for synthesizing a single-molecule guide RNA (sgRNA) conjugated with an RNA-guided endonuclease, the method comprising: providing a complex formed between a first RNA fragment, a second RNA fragment, a third RNA fragment, a first splice oligonucleotide, and a second splice oligonucleotide; and a ligase, wherein (a) the first RNA fragment comprises (i) a terminal region containing a 3' hydroxyl group; (b) the second RNA fragment comprises (i) a first terminal region containing a 5' phosphate moiety, and (ii) a second terminal region containing a 3' hydroxyl group; (c) the third RNA fragment comprises (i) a terminal region containing a 5' phosphate moiety; (d) the first splice oligonucleotide comprises (i) a first portion complementary to the terminal region containing the 3' hydroxyl group of the first RNA fragment, and (ii) a second portion complementary to the first terminal region containing the 5' phosphate moiety of the second RNA fragment; and (e) the second splice oligonucleotide comprises (i) a first portion complementary to the terminal region containing the 3' hydroxyl group of the second ...ii) the second splice oligonucleotide comprises (i) a first portion complementary to the terminal region containing the 3' hydroxyl group of the second RNA fragment, and (ii) a second portion complementary to the first terminal region containing the 5' phosphate moiety of the second RNA fragment; and (iii) the second splice oligonucleotide comprises (i) a first portion complementary to the terminal region containing the 3' hydroxyl group of the second RNA fragment, and (ii) a second portion complementary to the first terminal region containing the 5' phosphate moiety of the second RNA fragment. The complex is formed by hybridization of (a)(i) and (d)(i), (b)(i) and (d)(ii), (b)(ii) and (e)(i), and (c)(i) and (e)(ii), wherein the complex has a first linker site present between the 3' hydroxyl group of the first RNA fragment and the 5' phosphate group of the second RNA fragment, and a second linker site present between the 3' hydroxyl group of the second RNA fragment and the 5' phosphate group of the third RNA fragment, wherein a ligase induces ligation at the first linker site and ligation at the second linker site to form an sgRNA comprising, from 5' to 3': a spacer sequence and an invariant sequence for binding RNA-guided endonuclease; the invariant sequence comprising a stem loop formed between a crRNA repeat sequence and a tracrRNA anti-repeat sequence, and a 3' tracrRNA sequence comprising at least one stem loop, thereby synthesizing an sgRNA for use with an RNA-guided endonuclease.

[0011] In any of the foregoing or related aspects, the first linker site corresponds to a site in a stem loop formed between the crRNA repeat sequence and the tracrRNA anti-repetitive sequence. In some embodiments, the first linker site corresponds to a site in the 5' of the stem loop, the fourth loop of the stem loop, or the 3' stem of the stem loop. In some embodiments, the 3' tracrRNA sequence comprises a first stem loop, a second stem loop, and a third stem loop. In some embodiments, the 3' tracrRNA sequence consists of a first stem loop, a second stem loop, and a third stem loop. In some embodiments, the second linker site corresponds to a site in the first stem loop, the second stem loop, or the third stem loop. In some embodiments, the second linker site corresponds to a site in the second stem loop. In some embodiments, the site is in the 5' stem of the second stem loop, in the fourth loop of the second stem loop, or in the 3' stem of the second stem loop. In some embodiments, the second linker site corresponds to a site adjacent to the 5' base of the second stem-loop (e.g., ±1 nt, ±2 nt, ±3 nt from the 5' base of the second stem-loop) or to the 3' base of the second stem-loop (e.g., ±1 nt, ±2 nt, ±3 nt from the 3' base of the second stem-loop). In some embodiments, the first RNA fragment comprises a nucleotide sequence as the 5' first linker site. In some embodiments, the second RNA fragment comprises a nucleotide sequence located between the first and second linker sites. In some embodiments, the third RNA fragment comprises a nucleotide sequence from the 3' to the second linker site.

[0012] In any of the foregoing or related aspects, the terminal region of (a)(i) comprises a nucleotide sequence of about 10 to about 30 nucleotides located at the 3' end of the first RNA fragment. In some embodiments, the terminal region of (a)(i) comprises a spacer region sequence of sgRNA. In some embodiments, the terminal region of (a)(i) does not comprise a spacer region sequence of sgRNA. In some embodiments, the 5' end of the spacer region sequence is aligned with the 5' end of the first RNA fragment, wherein the terminal region of (a)(i) comprises a spacer region sequence of sgRNA. In some embodiments, the terminal region of (a)(i) extends from the 3' end of the first RNA fragment to include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nt upstream of the 3' end of the spacer region sequence. In some embodiments, the terminal region of (a)(i) extends from the 3' end of the first RNA fragment to the 3' end of the immediately adjacent spacer region sequence. In some embodiments, the first portion of (d)(i) is completely complementary to the terminal region of (a)(i). In some embodiments, the first portion of (d)(i) has one, two, or three mismatches relative to the terminal region of (a)(i). In some embodiments, the terminal region of (b)(i) comprises a nucleotide sequence of about 10 to about 30 nucleotides located at the 5' end of the second RNA fragment. In some embodiments, the second portion of (d)(ii) is completely complementary to the terminal region of (b)(i). In some embodiments, the second portion of (d)(ii) has one, two, or three mismatches relative to the terminal region of (d)(ii). In some embodiments, the terminal region of (b)(ii) comprises a nucleotide sequence of about 10 to about 30 nucleotides located at the 3' end of the second RNA fragment. In some embodiments, the first portion of (e)(i) is completely complementary to the terminal region of (b)(ii). In some embodiments, the first portion of (e)(i) has one, two, or three mismatches relative to the terminal region of (b)(ii). In some embodiments, the terminal region of (c)(i) comprises a nucleotide sequence of about 10 to about 40 nucleotides located at the 5' end of the third RNA fragment. In some embodiments, the second portion of (e)(ii) is completely complementary to the terminal region of (c)(i). In some embodiments, the second portion of (e)(ii) has one, two, or three mismatches relative to the terminal region of (c)(i).

[0013] In any of the foregoing or related aspects, the lengths of the first RNA fragment, the second RNA fragment, and the third RNA fragment are each independently about 10 to about 90 nucleotides, about 10 to about 60 nucleotides, about 10 to about 50 nucleotides, about 10 nucleotides to about 40 nucleotides, about 20 to about 40 nucleotides, and about 30 to about 40 nucleotides. In some embodiments, the first splice oligonucleotide is a DNA or RNA oligonucleotide, and the second splice oligonucleotide is a DNA or RNA oligonucleotide. In some embodiments, the lengths of the first splice oligonucleotide and the second splice oligonucleotide are each independently about 20 to about 100 nucleotides, about 20 to about 90 nucleotides, about 20 to about 80 nucleotides, about 20 to about 70 nucleotides, about 20 to about 60 nucleotides, about 30 to about 60 nucleotides, or about 30 to about 50 nucleotides.

[0014] In any of the foregoing or related aspects, the gRNA or sgRNA contains a spacer sequence complementary to a sequence in the target DNA. In some embodiments, the target DNA is mammalian DNA or human DNA. In some embodiments, the RNA-guided endonuclease is a small Cas nuclease or a small RNA-guided endonuclease. In some embodiments, the RNA-guided endonuclease is selected from the group consisting of Cas9, Cas12, aCas13, and variants thereof. In some embodiments, the RNA-guided endonuclease is Streptococcus pyogenes Cas9 (SpyCas9) or Staphylococcus aureus (SaCas9). In some embodiments, the RNA-guided endonuclease is a variant of Cas9, and the variant of Cas9 is selected from the group consisting of small Cas9, dead Cas9 (dCas9), and Cas9 cleavage enzyme.

[0015] In any of the foregoing or related aspects, the RNA-guided endonuclease is SpyCas9. In some embodiments, the invariant sequence comprises the nucleotide sequence of SEQ ID NO:17. In some embodiments, the invariant sequence comprises a nucleotide sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide deletions, insertions, or substitutions relative to SEQ ID NO:17. In some embodiments, the first RNA fragment, the second RNA fragment, and the third RNA fragment are each selected from nucleotide sequences comprising: (a)(i)N 15-30 GUUUUAGAGCUAG(SEQ ID NO:56), where N 15-30 Corresponding to the spacer sequence; (ii) SEQ ID NO:3; and (iii) SEQ ID NO:4; (b)(i) N 15-30 GUUUUAGAGCUAGA(SEQ ID NO:57), where N 15-30Corresponding to the spacer sequence; (ii) SEQ ID NO:40; (iii) SEQ ID NO:42; (c)(i) N 15-30 GUUUUAGAGCUAG(SEQID NO:56), where N 15-30 Corresponding to the spacer sequence; (ii) SEQ ID NO:58; (iii) SEQ ID NO:42; or (d)(i)N 15-30 GUUUUAGAGCUAGA(SEQ ID NO:57), where N 15-30 Corresponding to the spacer region sequence; (ii) SEQ ID NO:59; (iii) SEQ ID NO:4. In some embodiments, the spacer region sequence targets a target site in a target nucleic acid molecule (e.g., genomic DNA). In some embodiments, the spacer region sequence is about 10 to about 30 nucleotides in length. In some embodiments, the spacer region sequence is 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the spacer region sequence is 19 nucleotides in length. In some embodiments, the spacer region sequence is 20 nucleotides in length. In some embodiments, the spacer region sequence is 21 nucleotides in length. In some embodiments, the spacer region sequence is 22 nucleotides in length. In some embodiments, the first splint oligonucleotide comprises the nucleotide sequence listed in SEQ ID NO:60; SEQ ID NO:44; or SEQ ID NO:61. In some embodiments, no portion of the first splint oligonucleotide is complementary to the spacer region sequence. In some embodiments, the first splint oligonucleotide further includes a 3' end having a nucleotide sequence complementary to the spacer sequence or to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides present at the 3' end of the spacer sequence.

[0016] In any of the foregoing or related aspects, the first RNA fragment, the second RNA fragment, and / or the third RNA fragment comprises at least one secondary structure, wherein the complex formed by hybridizing the first, second, and third RNA fragments with the first and second splice oligonucleotides has a free energy lower than the free energy of the secondary structure having the lowest free energy. In some embodiments, providing the first, second, and third RNA fragments comprises synthesizing the RNA fragments using enzymatic synthesis or phosphoramidite chemistry. In some embodiments, providing the RNA fragments further comprises purifying the RNA fragments after synthesis. In some embodiments, synthesizing the RNA fragments using phosphoramidite chemistry comprises synthesizing the first RNA fragment, the second RNA fragment, and the third RNA fragment, respectively, in a 5' to 3' or 3' to 5' orientation. In some embodiments, synthesizing the RNA fragments using phosphoramidite chemistry comprises synthesizing the first RNA fragment in a 5' to 3' or 3' to 5' orientation, and synthesizing the second and third RNA fragments, respectively, in a 3' to 5' orientation. In some embodiments, providing the first and second splice oligonucleotides comprises synthesizing the oligonucleotides using enzymatic synthesis or phosphoramidite chemistry. In some embodiments, providing the splice oligonucleotides further comprises purifying the oligonucleotides after synthesis. In some embodiments, the first, second, and / or third RNA fragments contain at least one modification in the RNA backbone. In some embodiments, the modification is selected from the group consisting of: 2'-methoxy (2'OMe), 2'-fluoro, 2'-O-methoxy-ethyl (MOE), locked nucleic acid (LNA), unlocked nucleic acid (UNA), bridging nucleic acid, 2'-deoxynucleic acid (DNA), and peptide nucleic acid (PNA). In some embodiments, the modification is 2'-O-methylation of one or more nucleotides present in the RNA backbone. In some embodiments, the first, second, and / or third RNA fragments contain at least one base modification. In some embodiments, the base modification is selected from the group consisting of: 2-aminopurine, inosine, thymine, 2,6-diaminopurine, 2-pyrimidinone, and 5-methylcytosine. In some embodiments, the first, second, and / or third RNA fragments contain at least one phosphate thioester bond. In some embodiments, hybridization is performed in solution. In some embodiments, hybridization is performed without an annealing step. In some implementations, hybridization is performed with an annealing step.In some embodiments, the annealing step includes (i) heating the solution to about 80°C to about 95°C for a period of less than about 10 minutes (e.g., 1, 2, 3, 4, or 5 minutes); and (ii) cooling the solution to the temperature for ligation (e.g., about 15°C to about 40°C, or about 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C) at a rate of about 0.1°C / second to about 2°C / second (e.g., 1°C / second). In some embodiments, the concentrations of the first splice oligonucleotide, the second splice oligonucleotide, the first RNA fragment, the second RNA fragment, and the third RNA fragment in the solution are approximately equal. In some embodiments, the concentration is about 5 μM to about 50 μM. In some embodiments, the concentration is about 5 μM, about 10 μM, about 15 μM, about 20 μM, or about 25 μM. In some embodiments, ligation is performed at about 15°C to about 45°C, or about 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C. In some embodiments, ligation is performed for about 0.1 to about 48 hours, or about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 hours. In some embodiments, the ligation of the first and second RNA fragments further includes the use of a protease or a chelating agent. In some embodiments, the chelating agent is EDTA, EGTA, or a combination of both. In some embodiments, the ligation of the first and second RNA fragments further includes the use of one or more crowding agents. In some embodiments, one or more crowding agents include polyethylene glycol (PEG). Ethylene glycol, dextran, or any combination thereof. In some embodiments, the ligation is performed to at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% completion. In some embodiments, the method further includes purifying the gRNA or sgRNA after synthesis. In some embodiments, purifying the gRNA or sgRNA includes purification using chromatographic methods. In some embodiments, the chromatographic methods are reversed-phase HPLC, ion-exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, affinity chromatography, or polyacrylamide gel purification, or any combination thereof.

[0017] On the other hand, this document provides a method for generating a bimolecular gRNA comprising crRNA and tracrRNA, the method comprising: providing a first RNA fragment comprising a terminal region containing a 5' phosphate moiety, and a second RNA fragment comprising a terminal region containing a 3' hydroxyl group, wherein the first RNA fragment, the second RNA fragment, or both comprise at least one sequence portion capable of binding to an RNA-guided endonuclease; providing a splice oligonucleotide comprising a first portion of a terminal region complementary to the first RNA fragment, the terminal region comprising a 5' phosphate moiety, and a second portion of a terminal region complementary to the second RNA fragment, the terminal portion comprising a 3' hydroxyl group; hybridizing the first RNA fragment, the second RNA fragment, and the splice oligonucleotide together to form a complex; ligating the first and second RNA fragments at a ligation site present between the RNA complexes using a ligase, thereby synthesizing tracrRNA; providing crRNA comprising a sequence complementary to a sequence in a target DNA; and hybridizing tracrRNA and crRNA to generate a bimolecular gRNA. In some embodiments, providing crRNA comprises synthesizing crRNA by enzymatic synthesis or by phosphoramidite chemistry.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While similar or equivalent methods and materials to those described herein may be used in the practice or testing of this invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of conflict, this specification (including definitions) shall prevail. Furthermore, materials, methods, and examples are illustrative only and are not intended to be limiting.

[0019] Other features and advantages of the invention will become clear from the following detailed description and claims. Attached Figure Description

[0020] Figure 1 This is a schematic diagram illustrating the synthesis of gRNA molecules by using a splice oligonucleotide to link two RNA fragments.

[0021] Figure 2 This is a schematic diagram illustrating the synthesis of gRNA molecules by linking three RNA fragments using two splice oligonucleotides.

[0022] Figure 3 This is a schematic diagram showing the use of splice oligonucleotides to generate an invariant RNA construct, which is then linked to another RNA fragment to produce a gRNA molecule.

[0023] Figure 4This is an illustrative diagram showing the positions used to split an exemplary gRNA molecule (SEQ ID NO:16) into RNA fragments (RNA fragment 1, RNA fragment 2, and RNA fragment 3, having the nucleotide sequences listed in SEQ ID NO:56, 3, and 4, respectively).

[0024] Figure 5 This is a chromatogram showing the HPLC analysis results of splint-mediated RNA fragment 1-3 ligation before and after the ligation.

[0025] Figure 6 The image shows the cleavage of a plasmid containing the target DNA sequence by SpCas9 and the resulting gRNA generated using splint-mediated ligation.

[0026] Figure 7A and 7B Examples of splint-mediated ligation of RNA modification and modified RNA fragments are shown. Figure 7A The structures of unmodified ribonucleotides, 2'-O-methyl ribonucleotides, and phosphate thioester modified ribonucleotides are shown. Figure 7B This is a chromatogram showing the HPLC analysis results of the splice-mediated linkage products.

[0027] Figure 8 This is a chromatogram showing the HPLC analysis results of the splice-mediated ligation products before and after purification.

[0028] Figures 9A-9B A clamp-mediated ligation between three modified RNA fragments (RNA 1, RNA 2, and RNA 3) and two DNA clamp oligonucleotides (DNA clamp 1 and DNA clamp 2) was provided to generate a modified single-molecule gRNA (sgRNA) for use with Streptococcus pyogenes Cas9 (SpyCas9) to target the human G6PC gene. The sequences of RNA 1 (SEQ ID NO:11), RNA 2 (SEQ ID NO:12), and RNA 3 (SEQ ID NO:13) and the ligation site between these RNA fragments are shown in [the diagram]. Figure 9B middle.

[0029] Figure 10 It is shown in Figures 9A-9B The HPLC analysis results of the clamp-mediated ligation reaction are shown. For (i) before the addition of ligase (“before ligation”); (ii) after the addition of ligase with or without magnesium salt (“with Mg salt”). 2+ "After connection" and "without Mg" 2+(iii) After ligation, the mixture was analyzed ("non-annealed ligation"), or after the addition of ligase without prior annealing of the RNA fragment and DNA clip ("non-annealed ligation"). The full-length sgRNA (SEQ ID NO:20) corresponded to a peak with a retention time of 30.58 minutes.

[0030] Figure 11 Alignment of three RNA fragments and two DNA clips forming an RNA / DNA complex for clip-mediated ligation to synthesize sgRNA is provided. The 5' to 3' nucleotide sequences are the DNA version of the final sgRNA product (SEQ ID NO:19). sgRNA is synthesized by hybridization of RNA 1, RNA 2, and RNA 3 with DNA clips 1 and 2, followed by ligation at a ligation site between the 3' end of RNA 1 and the 5' end of RNA 2, and between the 3' end of RNA 2 and the 5' end of RNA 3. The nucleotide sequences of DNA clips 1 and 2 are shown in the 3' to 5' orientation, corresponding to SEQ ID NO:52 and 53, respectively. Detailed Implementation

[0031] This disclosure provides a method for synthesizing RNA, particularly intermediate-length RNA (mlRNA), such as guide RNA (gRNA), by ligating RNA fragments using one or more splice oligonucleotides and a ligase. In some cases, one or more RNA fragments include at least a portion of a sequence capable of binding to an RNA-guided endonuclease. In some embodiments, one or more RNA fragments contain a spacer region sequence for targeting a target sequence in a target DNA (e.g., a genomic DNA molecule).

[0032] For example, current methods for synthesizing mlRNA include intracellular transcription of exogenous plasmids or solid-phase synthesis using phosphoramide chemistry. One limitation of the chemical synthesis of mlRNA is the length of the resulting single-stranded RNA. For example, if the phosphoramide chemistry method used has a length of approximately 0.99... XGiven the coupling efficiency (where X is the number of nucleotides), when synthesizing RNA of approximately 100 nucleotides in length, the entire synthesis process is expected to produce approximately 30-40% full-length product (FLP). For RNA molecules of approximately 100 nucleotides in length, it is currently impossible to completely separate the FLP from the residual byproducts formed by incomplete coupling (truncated products) and deprotection using standard purification methods (e.g., chromatography). This disclosure provides a more efficient method for synthesizing mlRNA that increases the yield of full-length products and reduces the amount of truncated products produced. Furthermore, this document demonstrates that the method of this disclosure provides the synthesis of unmodified mlRNA (e.g., gRNA or sgRNA) and mlRNA containing one or more chemical modifications, such as backbone modifications (e.g., phosphate thioester bonds) and / or nucleoside modifications (e.g., 2'-O-methylation).

[0033] This disclosure is also based, at least in part, on the discovery that single-molecule gRNAs (sgRNAs) conjugated with RNA-guided endonucleases (e.g., Cas9) are efficiently synthesized using the splint-mediated ligation method described herein, such as by ligating two or three RNA fragments using one or more splint oligonucleotides and a ligase. In some aspects, the ligation comprises two RNA fragments, a splint oligonucleotide, and a ligase, wherein the two RNA fragments hybridize with the splint oligonucleotide to form a complex containing a ligation site, and wherein the ligase induces a ligation at the ligation site to form an sgRNA conjugated with an RNA-guided endonuclease. In some aspects, the sgRNA comprises a nucleotide sequence at a 5' ligation site and a nucleotide sequence at a 3' ligation site, wherein a first RNA fragment corresponds to the nucleotide sequence at the 5' ligation site, and a second RNA fragment corresponds to the nucleotide sequence at the 3' ligation site, wherein the ligation at the ligation site is capable of joining the first and second RNA fragments to form the nucleotide sequence of the sgRNA.

[0034] In some aspects, the ligation comprises three RNA fragments, two splice oligonucleotides, and a ligase, wherein the three RNA fragments hybridize with the two splice oligonucleotides to form a complex containing first and second linkage sites, and wherein the ligase induces ligation at the first and second linkage sites to form an sgRNA conjugated with an RNA-guided endonuclease. In some aspects, the sgRNA comprises nucleotide sequences of a 5' first linkage site, 3' first linkage sites, 5' second linkage sites, and 3' second linkage sites, wherein the first RNA fragment corresponds to the nucleotide sequence of the 5' first linkage site, the second RNA fragment corresponds to the nucleotide sequences of the 3' first linkage site and 5' second linkage site, and the third RNA fragment corresponds to the nucleotide sequence of the 3' second linkage site, wherein ligation at the first and second linkage sites enables the first, second, and third RNA fragments to be joined to form the nucleotide sequence of the sgRNA.

[0035] In some aspects, the nucleotide sequence of sgRNA comprises 5' to 3': a spacer region sequence for targeting a target site in a nucleic acid molecule (e.g., a genomic DNA molecule) and an invariant sequence for binding RNA-guided endonucleases, said invariant sequence comprising 5' to 3': a stem-loop formed between a CRISPR repeat sequence and a tracrRNA anti-repetition sequence, and a tracrRNA containing at least one stem-loop. In some aspects, the splint-mediated ligation method provides at least one RNA fragment, or a combination of RNA fragments, comprising the spacer region sequence; and at least one RNA fragment, or a combination of RNA fragments, comprising the invariant sequence.

[0036] In some respects, splint-mediated ligation methods involve placing a ligation site in the sgRNA within or near a stem-loop in an invariant sequence of the sgRNA (e.g., within or near a stem-loop formed between a CRISPR repeat and a tracrRNA anti-repetition sequence; e.g., within or near a stem-loop in tracrRNA). As described herein, placing the ligation site in a stem-loop prevents the formation of secondary structures in the RNA fragment (i.e., the RNA fragment ligated at the ligation site), which would prevent or be detrimental to hybridization of the RNA fragment with the splint oligonucleotide. For example, disruption of the stem-loop at the ligation site provides an RNA fragment with (i) minimal secondary structure; and / or (ii) a secondary structure with a higher free energy than that produced by hybridization of the RNA fragment and the splint oligonucleotide (i.e., the RNA fragment ligated at the ligation site).

[0037] In some aspects, the splint-mediated ligation method includes placing first and second ligation sites in sgRNA, each located within or near a stem-loop in an invariant sequence of the sgRNA (e.g., a stem-loop formed between a CRISPR repeat sequence and a tracrRNA anti-repetitive sequence; e.g., a stem-loop of tracrRNA), such that placing the first ligation site disrupts the formation of stem-loops in RNA fragments containing nucleotide sequences 5' and 3'; placing the second ligation site disrupts the formation of stem-loops in RNA fragments containing nucleotide sequences 5' and 3', such that the formation of secondary structures in each or all RNA fragments used in the splint-mediated ligation reaction is disadvantageous relative to hybridization with splint oligonucleotides.

[0038] In the following detailed description, reference is made to the accompanying drawings. In the drawings, similar designations generally indicate similar components unless the context otherwise requires. The illustrative alternatives described in the detailed description, drawings, and claims are not intended to be limiting. Other alternatives may be used and other changes may be made without departing from the spirit or scope of the subject matter set forth herein. It will be readily understood that aspects as generally described herein and illustrated in the figures can be arranged, substituted, combined, and designed in a variety of different configurations, all of which are explicitly considered and form part of this application.

[0039] Unless otherwise defined, all technical terms, symbols, and other scientific terms used herein are intended to have the meaning commonly understood by one of ordinary skill in the art to which this application pertains. In some cases, terms with generally understood meanings are defined herein for clarity and / or for ease of reference, and such definitions included herein are not necessarily to be construed as indicating a material difference from what is commonly understood in the art.

[0040] "Medium-length RNA (mlRNA)" refers to RNA molecules with a length of approximately 30 to 160 nucleotides (e.g., approximately 30 to 150, approximately 30 to 140, approximately 30 to 130, approximately 30 to 120, approximately 30 to 110, approximately 30 to 100, approximately 30 to 90, approximately 30 to 80, approximately 30 to 70, approximately 30 to 60, approximately 30 to 50, approximately 30 to 40, approximately 40 to 160, approximately 40 to 150, approximately 40 to 140, approximately 40 to 130, approximately 40 to 120, approximately 40 to 110, approximately 40 to 100, approximately 40 to 90). Approximately 40 to approximately 80, approximately 40 to approximately 70, approximately 40 to approximately 60, approximately 40 to approximately 50, approximately 50 to approximately 160, approximately 50 to approximately 150, approximately 50 to approximately 140, approximately 50 to approximately 130, approximately 50 to approximately 120, approximately 50 to approximately 110, approximately 50 to approximately 100, approximately 50 to approximately 90, approximately 50 to approximately 80, approximately 50 to approximately 70, approximately 50 to approximately 60, approximately 60 to approximately 160, approximately 60 to approximately 150, approximately 60 to approximately 140, approximately 60 to approximately 130, approximately 60 to approximately 120, approximately 60 to approximately 110, approximately 60 to approximately 100, approximately 60 to approximately 90, approximately 60 to approximately 80, approximately 60 to approximately 70. Approximately 70 to approximately 160, approximately 70 to approximately 150, approximately 70 to approximately 140, approximately 70 to approximately 130, approximately 70 to approximately 120, approximately 70 to approximately 110, approximately 70 to approximately 100, approximately 70 to approximately 90, approximately 70 to approximately 80, approximately 80 to approximately 160, approximately 80 to approximately 150, approximately 80 to approximately 140, approximately 80 to approximately 130, approximately 80 to approximately 120, approximately 80 to approximately 110, approximately 80 to approximately 100, approximately 80 to approximately 90, approximately 90 to approximately 160, approximately 90 to approximately 150, approximately 90 to approximately 140, approximately 90 to approximately 130, approximately 90 to approximately 120, approximately 90 to approximately 110, approximately 90 to approximately 100, approximately 100 to about 160, about 100 to about 150, about 100 to about 140, about 100 to about 130, about 100 to about 120, about 100 to about 110, about 110 to about 160, about 110 to about 150, about 110 to about 140, about 110 to about 130, about 110 to about 120, about 120 to about 160, about 120 to about 150, about 120 to about 140, about 120 to about 130, about 130 to about 160, about 130 to about 150, about 130 to about 140, about 140 to about 160, about 140 to about 150, or about 150 to about 160 nucleotides).

[0041] "RNA-guided endonuclease" refers to a polypeptide capable of binding RNA (e.g., gRNA) to form a complex that targets a specific DNA sequence (e.g., within the target DNA). An exemplary RNA-guided endonuclease is a Cas polypeptide (e.g., a Cas endonuclease, such as Cas9 endonuclease). Therefore, in some embodiments, RNA-guided endonucleases as described herein target a specific DNA sequence within the target DNA via an RNA molecule with which they bind. The RNA molecule includes a sequence complementary to and capable of hybridizing with the target sequence within the target DNA, thereby allowing the bound polypeptide to be targeted to a specific location within the target DNA.

[0042] As used herein, "guide RNA" or "gRNA" is a site-specific target RNA that can bind to an RNA-guided endonuclease to form a complex, directing the activity of the bound RNA-guided endonuclease (e.g., Cas endonuclease) to a specific target sequence within the target nucleic acid. Guide RNA may comprise one or more RNA molecules.

[0043] As used in this article, the “secondary structure” of a nucleic acid molecule (e.g., an RNA fragment or gRNA) refers to the base-pairing interactions within the nucleic acid molecule.

[0044] As used herein, “target DNA” is DNA that includes a “target site” or a “target sequence.” The term “target sequence” as used herein refers to a nucleic acid sequence present in the target DNA that can hybridize with a sequence or region of the gRNA’s target DNA (also referred to herein as a “spacer region”) provided sufficient hybridization conditions are present. For example, the target sequence 5'-GAGCATATC-3' within the target DNA may be targeted (or able to hybridize with or be complementary to) the RNA sequence 5'-GAUAUGCUC-3'. For example, hybridization between the gRNA’s target DNA sequence or region and the target sequence can be based on the Watson-Crick base pairing rules, which allows for programmability of the target DNA sequence or region. For example, the gRNA’s target DNA sequence or region can be designed to hybridize with any target sequence.

[0045] As used herein, the terms “Cas endonuclease” or “Cas nuclease” include, but are not limited to, for example, RNA-guided DNA endonucleases associated with the CRISPR adaptive immune system.

[0046] Unless otherwise stated, “nuclease” and “endonuclease” are used interchangeably in this document and refer to enzymes that have endonuclease catalytic activity for the cleavage of polynucleotides.

[0047] As used in this article, "cutting" refers to the breakage of the covalent backbone of a DNA molecule. Both single-strand and double-strand cuts are possible, and a double-strand cut can occur due to two different single-strand cutting events.

[0048] The term "domain" is used in this document to describe a segment of a protein or nucleic acid. Unless otherwise stated, a domain is not required to have any specific functional properties.

[0049] A "sandwich oligonucleotide" is an oligonucleotide that acts as a "sandwich" when hybridizing with other polynucleotides (e.g., RNA fragments), placing the ends of the polynucleotides adjacent to each other so that they can be linked together. A sandwich oligonucleotide can be any oligomer that hybridizes with polynucleotides via Watson-Crick base pairing interactions. Sandwich oligonucleotides can be DNA, RNA, non-natural or artificial nucleic acids (e.g., peptide nucleic acids). Sandwich oligonucleotides can include nucleotide sequences that are partially complementary to the nucleotide sequences of two or more different oligonucleotides. Typically, two nucleotide sequences can be linked together using RNA ligase, DNA ligase, or other types of ligases.

[0050] The "spacer region" or "variable region" of a gRNA comprises a nucleotide sequence complementary to a specific sequence within the target DNA (the complementary strand of the target DNA). In some respects, the spacer region confers gRNA targeting specificity in combination with an RNA-guided endonuclease, enabling the RNA-guided endonuclease to cleave at the target site targeted by the spacer region in the target DNA. As used herein, the term "spacer region" and the term "spacer region sequence" are used interchangeably.

[0051] The "invariant region" of the term gRNA refers to the nucleotide sequence of the gRNA that associates with an RNA-guided endonuclease. In some aspects, gRNA comprises crRNA and trans-activating crRNA (tracrRNA), wherein the crRNA and tracrRNA hybridize to form a double strand. In some aspects, the crRNA comprises a 5' to 3' spacer sequence and a minimal CRISPR repeat sequence (also referred to herein as the "crRNA repeat sequence"); the tracrRNA comprises a minimal tracrRNA sequence complementary to the minimal CRISPR repeat sequence (also referred to herein as the "tracrRNA anti-repetitive sequence") and a 3' tracrRNA sequence. In some aspects, the invariant region of gRNA refers to a portion of the minimal CRISPR repeat sequence of the crRNA and the tracrRNA.

[0052] The terms “polynucleotide” and “nucleic acid” are used interchangeably herein and refer to polymers of nucleotides (ribonucleotides or deoxyribonucleotides) of any length. Therefore, this term includes, but is not limited to: single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids / triple helices, or polymers comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derived nucleotide bases.

[0053] As used herein, “binding” refers to a non-covalent interaction between macromolecules (e.g., between proteins and nucleic acids). When macromolecules are in a non-covalent interaction state, they are referred to as “associated,” “interacting,” or “binding” (e.g., when molecule X interacts with molecule Y, this means that molecule X binds to molecule Y in a non-covalent manner). A general characteristic of binding interactions is that the dissociation constant (Kd) is less than 10. -6 M, less than 10 -7 M, less than 10 -8 M, less than 10 -9 M, less than 10 -10 M, less than 10 -11 M, less than 10 -12 M, less than 10 -13 M, less than 10 -14 M, or less than 10 -15 M. Kd depends on environmental conditions, such as pH and temperature, which are known to those in the art. “Affinity” refers to the strength of binding, and increased binding affinity is associated with lower Kd.

[0054] The term "hybridizing" or "hybridizing" refers to the pairing of substantially complementary or complementary nucleic acid sequences within two different molecules. Pairing can be achieved through any process in which nucleic acid sequences are linked to substantially or completely complementary sequences by base pairing to form a hybrid complex. In some embodiments, "hybridizing" or "hybridizing" includes denaturing the molecule to disrupt its intramolecular structure (e.g., secondary structure). In some embodiments, denaturing the molecule includes heating the solution containing the molecule to a temperature sufficient to disrupt the molecule's intramolecular structure. In some cases, denaturing the molecule includes adjusting the pH of the solution containing the molecule to a pH sufficient to disrupt the molecule's intramolecular structure. For hybridization purposes, two nucleic acid sequences or segments of sequences are considered "substantially complementary" if at least 80% of their individual bases are complementary to each other. For example, a splice oligonucleotide sequence typically has no more than about 50% identity with one of two polynucleotides (e.g., RNA fragments) that it is designed to be complementary to. However, the complementary parts of each sequence may be referred to as “segments” in this paper, and if segments have 80% or more identity, they are essentially complementary.

[0055] Where a numerical range is provided, it should be understood that every intermediate value between the upper and lower limits of the range (unless otherwise expressly specified by the context, one-tenth of the unit to the lower limit) and any other stated or intermediate values ​​within the range are included within this disclosure. The upper and lower limits of these smaller ranges may be independently included within the smaller range and also included within this disclosure, subject to any specifically excluded limit values ​​within the stated range. Where the range includes one or both limits, the range excluding any of those included limits is also included in this disclosure.

[0056] Certain ranges are presented herein with numerical values ​​beginning with the term “about”. The term “about” is used herein to provide literal support for the exact number preceding it and for numbers that are close to or approximate to the number preceding the term. In determining whether a number is close to or approximate to a specifically listed number, the unlisted number that is close to or approximates may be a substantially equivalent number to the specifically listed number provided in its presented context.

[0057] It should be understood that certain features of this disclosure described in the context of individual embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of this disclosure described in the context of individual embodiments for brevity may also be provided individually or in any suitable sub-combination. All combinations of embodiments relating to this disclosure are specifically included in this disclosure and disclosed herein as if each combination were disclosed separately and explicitly. Furthermore, all sub-combinations of various embodiments and their elements are also specifically covered by this disclosure and disclosed herein as if each such sub-combination were disclosed separately and explicitly herein.

[0058] I. Splint oligonucleotides

[0059] This disclosure provides a method for synthesizing RNA, particularly mlRNA, such as gRNA, using splint-mediated ligation of two or more RNA fragments. In some embodiments, the method includes the use of splint-mediated ligation of two, three, or more (e.g., four, five, six, seven, or eight) RNA fragments. Indispensable in these methods are splint oligonucleotides. These hybridize with a first RNA fragment and a second RNA fragment to form a complex, which facilitates the ligation of the first and second RNA fragments at a ligation site present between the RNA fragments. In some embodiments, the method includes the use of a single splint oligonucleotide for splint-mediated ligation of two RNA fragments. In some embodiments, the method includes the use of two splint oligonucleotides for splint-mediated ligation of three RNA fragments. In some embodiments, the method includes the use of a suitable number of splint oligonucleotides required for ligating the RNA fragments for splint-mediated ligation of more than three RNA fragments (e.g., four, five, six, seven, or eight RNA fragments).

[0060] For example, the splice oligonucleotide includes a first portion in a terminal region complementary to a first RNA fragment, said terminal region including a 5' phosphate moiety. It further includes a second portion in a terminal region complementary to a second RNA fragment, said terminal region containing a 3' hydroxyl group. The splice oligonucleotide can hybridize with the first and second RNA fragments to form a complex. In the complex, the RNA fragments are advantageously positioned to connect at linking sites existing between the RNA fragments.

[0061] The splice oligonucleotide may include a sequence in its terminal region that is either continuous or non-continuously complementary to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in the first RNA fragment, wherein the terminal region contains a 5' phosphate moiety. The splice oligonucleotide may also include a sequence in its terminal region that is either continuous or non-continuously complementary to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in the second RNA fragment, wherein the terminal region contains a 3' hydroxyl group. Splint oligonucleotides may include sequences complementary to 1 to 20 nucleotides, 21 to 40 nucleotides, 41 to 60 nucleotides, or 61 to 80 nucleotides of a second RNA fragment, wherein the nucleotides may be continuous or discontinuous.

[0062] The lengths of the sequences complementary to the first RNA fragment and the sequences complementary to the second RNA fragment in the splice oligonucleotide can be the same or different.

[0063] Splint oligonucleotides can be programmed to preferentially promote complex formation between RNA fragments and splint oligonucleotides, rather than intramolecular structures (e.g., secondary structures) present in the RNA fragments and / or splint oligonucleotides. Minimum free energy prediction algorithms can be used to design suitable splint oligonucleotides provided by the methods of this disclosure. Theoretically, the lower the free energy, the greater the likelihood of complex formation between the RNA fragment and the splint oligonucleotide. The minimum free energy structure of a sequence is the secondary structure calculated to have the lowest free energy value (and therefore is theoretically the most likely to form). For example, minimum free energy prediction algorithms can be used to calculate the free energy of the secondary structure of an RNA fragment, given by ΔG. intra This represents the free energy of intermolecular hybridization between RNA fragments and splint oligonucleotides, expressed as ΔG. inter Indication. In some cases, Nearest-Neighbor approximation is used. The melting temperature (Tm) of the secondary structure of an RNA fragment is determined by T. m-intra Indicated. The melting temperature of RNA fragments and splint oligonucleotide hybrids is determined by T. m-inter This indicates that the length of the splint oligonucleotide can be designed to ensure ΔG. intra Greater than ΔG inter , and / or T m-inter Greater than T m-intra An exemplary free energy prediction algorithm can be obtained from the URL:: / / unafold.rna.albany.edu / ?q=mfold.

[0064] When the first RNA fragment, the second RNA fragment, or both contain at least one secondary structure, hybridizing the first RNA fragment, the second RNA fragment, and the splice oligonucleotide produces a free energy lower than the free energy associated with one or more of the at least one secondary structure. For example, hybridizing the first RNA fragment, the second RNA fragment, and the splice oligonucleotide may also produce a free energy lower than the free energy of a secondary structure having the lowest free energy (or minimum free energy) of the first RNA fragment, the second RNA fragment, or both.

[0065] One or more splice oligonucleotides can be used to hybridize with RNA fragments to mediate the ligation of RNA fragments. The number of splice oligonucleotides used to mediate ligation can be less than the number of RNA fragments to be ligated. For example, the number of splice oligonucleotides used to mediate ligation can be one less than the number of RNA fragments to be ligated, i.e., if the number of RNA fragments to be ligated is n, then the number of splice oligonucleotides is n-1.

[0066] The length of the splice oligonucleotide can be 20 to 100 nucleotides (e.g., 20 to 95, 20 to 90, 20 to 85, 20 to 80, 20 to 75, 20 to 70, 20 to 65, 20 to 60, 20 to 55, 20 to 50, 20 to 45, 20 to 40, 20 to 35, 20 to 30, 20 to 25, 25 to 100, 25 to 95, 25 to 90, 25 to 85, 25 to 80, 25 to 75, 25 to 70, 25 to 65, 25 to 60, 25 to 55, 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 30 to 10). 0, 30 to 95, 30 to 90, 30 to 85, 30 to 80, 30 to 75, 30 to 70, 30 to 65, 30 to 60, 30 to 55, 30 to 50, 30 to 45, 30 to 40, 30 to 35, 35 to 100, 35 to 95, 35 to 90, 35 to 85, 35 to 80, 35 to 75, 35 to 70, 35 to 65, 35 to 60, 35 to 55, 35 to 50, 35 to 45, 35 to 40, 40 to 100, 40 to 95, 40 to 90, 40 to 85, 40 to 80, 40 to 75, 40 to 70, 40 to 65, 40 to 6 0, 40 to 55, 40 to 50, 40 to 45, 45 to 100, 45 to 95, 45 to 90, 45 to 85, 45 to 80, 45 to 75, 45 to 70, 45 to 65, 45 to 60, 45 to 55, 45 to 50, 50 to 100, 50 to 95, 50 to 90, 50 to 85, 50 to 80, 50 to 75, 50 to 70, 50 to 65, 50 to 60, 50 to 55, 55 to 100, 55 to 95, 55 to 90, 55 to 85, 55 to 80, 55 to 75, 55 to 70, 55 to 65, 55 to 60, 60 to 100, 60 Up to 95, 60 to 90, 60 to 85, 60 to 80, 60 to 75, 60 to 70, 60 to 65, 65 to 100, 65 to 95, 65 to 90, 65 to 85, 65 to 80, 65 to 75, 65 to 70, 70 to 100, 70 to 95, 70 to 90, 70 to 85, 70 to 80, 70 to 75, 75 to 100, 75 to 95, 75 to 90, 75 to 85, 75 to 80, 80 to 100, 80 to 95, 80 to 90, 80 to 85, 85 to 100, 85 to 95, 85 to 90, 90 to 100 or 90 to 95 nucleotides).

[0067] Splice oligonucleotides can be attached to a support. Various techniques can be used to attach splice oligonucleotides to a support. For example, splice oligonucleotides can be directly attached to the support or immobilized to the support via chemical fixation. For instance, chemical fixation can occur between functional groups on the support and corresponding functional elements in the splice oligonucleotide. These corresponding functional elements in the splice oligonucleotide can be intrinsic chemical groups of the splice oligonucleotide, such as hydroxyl groups, or additionally introduced. One example of such functional group is an amino group. Typically, the splice oligonucleotide to be immobilized includes or is chemically modified to include functional amino groups. The means and methods of such chemical modification are known in the art.

[0068] The positioning of functional groups within the splice oligonucleotide to be immobilized can be used to control and shape the binding behavior and / or orientation of the splice oligonucleotide. For example, functional groups can be placed at the 5' or 3' end of the splice oligonucleotide or within the sequence of the splice oligonucleotide. Typical vectors for the splice oligonucleotide to be immobilized include moieties capable of binding such splice oligonucleotides, for example, binding amine-functionalized nucleic acids. Non-limiting examples of such vectors include carboxyl, aldehyde, and epoxy vectors.

[0069] The carrier on which splice oligonucleotides can be immobilized can be chemically activated, for example, by activating functional groups available on the carrier. The term "activated substrate" refers to a material in which interacting or reactive chemical functional groups are established or enabled through a chemical modification process. For example, a carrier including a carboxyl group can be activated prior to use. Furthermore, some carriers contain functional groups that can react with specific portions already present in the splice oligonucleotide.

[0070] The covalent linking used to couple splice oligonucleotides to a carrier can be considered both direct and indirect. This is because, although the splice oligonucleotides are attached via “direct” covalent bonds, there may be a chemical portion or linker that separates the “first” nucleotide of the splice oligonucleotide from the support, i.e., an indirect linking. In some cases, splice oligonucleotides immobilized to the carrier via covalent bonds and / or chemical linkers are generally considered to be directly immobilized or attached to the carrier. Splice oligonucleotides may not bind directly to the carrier but interact indirectly, for example, by binding to molecules that are directly or indirectly bound to the carrier. Splice oligonucleotides can also be indirectly attached to the carrier (e.g., via a solution comprising a polymer).

[0071] In cases where the splint oligonucleotide is indirectly immobilized on a vector, for example by hybridization with a surface oligonucleotide capable of binding the splint oligonucleotide, the splint oligonucleotide may further include an upstream sequence (5' to a sequence that hybridizes with two or more RNA fragments as described herein) capable of hybridizing with the 5' end of the surface oligonucleotide.

[0072] Splice oligonucleotides can be attached to a vector via their 5' or 3' ends. Splice oligonucleotides attached to the vector can be synthesized in situ on the vector.

[0073] II. Methods for synthesizing RNA

[0074] Currently disclosed methods for synthesizing mlRNA typically involve providing a first RNA fragment, a second RNA fragment, and a splice oligonucleotide. The first RNA fragment, the second RNA fragment, and the splice oligonucleotide hybridize together to form a complex. This complex positions the first and second RNA fragments in close proximity to facilitate ligation. A ligase is then used to ligate the first and second RNA fragments through the ligation site, thereby synthesizing mlRNA.

[0075] In some embodiments, the method includes providing a first RNA fragment, a second RNA fragment, a third RNA fragment, a first splice oligonucleotide, and a second oligonucleotide. The first RNA fragment, the second RNA fragment, and the first splice oligonucleotide hybridize together; and the second RNA fragment, the third RNA fragment, and the second splice oligonucleotide hybridize together to form a complex comprising the first, second, and third RNA fragments and the first and second splice oligonucleotides. The formation of this complex positions (i) the 3' hydroxyl group of the first RNA fragment and the 5' phosphate moiety of the second RNA fragment closely adjacent to each other to provide a first linkage site; and (ii) positions the 3' hydroxyl group of the second RNA fragment and the 5' phosphate moiety of the third RNA fragment closely adjacent to each other to provide a second linkage site. The method also provides a ligase for ligating the first and second RNA fragments at the first linkage site and for ligating the second and third RNA fragments at the second linkage site to synthesize mlRNA.

[0076] Hybridization of the first RNA fragment, the second RNA fragment, and the splice oligonucleotide can be performed in solution. In some embodiments, hybridization further includes a third RNA fragment and a second splice oligonucleotide, which are performed in solution. When hybridizing in solution, the concentration of the first RNA fragment can, for example, be approximately equal to the concentration of the second RNA fragment. In some embodiments, where hybridization further includes a third RNA fragment, the concentrations of the first and second RNA fragments are each approximately equal to the concentration of the third RNA fragment. Depending on the method, fragments, and splice oligonucleotides used, the concentration of the splice oligonucleotide in solution can be approximately equal to, greater than, or less than the concentration of the first RNA fragment in solution, or the concentration of the second RNA fragment in solution. For example, the concentrations of the splice oligonucleotide, the first RNA fragment, and the second RNA fragment can be substantially equal. In some embodiments, the method includes first, second, and third RNA fragments, and first and second splice oligonucleotides, wherein the concentrations of the first splice oligonucleotide, the second splice oligonucleotide, the first RNA fragment, the second RNA fragment, and the third RNA fragment in solution are substantially equal.

[0077] In some cases, for hybridization, the RNA fragment and / or splice oligonucleotides are denatured, i.e., the intramolecular structure of the RNA fragment and / or splice oligonucleotides is disrupted to allow annealing between the RNA fragment and the splice oligonucleotides. For example, this can be achieved by heating a solution containing the RNA fragment and splice oligonucleotides to at least about 37°C (e.g., at least about 37°C, 38°C, 39°C, 40°C, 42°C, 44°C, 46°C, 48°C, 50°C, 52°C, 54°C, 56°C, 58°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, or at least about 100°C). In some cases, hybridization involves heating the solution to a temperature of approximately 80°C to approximately 100°C, for example, approximately 82°C to approximately 98°C, approximately 84°C to approximately 96°C, approximately 86°C to approximately 94°C, or approximately 88°C to approximately 92°C (e.g., approximately 81°C, approximately 82°C, approximately 83°C, approximately 84°C, approximately 85°C, approximately 86°C, approximately 87°C, approximately 88°C, approximately 89°C, approximately 90°C, approximately 91°C, approximately 92°C, approximately 93°C, approximately 94°C, approximately 95°C, approximately 96°C, approximately 97°C, approximately 98°C, or approximately 99°C). In other cases, hybridization does not involve heating the solution.

[0078] In some cases, hybridization includes cooling the solution to a temperature of approximately 20°C to approximately 45°C after heating, for example, approximately 22°C to approximately 43°C, approximately 25°C to approximately 40°C, or approximately 27°C to approximately 38°C (e.g., approximately 21°C, approximately 22°C, approximately 23°C, approximately 24°C, approximately 25°C, approximately 26°C, approximately 27°C, approximately 28°C, approximately 29°C, approximately 30°C, approximately 31°C, approximately 32°C, approximately 33°C, approximately 34°C, approximately 35°C, approximately 36°C, approximately 37°C, approximately 38°C, approximately 39°C, approximately 40°C, approximately 41°C, approximately 42°C, approximately 43°C, or approximately 44°C). For example, in some cases, hybridization includes cooling the solution to approximately 37°C after heating. Hybridization may include cooling the solution to a temperature at which the ligase used in the currently described method retains sufficient ligase activity to ligate the first and second RNA fragments, and / or below the melting temperature of the complex formed by the RNA fragments and the splice oligonucleotides during hybridization. In cases where hybridization does not involve heating the solution, hybridization can be performed at temperatures below the melting temperature of the complex formed by the RNA fragment and the splice oligonucleotide during hybridization. Depending on the specific method used, cooling the solution after heating may involve decreasing the solution temperature at a constant rate or at an uncontrolled rate.

[0079] The method described in this disclosure includes ligating first and second RNA fragments at a ligation site using a ligase. Ligation may include linking a 5' phosphate group at the end of the first RNA fragment to a 3' hydroxyl group at the end of the second RNA fragment. Catalyzed by the ligase, the 5' phosphate group and the 3' hydroxyl group can react to form a phosphodiester bond. The ligation site may be a site where a phosphodiester bond is formed between the 5' phosphate group and the 3' hydroxyl group.

[0080] In some embodiments, the method includes using a ligase to ligate a first RNA fragment and a second RNA fragment at a first ligation site, and using a ligase to ligate the second RNA fragment and a third RNA fragment at a second ligation site. In some embodiments, the ligation includes linking a 3' hydroxyl group at the end of the first RNA fragment to a 5' phosphate group at the end of the second RNA fragment; and linking a 3' hydroxyl group at the end of the second RNA fragment to a 5' phosphate group at the end of the third RNA fragment, each ligation causing the formation of a phosphodiester bond.

[0081] Typically, the ligation of the first and second RNA fragments can be performed at approximately 15°C to approximately 45°C, for example, approximately 17°C to approximately 43°C, approximately 20°C to approximately 40°C, or approximately 22°C to approximately 38°C (e.g., approximately 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, or approximately 45°C). For example, the ligation of the first and second RNA fragments can be performed at 37°C. Depending on the method used, the ligation of the first and second RNA fragments can take place for different time periods, for example, about 0.1 to about 48 hours, or about 0.3 to about 45 hours, about 0.5 to about 40 hours, about 0.7 to about 35 hours, about 1 to about 30 hours, or about 1.5 to about 25 hours (e.g., about 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, or about 45 hours). In some embodiments, the temperature and / or reaction time for the splint-mediated ligation reaction are independent of the number of RNA fragments used in the reaction; for example, the reaction temperature and / or reaction time suitable for a splint-mediated ligation reaction containing two RNA fragments are suitable for a splint-mediated ligation reaction containing three or more RNA fragments.

[0082] In some cases, quenching the ligation reaction after gRNA synthesis is useful. For example, a protease or a chelating agent can be used to quench the ligation reaction. Non-limiting examples of proteases include proteinase K. Non-limiting examples of chelating agents include EDTA and EGTA, or a combination of both.

[0083] In some cases, linking the first and second RNA fragments also includes the use of one or more constricting agents. Non-limiting examples of constricting agents include: polyethylene glycol (PEG), Ethylene glycol and dextran, or any combination thereof. In some embodiments, the use of one or more congesting agents is suitable for splint-mediated ligation reactions involving two, three, or more RNA fragments.

[0084] A variety of ligases can be used in the methods described herein. For example, the ligase can be T4 DNA ligase, T4 RNA ligase I, T4 RNA ligase II, RtcB ligase, T3 DNA ligase, T7 DNA ligase, Taq DNA ligase, PBCV-1 DNA ligase, thermostable DNA ligase (e.g., 5' App DNA / RNA ligase), or ATP-dependent DNA ligase. In some cases, any combination of two or more of these ligases can be used.

[0085] T4 RNA ligase II is particularly useful in the methods described herein. In some cases, T4 RNA ligase II can be modified. For example, T4 RNA ligase II can be truncated and / or contain mutations. For example, T4 RNA ligase II can contain K227Q mutations and / or R55K mutations. In some cases, T4 RNA ligase II can be truncated and have K227Q and / or R55K mutations. Also useful in the methods described herein is PBCV-1 DNA ligase (i.e., Chlorella virus DNA ligase; Ligase). In some cases, the ligase can be a DNA ligase (e.g., DNA ligase.

[0086] In some of the methods described herein, three or more RNA fragments (e.g., three, four, or five) can be ligated to synthesize mlRNA. The ligation of three or more RNA fragments can be performed in the same step or in different steps (e.g., in a stepwise manner).

[0087] The methods described herein may also include purifying ml RNA after synthesis. Purification can isolate the full-length RNA product from unreacted RNA fragments and / or splice oligonucleotides. For example, purification may include enzymatic degradation of unreacted RNA fragments using an exonuclease (e.g., an exonuclease specific for RNA containing a 5'-monophosphate). An exemplary exonuclease is XRN-1.

[0088] Full-length RNA products can also be purified from unreacted RNA fragments and / or splice oligonucleotides using ultrafiltration or chromatography. Non-limiting examples of chromatographic methods include: reversed-phase HPLC, ion-exchange chromatography (e.g., strong anion-exchange HPLC or weak anion-exchange HPLC), size exclusion chromatography, hydrophobic interaction chromatography, affinity chromatography, liquid chromatography-mass spectrometry (LCMS), capillary electrophoresis (CE), capillary gel electrophoresis (CGE), and polyacrylamide gel purification.

[0089] This document also provides methods for synthesizing RNA molecules comprising all or part of the tracrRNA sequence using any of the methods described herein. The RNA molecule may optionally be purified and used to generate full-length gRNA (e.g., sgRNA). To generate full-length sgRNA, an additional RNA fragment comprising a spacer region and a minimal CRISPR repeat sequence (e.g., crRNA) is ligated to a previously synthesized RNA molecule using splice oligonucleotides. For example, these methods can generate full-length gRNA specific to any target DNA sequence by ligating an RNA fragment containing the corresponding spacer region sequence to a previously synthesized RNA molecule containing all or part of the tracrRNA sequence. Figure 3This is a representative schematic diagram illustrating a three-segment system, where two RNA fragments (fragments II and III) are first ligated using a splice oligonucleotide to form an invariant RNA construct that includes a portion of the tracrRNA sequence. For example, this RNA product can be purified and stored for later use. RNA fragment I', containing a sequence complementary to a specific target DNA, can then be combined with the previously synthesized RNA construct to form a full-length gRNA.

[0090] III. RNA fragments

[0091] The method for synthesizing mlRNA as described in this disclosure includes providing a first RNA fragment comprising a terminal region including a 5' phosphate moiety, and a second RNA fragment comprising a terminal region including a 3' hydroxyl group, wherein the RNA is synthesized by linking the first and second RNA fragments. When synthesizing gRNA, the first RNA fragment, the second RNA fragment, or both include at least a portion of a sequence that can bind to an RNA-guided endonuclease. An exemplary mlRNA synthesized by the method may include a second RNA fragment from 5' to 3' followed by the first RNA fragment. The second RNA fragment may not include the 5' phosphate moiety. The 5' phosphate moiety may be, for example, a 5'-phosphate or a 5'-thiophosphate. The first fragment, the second RNA fragment, or both may include a sequence or a portion of a sequence complementary to a sequence in the target DNA. In some cases, the second RNA fragment contains a sequence complementary to a sequence in the target DNA.

[0092] mlRNA can be synthesized by linking three or more (e.g., three, four, five or six) RNA fragments. Figure 2 This is a schematic diagram illustrating the ligation of three RNA fragments using two splice oligonucleotides. In some cases, RNA is synthesized by ligating fewer than six RNA fragments. As an example, for RNA synthesized by ligating RNA fragments A, B, and C (listed in 5' to 3' order), prior to ligation, RNA fragment A may include a 3' hydroxyl group and may not contain a 5' phosphate moiety; RNA fragment B may include a 3' hydroxyl group and a 5' phosphate moiety; and RNA fragment C may include a 5' phosphate moiety and may or may not include a 3' hydroxyl group. Ligating RNA fragments A, B, and C may involve forming a phosphodiester bond between the 3' hydroxyl group of A and the 5' phosphate moiety of B, and between the 3' hydroxyl group of B and the 5' phosphate moiety of C.

[0093] The length of any RNA fragment can be 10 to 90 nucleotides (e.g., 10 to 85, 10 to 80, 10 to 75, 10 to 70, 10 to 65, 10 to 60, 10 to 55, 10 to 50, 10 to 45, 10 to 40, 10 to 35, 10 to 30, 10 to 25, 10 to 20, 10 to 15, 15 to 90, 15 to 85, 15 to 80, 15 to 75, 15 to 70, 15 to 65, 15 to 60, 15 to 55, 15 to 50, 15 to 45, 15 to 40, 15 to 35, 15 to 30, 15 to 25, 15 to 20, 20 to 90). 0, 20 to 85, 20 to 80, 20 to 75, 20 to 70, 20 to 65, 20 to 60, 20 to 55, 20 to 50, 20 to 45, 20 to 40, 20 to 35, 20 to 30, 20 to 25, 25 to 90, 25 to 85, 25 to 80, 25 to 75, 25 to 70, 25 to 65, 25 to 60, 25 to 55, 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 30 to 90, 30 to 85, 30 to 80, 30 to 75, 30 to 70, 30 to 65, 30 to 60, 30 to 55, 30 to 5 0, 30 to 45, 30 to 40, 30 to 35, 35 to 90, 35 to 85, 35 to 80, 35 to 75, 35 to 70, 35 to 65, 35 to 60, 35 to 55, 35 to 50, 35 to 45, 35 to 40, 40 to 90, 40 to 85, 40 to 80, 40 to 75, 40 to 70, 40 to 65, 40 to 60, 40 to 55, 40 to 50, 40 to 45, 45 to 90, 45 to 85, 45 to 80, 45 to 75, 45 to 70, 45 to 65, 45 to 60, 45 to 55, 45 to 50, 50 to 90, 50 to 8 5. 50 to 80, 50 to 75, 50 to 70, 50 to 65, 50 to 60, 50 to 55, 55 to 90, 55 to 85, 55 to 80, 55 to 75, 55 to 70, 55 to 65, 55 to 60, 60 to 90, 60 to 85, 60 to 80, 60 to 75, 60 to 70, 60 to 65, 65 to 90, 65 to 85, 65 to 80, 65 to 75, 65 to 70, 70 to 90, 70 to 85, 70 to 80, 70 to 75, 75 to 90, 75 to 85, 75 to 80, 75 to 80, 80 to 90, 80 to 85, or 85 to 90 nucleotides. For example, the length of each of the first and second RNA fragments can be 10 to 90 nucleotides. The length of the second RNA fragment can be about 40 nucleotides or shorter (e.g., about 35, 30, 25, 20, 15, or about 10 nucleotides). In some cases, the length of the second RNA fragment can be about 20 nucleotides, while the length of the first RNA fragment can be about 80 nucleotides.

[0094] RNA fragments may include one or more secondary structures. The secondary structures of an RNA molecule (e.g., an RNA fragment or mlRNA) may include stems and loops, or combinations thereof. Non-limiting examples of secondary structures of RNA molecules include stem-loops, hairpins, hairpin loops, tetraloops, inner loops, protrusions, pseudoknots, and cloverleaf structures. In some cases, the RNA fragment does not include any secondary structures (e.g., stem-loops). RNA synthesized by the methods provided herein may include one or more secondary structures, such as, but not limited to, one or more stem-loop structures formed during the ligation of RNA fragments. In some cases, the ligation sites present between RNA fragments correspond to sites in the secondary structures (e.g., stem-loop structures) of the synthesized RNA. Ligation sites may correspond to sites in a portion of the secondary structure, including but not limited to the tetraloop or helical portion of a stem-loop structure.

[0095] Based on the sequence of an RNA fragment, the methods disclosed herein may include predicting the secondary structure of the RNA fragment and / or the free energy associated with the secondary structure. Methods for predicting RNA secondary structure are known in the art, including those described in Zuker and Stiegler (1981) Nucleic Acids Research, 9(1):133-148, Reuter and Mathews (2010) BMC Bioinformatics 11:129; and Xia et al. (1998) Biochemistry, 37:14719-14735. RNA secondary structure can be predicted from the RNA sequence by minimizing the free energy, for example, those described in Mathews and Turner (2006) Current Opinion in Structural Biology, 16:270-278. Non-limiting embodiments of software for predicting RNA secondary structure are available at: en.wikipedia.org / wiki / List_of_RNA_structure_prediction_software.

[0096] Modification

[0097] RNA fragments may include one or more modifications. For example, an RNA fragment may include at least one modification in the RNA backbone. Non-limiting examples of backbone modifications include: 2'-methoxy (2'OMe), 2'-fluoro (2'fluoro), 2'-O-methoxy-ethyl (MOE), locked nucleic acid (LNA), unlocked nucleic acid (UNA), bridging nucleic acid, 2'-deoxynucleic acid (DNA), and peptide nucleic acid (PNA). Alternatively or additionally, RNA fragments may include at least one base modification. Non-limiting examples of base modifications include: 2-aminopurine, inosine, thymine, 2,6-diaminopurine, 2-pyrimidinone, and 5-methylcytosine. In some cases, the RNA fragment contains at least one phosphate-thioester bond.

[0098] Modifications to RNA fragments can be used, for example, to enhance stability, reduce the likelihood or extent of an innate immune response, and / or enhance other properties; and new types of modifications are developed regularly. By way of example, various types of modifications can include one or more nucleotides modified at the 2' position of the sugar, such as, but not limited to, nucleotides modified with 2'-O-alkyl, 2'-O-alkyl-O-alkyl, or 2'-fluorine. DNA (2'-deoxy-)nucleotide substitutions are also considered. Non-limiting examples of RNA modifications include 2'-fluorine, 2'-amino, or 2'-O-methyl modifications on the ribose of the pyrimidine, and basic residues or reverse bases at the 3' end of the RNA. Such modifications are often incorporated into oligonucleotides, and these oligonucleotides have been shown to have higher T values ​​than 2'-deoxy oligonucleotides targeting a given target. m (i.e., higher target binding affinity). In some embodiments, modifications of the RNA fragments disclosed herein include 2'O-methyl modifications of one or more nucleotides in the RNA fragment.

[0099] RNA fragments according to any of the embodiments described herein may include modifications, for example, to increase resistance to nuclease digestion compared to native nucleic acids. In some cases, the modified nucleic acid comprises a modified backbone selected from, for example, thiophosphates, triphosphates, methyl phosphonates, short-chain alkyl or cycloalkyl sugar bonds, and short-chain heteroatom or heterocyclic sugar bonds. The nucleic acid may have a thiophosphate backbone or a heteroatom backbone, such as CH2-NH-O-CH2, CH, -N(CH3)-O-CH2 (referred to as a methylene (methylimino) or MMI backbone), CH2-ON(CH3)-CH2, CH2-N(CH3)-N(CH3)-CH2, and ON(CH3)-CH2-CH2 backbones; amide backbones (see De... Mesmaeker et al. (1995) Acc. Chem. Res., 28(9): 366-374); Morpholinized backbone structure (see Summerton and Weller, U.S. Patent No. 5,034,506); Peptide nucleic acid (PNA) backbone (in which the phosphodiester backbone of the oligonucleotide is replaced by a polyamide backbone, and the nucleotide is directly or indirectly bonded to the aza-nitrogen atom of the polyamide backbone, see Nielsen et al. (1991) Science, 254(5037): 1497-1500). Phosphorus-containing bonds include, but are not limited to: thiophosphates, chiral thiophosphates, dithiophosphates, phosphate triesters, aminoalkyl phosphate triesters, methyl and other alkylphosphonates (including 3'-alkylene phosphonates and chiral phosphonates), hypophosphonates, phosphoramidites (including 3'-aminophosphatides and aminoalkylphosphatides), thiocarbonylphosphatides, thiocarbonylalkylphosphonates, thiocarbonylalkyl phosphate triesters, and borophosphates having a normal 3'-5' linkage, 2'-5' linkage analogs of these esters, and those esters having reverse polarity, wherein adjacent pairs of nucleoside units are linked in 3'-5' to 5'-3' or 2'-5' to 5'-2' configurations; see, for example, U.S. Patent Nos. 3,687,808, 4,469,863, and 4,476,301. No.; No. 5,023,243; No. 5,177,196; No. 5,188,897; No. 5,264,423; No. 5,276,019; No. 5,278,302; No. 5,286,717; No. 5,321,131; No. 5,399,676; No. 5,405,939; No. 5,453,496 No. 5,455,233; No. 5,466,677; No. 5,476,925; No. 5,519,126; No. 5,536,821; No. 5,541,306; No. 5,550,111; No. 5,563,253; No. 5,571,799; No. 5,587,361 and No. 5,625,050.In some embodiments, the modifications to the RNA fragments disclosed herein include one or more phosphate thioester bonds in the backbone of the RNA fragment.

[0100] Morpholin-based oligomers have been described in Braasch et al. (2002) Biochem., 41(14):4503-4510; Genesis, Volume 30, Issue 3, (2001) Wiley Online Library; Heasman (2002) Dev. Biol., 243(2):209-214; Nasevicius et al. (2000) Nat. Genet., 26(2):216-220; Lacera et al. (2000) Proc. Natl. Acad. Sci. USA, 97(17):9591-9591; and U.S. Patent No. 5,034,506. Cyclohexenyl nucleic acid oligonucleotide mimics have been described in Wang et al. (2000) J. Am. Chem. Soc., 122(36):8595-8602.

[0101] RNA fragments according to any of the embodiments described herein may include a backbone that does not include phosphorus atoms, for example, a backbone formed by short-chain alkyl or cycloalkyl nucleoside bonds, mixed heteroatoms and alkyl or cycloalkyl nucleoside bonds, or one or more short-chain heteroatoms or heterocyclic nucleoside bonds. These backbones include: backbones containing morpholino bonds (partially formed from the sugar moiety of a nucleoside); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; olefin-containing backbones; aminosulfonate backbones; methyleneimino and methylenehydrazine backbones; sulfonate and sulfonamide backbones; amide backbones; and backbones that incorporate N, O, S, and CH2 component moieties; see U.S. Patents 5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, and 5,235,033. No. 5,264,562, No. 5,264,564, No. 5,405,938, No. 5,434,257, No. 5,466,677, No. 5,470,967, No. 5,489,677, No. 5,541,307, No. 5,561,225, No. 5,596,086, No. 5,60 Nos. 2,240, 5,610,289, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437 and 5,677,439.

[0102] RNA fragments according to any embodiment described herein may include one or more substituted sugar moieties, including, for example, one of the following at the 2' position: OH, SH, SCH3, F, OCN, OCH3, OCH3 O(CH2)n CH3, O(CH2)nNH2, or O(CH2)n CH3, where n is 1 to 10; C1 to C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkylaryl, or aryl; Cl; Br; CN; CF3; OCF3; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH3; SO2CH3; ONO2; NO2; N3; NH2; heterocyclic alkyl; heterocyclic alkylaryl; aminoalkylamino; polyalkylamino; substituted silyl; RNA cleavage group; reporter group; intercalator; group for improving the pharmacokinetic properties of oligonucleotides; or group for improving the pharmacodynamic properties of oligonucleotides, and other substituents having similar properties. For example, modifications can include 2'-methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl)) (Martin et al. (1995) Helv. Chim. Acta, 78(2):486-504). Other modifications include 2'-methoxy (2'-O-CH3), 2'-propoxy (2'-OCH2CH2CH3), and 2'-fluorinated (2'-F). Similar modifications can also be made at other positions on the oligonucleotide, particularly at the 3' position of the sugar on the 3' terminal nucleotide and at the 5' position of the 5' terminal nucleotide. Oligonucleotides can also have sugar mimics, such as a cyclobutyl group replacing the pentofuranose group. In some cases, both the sugar and the nucleotide internucleotide bond (i.e., the backbone) are replaced by novel groups. Maintaining hybridization of the base unit with a suitable nucleic acid target compound. One such oligomer, an oligonucleotide mimic that has shown excellent hybridization properties, is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of the oligonucleotide is replaced with an amide backbone, such as an aminoethylglycine backbone. The nucleobases are retained and directly or indirectly bound to the nitrogen atom of the amide portion of the backbone. Representative U.S. patents teaching the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082, 5,714,331, and 5,719,262. Other teachings on PNA compounds can be found in Nielsen et al. (1991) Science, 254(5037):1497-1500. RNA fragments as described herein may include 2'-O-thiocarbamate MP (2'-O-methyl-3'-phosphonoacetate) and MSP (O-methyl-3'-thiophosphonoacetate) (Ryan et al. (2017) Nuc. Acids Res. 46(2):792-803).

[0103] As described herein, RNA fragments may include one or more modifications selected from the group consisting of: pseudouridine, N... 1 -Methylpseudouridine and 5-methoxyuridine. For example, one or more N... 1 -Methylpseudouridine is incorporated into RNA fragments to provide enhanced RNA stability and reduced immunogenicity in animal cells such as mammalian cells (e.g., human and mouse cells). 1 5-methylpseuuridine modification can also be integrated with one or more 5-methylcytidines.

[0104] Modified RNA is available from numerous commercial suppliers, including companies such as Trilink Biotech, AxoLabs, Bio-Synthesis Inc., and Dharmacon. For example, as described by Trilink, 5-methyl-CTP can be used to confer desired characteristics, such as enhanced nuclease stability or reduced interaction of innate immune receptors with RNA transcribed in vitro. 5'-methylcytidine-5'-triphosphate (5-methyl-CTP), N6-methyl-ATP, as well as pseudo-UTP and 2-thio-UTP have also been shown to reduce innate immune stimulation in culture and in vivo, as illustrated by Kormann et al. (2011) Nat. Biotechnol., 29:154–157 and Warren et al. (2010) Cell Stem Cell, 7(5):618–630.

[0105] RNA fragments can be integrated with modifications engineered to bypass innate antiviral responses. See, for example, Warren et al. (2010) Cell Stem Cell, 7(5):618-630. For example, the RNA can be enzymatically synthesized RNA incorporating 5-methyl-CTP, pseudo-UTP, and / or an antiretroviral Cap analog (ARCA); see, for example, Warren et al. (2010) Cell Stem Cell, 7(5):618-630.

[0106] Various modifications have been developed and applied to enhance RNA stability, reduce innate immune responses, and / or achieve other benefits; see, for example, Whitehead et al. (2011) Ann. Rev. Chem. Biomolec. Eng., 2:77-96; Gaglione et al. (2010) Mini Rev. Med. Chem., 10(7):578-595; Chernolovskaya et al. (2010) Curr. Opin. Mol. Ther., 12(2):158-167; Deleavey et al. (2009) Curr. Protoc. Nucleic Acid Chem., 39(1):16.3.1-16.3.22; Behlke (2008) Oligonucleotides, 18(4):305-319; Fucini et al. (2012) Nucleic Acid Ther., 22(3):205–210; a review by Bremsen et al. (2012) Front. Genet., 3:154.

[0107] Simulation

[0108] RNA fragments can be nucleic acid mimics. The term "mimic" applied to polynucleotides is intended to include polynucleotides in which only the furanose ring or both the furanose ring and the intermolecular bonds are replaced by non-furanose groups. Substitution of only the furanose ring is also referred to in the art as a sugar surrogate. The heterocyclic base moiety or modified heterocyclic base moiety is retained for hybridization with a suitable target nucleic acid. One such nucleic acid, a polynucleotide mimic that has shown excellent hybridization properties, is called a peptide nucleic acid (PNA). In a PNA, the sugar backbone of the polynucleotide is replaced by an amide-containing backbone (particularly an aminoethylglycine backbone). The nucleotide is retained and directly or indirectly bound to the nitrogen atom of the amide moiety of the backbone. Representative U.S. patents describing the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082, 5,714,331, and 5,719,262. In some cases, the RNA fragments described herein are PNAs.

[0109] RNA fragments can be polynucleotide mimics based on linked morpholino units (morpholinonucleotides) having heterocyclic bases attached to the morpholino ring. Numerous linker groups have been reported that link morpholino monomer units in morpholinonucleotides. A class of linker groups has been selected to generate nonionic oligomers. Morpholino-based polynucleotides are nonionic mimics of oligonucleotides, which are less likely to form undesirable interactions with cellular proteins (Braasch et al. (2002) Biochemistry, 41(14):4503-4510). Morpholino-based polynucleotides are disclosed in U.S. Patent No. 5,034,506. Various compounds in the morpholino polynucleotide class have been prepared with linker groups having a variety of different linker monomer subunits.

[0110] RNA fragments can be polynucleotide mimics called cyclohexenyl nucleic acids (GeNA), in which the furanose ring, typically found in DNA / RNA molecules, is replaced by a cyclohexenyl ring. GeNA DMT-protected phosphoramide monomers have been prepared and used in the synthesis of oligomers following classical phosphoramide chemistry. Fully modified GeNA oligomers and oligonucleotides with specific sites modified by GeNA have been prepared and studied (see Wang et al. (2000) J. Am. Chem. Soc., 122(36): 8595-8602). NMR and circular dichroism indicate that studies integrating GeNA structures into native nucleic acid structures are readily conformationally adapted.

[0111] The RNA fragment can be a locked nucleic acid (LNA), in which a 2'-hydroxyl group is attached to the 4' carbon atom of the sugar ring, forming a 2'-C,4'-C-formaldehyde bond, thus forming a bicyclic sugar moiety. This bond can be a methylene (-CH2-) group. n A group that bridges the 2' oxygen atom and the 4' carbon atom, where n is 1 or 2 (Singh et al. (1998) Chem. Commun., 4:455-456). LNA and LNA analogs with complementary DNA and RNA (T m=+3 to +10℃) exhibits very high double-stranded thermal stability, stability against 3'-exonuclease degradation, and good solubility. Powerful and non-toxic antisense oligonucleotides containing LNA have been described (Wahlestedt et al. (2000) Proc. Natl. Acad. Sci. USA, 97(10):5633-5638). The synthesis and preparation of LNA monomers adenine, cytosine, guanine, 5-methylcytosine, thymine, and uracil, as well as their oligomerization and nucleic acid recognition properties, have been described (Koshkin et al. (1998) Tetrahedron, 54(14)):3607-3630). LNA and its preparation are described in WO98 / 39352 and WO 99 / 14226.

[0112] Modified sugar portion

[0113] RNA fragments may include one or more substituted sugar moieties, including, for example, sugar substituents selected from: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-ynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl, and ynyl groups may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and ynyl groups. O((CH2)) is particularly suitable. n O) m CH3, O(CH2) n OCH3、O(CH z ) n NH2, O(CH2)CH3, O(CH2) n ONH2 and O(CH2) n ON((CH2) n CH3)2, where n and m range from 1 to approximately 10. Other RNA fragments include suitable sugar substituents selected from the following: C1 to C2. 10Lower alkyl groups, substituted lower alkyl groups, alkenyl groups, alkynyl groups, aryl groups, O-alkaneyl or O-aryl groups, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocyclic alkyl groups, heterocyclic alkaneyl groups, aminoalkylamino groups, polyalkylamino groups, substituted silyl groups, RNA cleaving groups, reporter groups, intercalators, groups used to improve the pharmacokinetic properties of oligonucleotides or groups used to improve the pharmacodynamic characteristics of oligonucleotides, and other substituents with similar characteristics. Suitable modifications include 2'-methoxyethoxy2'-O-CH2-CH2OCH3, also known as -2'-O-(2-methoxyethyl) or 2'-MOE (Martin et al. (1995) Helv. Chim. Acta, 78(2):486-504), for example, alkoxyalkoxy groups. Further suitable modifications include 2'-dimethylaminooxyethoxy, for example, the O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described in the examples below, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), for example, 2'-O-CH2-O-CH2-N(CH3)2.

[0114] Other suitable sugar substituents include methoxy (-O-CH3), aminopropoxy (-O-CH2CH2CH2NH2), allyl (-CH2-CH=CH2), -O-allyyl (-O-CH2-CH=CH2), and fluorine (F). The 2'-sugar substituent can be located in the arabinose (top) or ribose (bottom) position. A suitable 2'-arabinose modification is 2'-F. Similar modifications can also be made at other positions in oligomers, particularly at the 3' position of the sugar in 3'-terminal nucleosides or 2'-5' linked oligonucleotides and the 5' position of the 5'-terminal nucleotide. Oligomers can also have sugar mimics, such as replacing the cyclobutyl moiety of the pentofuranosyl sugar.

[0115] Base modification and substitution

[0116] RNA fragments according to any of the embodiments described herein may additionally or alternatively include nucleobase (generally referred to in the art simply as "bases") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U). Modified nucleobases include those rarely or transiently found in native nucleic acids, such as hypoxanthine, 6-methyladenine, 5-Me pyrimidine (especially 5-methylcytosine (also known as 5-methyl-2'-deoxycytosine and often referred to as 5-Me-C in the field)), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentiobiose HMC, and synthetic nucleobases such as 2-aminoadenine, 2-(methylamino)adenine, 2-(indazolylmethyl)adenine, 2-(aminomethylamino)adenine or other heterosubstituted methyladenine, 2-thiouracil, 2-thiothymidine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6(6-aminohexyl)adenine and 2,6-diaminopurine (see Kornberg et al. (1980) DNA Replication (2 nd (ed.)(pp.75-77). San Francisco, CA: WH Freeman & Co.; Gebeyehu et al. (1987) Nucl. Acids Res., 15(11):4513-4534). It may also include “universal” bases known in the field, such as inosine. It has been shown that 5-Me-C substitution increases the stability of nucleic acid duplexes by 0.6 to 1.2 °C. (Sanghvi (1993). Antisense Research and Applications, (pp.276-278). Crooke, ST. and Lebleu, B., (Eds.), Boca Raton, FL: CRC Press) and are embodiments of base substitution.

[0117] Modified nucleobases include other synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C); 5-hydroxymethylcytosine; xanthine; hypoxanthine; 2-aminoadenine; 6-methyl and other alkyl derivatives of adenine and guanine; 2-propyl and other alkyl derivatives of adenine and guanine; 2-thiouracil; 2-thiothymidine and 2-thiocytosine; 5-halouracil and cytosine; 5-propynyluracil and cytosine; 6-azouracil and cytosine. And thymine; 5-uracil (pseudouracil); 4-thiouracil; 8-halogenated, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other α-substituted adenine and guanine; 5-halogenated, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracil and cytosine; 7-methylguanine and 7-methyladenine; 8-nitroguanine and 8-nitroadenine; 7-denitroguanine and 7-denitroadenine; and 3-denitroguanine and 3-denitroadenine.

[0118] Furthermore, the nucleobases include those disclosed in U.S. Patent No. 3,687,808, those disclosed in Kroschwitz (Ed.) (1990). The Concise Encyclopedia of Polymer Science and Engineering, (pp. 858-859). Hoboken, NJ: John Wiley & Sons, those disclosed in Englisch et al. (1991) Angewandte Chemie International Edition, 30(6): 613-722, and those disclosed in Sanghvi (1993) Chapter 15, Antisense Research and Applications, (pp. 289-302), Crooke, ST and Lebleu, B. (Eds), Boca Raton, FL: CRC Press. Certain of these nucleobases are particularly useful for enhancing the binding affinity of the oligomers disclosed herein. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and -O-6 substituted purines, comprising 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-methylcytosine substitution has been shown to increase the stability of nucleic acid duplexes by 0.6–1.2 oc (Sanghvi (1993) Antisense Research and Applications, (pp. 276–278). Crooke and Lebleu, (Eds.), Boca Raton, FL: CRC Press) and is an embodiment of base substitution, even more particularly when combined with 2'-O-methoxyethyl sugar modification. The modified nucleobases are described in the following: U.S. Patents 3,687,808 and 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,17 No. 7; No. 5,525,711; No. 5,552,540; No. 5,587,469; No. 5,596,091; No. 5,614,617; No. 5,681,941; No. 5,750,692; No. 5,763,588; No. 5,830,653; No. 6,005,096 and U.S. Patent Application Publication No. 2003 / 0158403.

[0119] RNA fragments containing nucleobase modifications or substitutions according to any of the embodiments described herein may not have all positions with consistent modifications. For example, an RNA fragment may have modifications that incorporate a single nucleotide.

[0120] IV. Synthesis of RNA Fragments and Splint Oligonucleotides

[0121] The RNA fragments and splint oligonucleotides provided by this invention can be synthesized by any method suitable for oligonucleotide synthesis described herein or known in the art. Non-limiting embodiments include enzymatic synthesis and chemical synthesis (e.g., phosphorous amide chemistry).

[0122] Methods for synthesizing RNA from DNA templates are known in the art. For example, RNA fragments and splice oligonucleotides can be synthesized in vitro using RNA polymerases (e.g., T7 polymerase, T3 polymerase, SP6 polymerase, etc.). Solid-phase synthesis using phosphoramide chemistry involves assembling monomers of protected 2'-deoxynucleosides (dA, dC, dG, and T), ribonucleosides (A, C, G, and US), or chemically modified nucleosides, such as LNA or BNA. The monomers are sequentially coupled to the growing oligonucleotide chain in the desired order of the product sequence. After chain assembly is complete, the product is released from the solid phase into solution, deprotected, and collected.

[0123] The RNA fragment and splice oligonucleotide can be synthesized in a 5' to 3' orientation or a 3' to 5' orientation. In some cases, the second RNA fragment is synthesized in a 5' to 3' orientation. The synthesized RNA fragment and splice oligonucleotide can be purified prior to ligation according to methods known in the art, such as, but not limited to, high-performance liquid chromatography (HPLC), reversed-phase HPLC, ion-exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, affinity chromatography, and polyacrylamide gel purification.

[0124] V. Medium-length RNA

[0125] Exemplary mlRNAs synthesized using any of the methods described herein are guide RNAs (gRNAs) (e.g., any gRNA described herein). gRNAs synthesized using the methods of this invention can be single-molecule gRNAs (sgRNAs) or bimolecular gRNAs. gRNAs provide target specificity by association with RNA-guided endonucleases, thereby directing the activity of RNA-guided endonucleases. The RNAs of this disclosure can be synthesized from two or more RNA molecules (referred to as RNA fragments) using one or more clamps. Exemplary bimolecular gRNAs comprise crRNA and trans-activating crRNA (tracrRNA), with the crRNA and tracrRNA hybridizing to form a double strand. Bimolecular gRNAs can also be double strands of two crRNAs. The gRNA double strand can bind to an RNA-guided endonuclease, thereby forming a complex between the gRNA and the RNA-guided endonuclease. The crRNA contains a spacer region sequence capable of hybridizing to a target nucleic acid sequence and a crRNA repeat sequence. The tracrRNA can be of any form (e.g., full-length tracrRNA or tracrRNA of an active portion) and can have different lengths. For example, the tracrRNA may comprise or consist of all or part of a wild-type tracrRNA sequence (e.g., approximately or at least 20, 26, 32, 45, 48, 54, 63, 67, 85 or more nucleotides of a wild-type tracrRNA sequence). Examples of wild-type tracrRNA sequences from *Streptococcus pyogenes* include versions of 171 nucleotides, 89 nucleotides, 75 nucleotides, and 65 nucleotides. See, for example, Deltcheva et al. (2011) *Nature* 471:602-607 and WO2014 / 093661. For example, the crRNA may have an optional spacer extension sequence, a pacer sequence, and a minimal CRISPR repeat sequence in the 5' to 3' orientation. The tracrRNA may have a minimal tracrRNA sequence (complementary to the minimal CRISPR repeat sequence), a 3' tracrRNA sequence, and an optional tracrRNA extension sequence. The optional tracrRNA extension may have elements that contribute additional function (e.g., stability) to the gRNA and may have one or more hairpin structures. The crRNA and tracrRNA hybridize to form the gRNA via a minimal CRISPR repeat sequence and a minimal tracrRNA sequence.

[0126] Exemplary sgRNAs comprise nucleotide sequences complementary to those in the target DNA, and nucleotide sequences capable of binding to RNA-guided endonucleases. For example, sgRNAs may have optional spacer extension sequences, spacer sequences, minimal CRISPR repeat sequences, single-molecule guide connectors, minimal tracrRNA sequences, 3' tracrRNA sequences, and optional tracrRNA extension sequences in the 5' to 3' direction. In some cases, sgRNAs may have both minimal CRISPR repeat sequences and spacer sequences in the 5' to 3' direction. A single-molecule guide connector links the minimal CRISPR repeat sequence and the minimal tracrRNA sequence to form a hairpin structure. In some embodiments, the single-molecule guide connector is a four-loop. For example, exemplary gRNAs are described in WO 2018 / 002719.

[0127] Generally, CRISPR repeat sequences include sequences sufficiently complementary to the tracr sequence to facilitate one or more of the following: (1) excision of a DNA target region flanked by the CRISPR repeat sequence in a cell containing the corresponding tracr sequence; and (2) formation of a CRISPR complex at the target sequence, wherein the CRISPR complex comprises a CRISPR repeat sequence hybridized to the tracr sequence. Generally, the degree of complementarity refers to the optimal alignment of the CRISPR repeat sequence and the tracr sequence along the shorter of the two sequences. Optimal alignment can be determined by any suitable alignment algorithm, and secondary structures such as self-complementarity within the tracr sequence or the CRISPR repeat sequence can be further considered. In some cases, when optimal alignment is achieved, the degree of complementarity between the tracr sequence and the CRISPR repeat sequence along 30 nucleotides of the shorter of the two is approximately or greater than 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher. For example, the length of the tracr sequence can be about or more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50 or more nucleotides.

[0128] The spacer region of the gRNA comprises a nucleotide sequence complementary to a sequence in the target DNA. In other words, the spacer region of the gRNA interacts with the target DNA in a sequence-specific manner through hybridization (e.g., base pairing). Therefore, the nucleotide sequence of the spacer region may vary and determine its location within the target DNA where the gRNA and target DNA will interact. The spacer region of the gRNA can be selected to hybridize with any desired sequence within the target DNA.

[0129] For example, the spacer region can have a length of 10 to 30 nucleotides (e.g., any length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides). For example, the spacer region can have a length of 13 to 25 nucleotides, 15 to 23 nucleotides, 18 to 22 nucleotides, or 20 to 22 nucleotides.

[0130] For example, the percentage of sequence complementarity between the spacer region of the gRNA and the target sequence of the target DNA can be at least about 60% (e.g., any one of at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%).

[0131] For example, the length of gRNA synthesized by the methods described herein can be from 30 to 160 nucleotides, such as 40 to 150, 50 to 140, 60 to 130, 70 to 120, 80 to 110, or 90 to 100 nucleotides (e.g., any length of 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 nucleotides). The gRNA synthesized by the methods described herein may include a spacer region. In some embodiments, the gRNA synthesized by the methods described herein includes a sequence complementary to a sequence in a target DNA, including but not limited to target mammalian DNA. For example, the target DNA may be human DNA.

[0132] Modifications in mlRNA

[0133] As described herein, mlRNAs may include one or more modifications for, for example, tracking, increasing stability, targeting RNA to specific subcellular sites, or reducing immunogenicity. Modifications to gRNAs can be used to enhance the formation or stability of DNA editing complexes comprising gRNA and RNA-guided endonucleases (e.g., Cas endonucleases, such as Cas9 endonucleases). Modifications to gRNAs can also, or alternatively, be used to enhance the initialization, stability, or kinetics of the interaction between the DNA editing complex and a target sequence in a target DNA, which can, for example, be used to improve on-target activity. Modifications to gRNAs can also, or alternatively, be used to enhance specificity, for example, the relative rate of DNA editing at on-target sites compared to effects at other (off-target) sites.

[0134] The modification can also be used, for example, to increase its resistance to degradation by ribonucleases (RNases) present in the cell, thereby increasing its half-life in the cell.

[0135] The mlRNA according to any of the embodiments described herein may include a segment at its 5' or 3' end that provides any of the features described above. Suitable segments include, for example, riboswitch sequences (e.g., for allowing regulation of stability and / or accessibility via proteins and protein complexes); stability control sequences; sequences forming dsRNA duplexes (e.g., hairpins); sequences enabling RNA targeting to subcellular locations (e.g., the nucleus, mitochondria, chloroplasts, etc.); modifications or sequences providing tracking (e.g., direct conjugation to fluorescent molecules, conjugation to portions promoting fluorescence detection, sequences allowing fluorescence detection, etc.); modifications or sequences providing a response to light or radiation (e.g., UV, vis, IR optogenetic elements); modifications or sequences providing binding sites for proteins (e.g., proteins acting on DNA, including transcriptional activators, transcriptional repressors, DNA methyltransferases, DNA demethylases, histone acetyltransferases, histone deacetylases, etc.); modifications or sequences providing enhanced, reduced, and / or controllable stability; and combinations thereof.

[0136] mlRNA according to any of the embodiments described herein may include modifications that reduce the likelihood or extent to which the RNA triggers an innate immune response when introduced into cells. Such responses, which have been well characterized in the context of RNA interference (RNAi) including small interfering RNA (siRNA) as described below and in the art, are often associated with a reduction in the half-life of the RNA and / or with the elicitation of cytokines or other factors associated with the immune response.

[0137] The mlRNA according to any of the embodiments described herein may also include one or more modifications selected from modifications that enhance RNA stability (e.g., by reducing degradation of it by RNases, e.g., in the cellular environment) and modifications that reduce the likelihood or extent to which the RNA triggers an innate immune response upon introduction into the cell. Combinations of modifications such as those described above and others may also be used.

[0138] Stability control sequence

[0139] The mlRNA according to any of the embodiments described herein may include stability control sequences that affect RNA stability. A non-limiting example of a suitable stability control sequence is a transcription terminator region (e.g., a transcription termination sequence). The total length of the transcription terminator region of RNA is from about 10 nucleotides to about 100 nucleotides, for example, from about 10 nucleotides (nt) to about 20 nt, from about 20 nt to about 30 nt, from about 30 nt to about 40 nt, from about 40 nt to about 50 nt, from about 50 nt to about 60 nt, from about 60 nt to about 70 nt, from about 70 nt to about 80 nt, from about 80 nt to about 90 nt, or from about 90 nt to about 100 nt. For example, the length of the transcription terminator region is from about 15 nucleotides (nt) to about 80 nt, from about 15 nt to about 50 nt, from about 15 nt to about 40 nt, from about 15 nt to about 30 nt, or from about 15 nt to about 25 nt.

[0140] Transcription termination sequences can be sequences that function in eukaryotic and / or prokaryotic cells.

[0141] Nucleotide sequences that can be included in stability control sequences (e.g., transcription termination segments, or any segment of RNA to provide increased stability) include, for example, Rho-independent trp termination sites.

[0142] Conjugate

[0143] The mlRNA according to any embodiment described herein may include modifications involving the chemical linking of one or more portions or conjugates that enhance RNA activity, cellular distribution, or cellular uptake to gRNA. These portions or conjugates may contain conjugation groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugation groups include, but are not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacokinetic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Suitable conjugation groups include, but are not limited to, cholesterol, lipids, phospholipids, biotin, phenazine, folic acid, phenidine, anthraquinones, acridine, fluorescein, rhodamine, coumarin, and dyes. Groups that enhance pharmacokinetic properties include those that improve uptake, enhance resistance to degradation, and / or enhance sequence-specific hybridization with target nucleic acids. Groups that enhance pharmacokinetic properties include those that improve nucleic acid uptake, distribution, metabolism, or excretion.

[0144] The mlRNA according to any of the embodiments described herein may include chemically linked conjugated portions, including but not limited to lipid portions such as cholesterol portions (Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA, 86(17): 6553-6556); bile acids (Manoharan et al. (1994) Bioorg. Med. Chem. Let., 4(8): 1053-1060); thioethers, for example, hexyl-S-triphenylmethylthiol (Manoharan et al. (1992) Ann. NY Acad. Sci., 660(1): 306-309 and Manoharan et al. (1993) Bioorg. Med. Chem. Let., 3(12): 2765-2770); thiocholesterol (Oberhauser et al. (1992) Nucl. Acids Res., 20(3): 533-538); aliphatic chains, for example, dodecanediol or undecyl residues (Saison-Behmoaras et al. (1991) EMBO J., 10(5): 1111-1118; (Kabanov et al. (1990) FEBS Lett., 259(2): 327-330 and Svinarchuk et al. (1993) Biochimie, 75(1-2): 49-54), phospholipids, for example, di-hexadecyl-racemic-glycerol or triethylammonium 1,2-di-O-hexadecyl-racemic-glycerol-3-H phosphonate (Manoharan et al. (1995) Tetrahedron Lett., 36(21): 3651-3654; Shea et al. (1990) Nucl. Acids Res., 18(13): 3777-3783), polyamine or polyethylene glycol chain (Manohoran et al. (1995) Nucleos. Nucleot. Nucl., 14(3-5): 969-973); adamantane acetic acid (Manoharan et al. (1995) Tetrahedron Lett., 36(21): 3651-3654); palmityl moiety (Mishra et al. (1995) Biochim. Biophys. Acta, 1264(2): 229-237); or octadecylamine or hexano-carbonyl-tertiary oxycholesterol moiety (Crooke et al. (1996) J. Pharmacol. Exp. Ther., 277(2): 923-937).

[0145] The mlRNA according to any of the embodiments described herein may include chemically linked conjugates comprising a “protein transduction domain” or PTD (also known as a cell-penetrating peptide, or CPP), which may refer to a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates passage through a lipid bilayer, micelles, cell membrane, organelle membrane, or vesicle membrane. A PTD attached to another molecule (ranging from small polar molecules to large macromolecules and / or nanoparticles) facilitates the molecule’s passage through membranes, e.g., from extracellular space to intracellular space, or from cytoplasm to organelles. The PTD may be covalently linked to gRNA. Exemplary PTDs include, but are not limited to, a minimum undecapeptide protein transduction domain (corresponding to residues 47-57 of HIV-1 TAT, containing YGRKKRRQRRR (SEQ ID NO:1); a polyarginine sequence containing a plurality of arginine residues sufficient to guide entry into the cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginine residues); and a VP22 domain (Zender et al. (2002) Cancer). GeneTher., 9(6):489-496); Drosophila antennal transduction domain (Noguchi et al. (2003) Diabetes, 52(7):1732-1737); truncated human calcitonin peptide (Tréhin et al. (2004) Pharm.Research, 21(7):1248-1256); polylysine (Wender et al. (2000) Proc.Natl.Acad.Sci.USA, 97(24):13003-13008). PTD can be an activatable CPP (ACPP). (Aguilera et al. (2009) Integr. Biol. (Camb), 1(5-6):371-381). ACPP comprises a polycationic CPP (e.g., Arg9 or "R9") connected to a matched polyanion (e.g., Glu9 or "E9") via a cleavable linker, which reduces the net charge to near zero, thereby inhibiting cell adhesion and uptake. Upon linker rupture, the polyanion is released, locally exposing the polyarginine and its inherent adhesiveness, thereby "activating" the ACPP to cross the membrane. PTD can be chemically modified to increase PTD bioavailability. Exemplary modifications are shown in... Published in Expert Opin. DrugDeliv., 6(11):1195-1205 (2009).

[0146] The mlRNA according to any of the embodiments described herein may also include applied conjugates that can enhance its delivery and / or uptake by cells, including, for example, cholesterol, tocopherol and folic acid, lipids, peptides, polymers, linkers and aptamers; see, for example, the review by Winkler (2013) Ther. Deliv., 4(7):791-809, and the references cited therein.

[0147] VI. RNA-guided endonucleases

[0148] Currently disclosed methods for synthesizing gRNA typically involve providing a first RNA fragment and a second RNA fragment, wherein the first RNA fragment, the second RNA fragment, or both contain at least a portion of a sequence that can bind to an RNA-guided endonuclease.

[0149] RNA-guided endonucleases can be naturally occurring or non-natural. Examples of such endonucleases include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cpf1 endonucleases and their functional derivatives. In some cases, the RNA-guided endonuclease is a Cas9 endonuclease. Cas9 endonucleases can be derived from, for example, *Streptococcus pyogenes* (SpyCas9), *SluCas9*, or *Staphylococcus aureus* (SaCas9). In some cases, the RNA-guided endonuclease is a variant of Cas9, and the Cas9 variant is selected from the group consisting of: small Cas9, dead Cas9 (dCas9), and Cas9 cleavage enzymes.

[0150] RNA-guided endonucleases can be small RNA-guided endonucleases. Small RNA-guided endonucleases can be partially engineered from RNA-guided endonucleases derived from any RNA-guided endonuclease described herein and known in the art. Small RNA-guided endonucleases can be, for example, small Cas endonucleases (e.g., PCT / US2018 / 065863; PCT / US2019 / 023044). In some cases, the length of small RNA-guided endonucleases is, for example, less than about 1,100 amino acids.

[0151] RNA-guided endonucleases can be mutated RNA-guided endonucleases. For example, an RNA-guided endonuclease can be a mutant of a naturally occurring RNA-guided endonuclease. A mutated RNA-guided endonuclease can also be a mutated RNA-guided endonuclease with altered activity compared to a naturally occurring RNA-guided endonuclease, such as altered endonuclease activity (e.g., altered or eliminated DNA endonuclease activity without a significant reduction in DNA binding affinity). This modification can allow the mutated RNA-guided endonuclease to target sequence-specific DNA for transcriptional regulation (e.g., activation or repression); through epigenetic or chromatin modifications such as methylation, demethylation, acetylation, or deacetylation, or any other modifications known in the art to DNA-binding and / or DNA-modifying proteins. In some cases, mutated RNA-guided endonucleases lack DNA endonuclease activity.

[0152] RNA-guided endonucleases can be either cleaving enzymes that cut the complementary strand of the target DNA but have reduced ability to cut the non-complementary strand, or cleaving enzymes that cut the non-complementary strand of the target DNA but have reduced ability to cut the complementary strand. In some cases, RNA-guided endonucleases have reduced ability to cut both the complementary and non-complementary strands of the target DNA.

[0153] VII. Methods for synthesizing sgRNA

[0154] In some embodiments, this disclosure provides a method for synthesizing sgRNA coupled with an RNA-guided endonuclease.

[0155] In some embodiments, the RNA-guided endonuclease is a Cas endonuclease. In some embodiments, the RNA-guided endonuclease is a Cas9 endonuclease. In some embodiments, the Cas9 endonuclease is a SpyCas9, SaCas9, or SluCas9 endonuclease. In some embodiments, the RNA endonuclease is a Cas9 endonuclease. In some embodiments, the RNA-guided endonuclease is a small RNA-guided endonuclease.

[0156] In some embodiments, the RNA-guided endonuclease is a small Cas endonuclease.

[0157] In some embodiments, the sgRNA comprises 5' to 3': crRNA and tracrRNA, wherein the crRNA and tracrRNA hybridize to form a double strand. In some embodiments, the crRNA comprises a spacer region sequence capable of targeting a target sequence in a target nucleic acid (e.g., a genomic DNA molecule) and a crRNA repeat sequence. In some embodiments, the tracrRNA comprises a tracrRNA anti-repetition sequence and a 3' tracrRNA sequence. In some embodiments, the 3' end of the crRNA repeat sequence is linked to the 5' end of the tracrRNA anti-repetition sequence, for example, via a four-loop, wherein the crRNA repeat sequence and the tracrRNA anti-repetition sequence hybridize to form the sgRNA. In some embodiments, the sgRNA comprises 5' to 3': a spacer region sequence, a crRNA repeat sequence, a four-loop, a tracrRNA anti-repetition sequence, and a 3' tracrRNA sequence. In some embodiments, the sgRNA further comprises a 5' spacer region extension sequence. In some embodiments, the sgRNA further comprises a 3' tracrRNA extension sequence. In some embodiments, the 3' tracrRNA comprises one or more stem-loops. In some embodiments, the 3'tracRNA comprises one, two, three, or more stem loops.

[0158] In some embodiments, the method includes synthesizing sgRNA using a splint-mediated ligation method, the method comprising two RNA fragments and a splint oligonucleotide. In some embodiments, the method includes providing a complex formed between a first RNA fragment, a second RNA fragment, and a splint oligonucleotide; and a ligase, wherein (a) the first RNA fragment includes a terminal region containing a 3' hydroxyl group; (b) the second RNA fragment includes a terminal region containing a 5' phosphate moiety; (c) the splint oligonucleotide includes (i) a first portion complementary to the terminal region containing the 3' hydroxyl group of the first RNA fragment, and (ii) a second portion complementary to the first terminal region containing the 5' phosphate moiety of the second RNA fragment; and wherein the complex is obtained by hybridization of (a) and (c)(i) and (b) and (c)(i) The hybridization formation (i) wherein the complex comprises a linker site present between a 3' hydroxyl group of a first RNA fragment and a 5' phosphate group of a second RNA fragment, wherein a ligase induces a linker at the linker site to form a phosphodiester bond between the 3' hydroxyl group of the first RNA fragment and the 5' phosphate group of the second RNA fragment, and the resulting sgRNA comprises a spacer sequence from 5' to 3' and an invariant sequence comprising a double strand formed between a crRNA repeat sequence and a tracrRNA anti-repetitive sequence, and a 3' tracrRNA sequence comprising at least one stem loop, thereby synthesizing sgRNA. In some embodiments, the linker site corresponds to a site in the double strand formed between the crRNA repeat sequence and the tracrRNA anti-repetitive sequence. In some embodiments, the linker site is in the crRNA repeat sequence, in a quadruplex linking the crRNA repeat sequence and the tracrRNA anti-repetitive sequence, or in the tracrRNA anti-repetitive sequence. In some embodiments, the linker site is within the stem loop of the 3' tracrRNA sequence. In some implementations, the first RNA fragment contains the nucleotide sequence of sgRNA, i.e., the 5' linker site; and the second RNA fragment contains the nucleotide sequence of sgRNA, i.e., the 3' linker site.

[0159] In some embodiments, the method includes synthesizing sgRNA using a splint-mediated ligation method, which comprises three RNA fragments and two splint oligonucleotides. In some embodiments, the method includes providing a complex formed between a first RNA fragment, a second RNA fragment, a third RNA fragment, a first splice oligonucleotide, and a second splice oligonucleotide; and a ligase, wherein (a) the first RNA fragment includes (i) a terminal region containing a 3' hydroxyl group; (b) the second RNA fragment includes (i) a first terminal region containing a 5' phosphate moiety, and (ii) a second terminal region containing a 3' hydroxyl group; (c) the third RNA fragment includes (i) a terminal region containing a 5' phosphate moiety; (d) the first splice oligonucleotide includes (i) a first portion complementary to the terminal region containing the 3' hydroxyl group of the first RNA fragment, and (ii) a second portion complementary to the first terminal region containing the 5' phosphate moiety of the second RNA fragment; and (e) the second splice oligonucleotide includes (i) a first portion complementary to the second terminal region containing the 3' hydroxyl group of the second RNA fragment, and (ii) a second portion complementary to the terminal region containing the 5' phosphate moiety of the third RNA fragment, wherein the complex is ligated via (a)(i) Hybridization of (d)(i), (b)(i) and (d)(ii), (b)(ii) and (e)(i) and (c)(i) and (e)(ii) forms a complex having a first linker site between a 3' hydroxyl group of the first RNA fragment and a 5' phosphate group of the second RNA fragment, and a second linker site between a 3' hydroxyl group of the second RNA fragment and a 5' phosphate group of the third RNA fragment, wherein a ligase induces ligation at the first linker site, forming a phosphodiester bond between the 3' hydroxyl group of the first RNA fragment and the 5' phosphate group of the second RNA fragment, and ligation at the second linker site, forming a phosphodiester bond between the 3' hydroxyl group of the second RNA fragment and the 5' phosphate group of the third RNA fragment, thereby forming sgRNA comprising a spacer sequence from 5' to 3' and an invariant sequence, the invariant sequence comprising a doublet formed between a crRNA repeat sequence and a tracrRNA anti-repetition sequence, and a 3' tracrRNA sequence comprising at least one stem loop, thereby synthesizing sgRNA.

[0160] In some embodiments, the first linker site corresponds to a site in the double strand formed between the crRNA repeat sequence and the tracrRNA anti-repetition sequence. In some embodiments, the first linker site is in the crRNA repeat sequence, in the quadruplex connecting the crRNA repeat sequence and the tracrRNA anti-repetition sequence, or in the tracrRNA anti-repetition sequence.

[0161] In some embodiments, the 3' tracrRNA sequence comprises a first, second, and third stem-loop. In some embodiments, the second linker site corresponds to a site in the first, second, or third stem-loop. In some embodiments, the second linker site corresponds to a site in the second stem-loop. In some embodiments, the second linker site corresponds to a site in the 5' stem of the second stem-loop, a site in the fourth loop of the second stem-loop, or a site in the 3' stem of the second stem-loop. In some embodiments, the second linker site is (i) immediately adjacent to a base in the 5' stem of the second stem-loop; (ii) close to a base in the 5' stem of the second stem-loop (e.g., ±1 nt, ±2 nt, or ±3 nt from the 5' stem); (iii) immediately adjacent to a base in the 3' stem of the second stem-loop; or (iv) close to a base in the 3' stem of the second stem-loop (e.g., ±1 nt, ±2 nt, or ±3 nt from the 3' stem).

[0162] In some embodiments, the first RNA fragment contains the nucleotide sequence of sgRNA, namely the 5' first linker site; the second RNA fragment contains the nucleotide sequence of sgRNA, namely the 3' first linker site and the 5' second linker site; and the third RNA fragment contains the nucleotide sequence of sgRNA, namely the 3' second linker site.

[0163] In some embodiments, the first RNA fragment comprises a 5' to 3':sgRNA spacer sequence and a portion of a crRNA repeat sequence. In some embodiments, the first RNA fragment comprises a 5' to 3':sgRNA spacer sequence and a crRNA repeat sequence. In some embodiments, the first RNA fragment comprises a 5' to 3':sgRNA spacer sequence, a crRNA repeat sequence, and a portion of a tetracyclic ring. In some embodiments, the first RNA fragment comprises a 5' to 3':sgRNA spacer sequence, a crRNA repeat sequence, a tetracyclic ring, and a portion of a tracRNA repeat sequence. In some embodiments, the first RNA fragment comprises a 5' to 3':sgRNA spacer sequence, a crRNA repeat sequence, a tetracyclic ring, and a portion of a tracRNA repeat sequence. In some embodiments, the terminal region of (a)(i), complementary to (d)(i) of the first splice oligonucleotide, comprises a nucleotide sequence of 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, or 34 nucleotides, wherein this nucleotide sequence is located at the 3' end of the first RNA fragment. In some embodiments, the terminal region of (a)(i) extends from the 3' end of the first RNA fragment to the 3' end of the spacer sequence (e.g., wherein the 5' end of the spacer sequence aligns with the 5' end of the first RNA fragment). In some embodiments, the terminal region of (a)(i) extends from the 3' end of the first RNA fragment to include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides present at the 3' end of the spacer region sequence. In some embodiments, the first portion (d)(i) of the first splice oligonucleotide is completely complementary to the terminal region of (a)(i). In some embodiments, the first portion (d)(i) of the first splice oligonucleotide has 1, 2, or 3 mismatches relative to the terminal region of (a)(i).

[0164] In some embodiments, the second RNA fragment comprises a 5' to 3' portion of the sgRNA crRNA repeat sequence, a tetracyclic ring, a tracrRNA repeat sequence, and a portion of the 3' tracrRNA sequence (i.e., the portion from 5' to the second linker site). In some embodiments, the second RNA fragment comprises a portion of the tracrRNA repeat sequence of the sgRNA and a portion of the 3' tracrRNA (i.e., the portion from 5' to the second linker site). In some embodiments, the terminal region (b)(i) complementary to the second portion (d)(ii) of the first splice oligonucleotide comprises a nucleotide sequence of 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, or 34 nucleotides and is located at the 5' end of the second RNA fragment. In some embodiments, the second portion (d)(ii) of the first splice oligonucleotide is completely complementary to the terminal region (b)(i), or has 1, 2, or 3 mismatches relative to the terminal region (d)(ii). In some embodiments, the terminal region (b)(ii) of the second RNA fragment complementary to the first portion (e)(i) of the second splice oligonucleotide comprises a nucleotide sequence of 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, or 34 nucleotides and is located at the 3' end of the second RNA fragment. In some embodiments, the first portion (e)(i) of the second splice oligonucleotide is completely complementary to the terminal region of (b)(ii), or has 1, 2, or 3 mismatches relative to the terminal region of (b)(ii).

[0165] In some embodiments, the third RNA fragment comprises a portion of the 3' tracrRNA sequence of the sgRNA (i.e., 3' to the second linker site). In some embodiments, the terminal region (c)(i) of the third RNA fragment, complementary to the second portion (e)(ii) of the second splice oligonucleotide, comprises a nucleotide sequence of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 nucleotides and is located at the 5' end of the third RNA fragment. In some embodiments, the first portion (e)(ii) of the second splice oligonucleotide is completely complementary to the terminal region (c)(i), or has 1, 2, or 3 mismatches relative to the terminal region (c)(i).

[0166] In some embodiments, the invariant sequence of the sgRNA comprises the nucleotide sequence of SEQ ID NO:17, or a nucleotide sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide deletions, insertions, or substitutions relative to SEQ ID NO:17. In some embodiments, the sgRNA is used in conjunction with the SpyCas9 endonuclease, wherein the invariant sequence of the sgRNA comprises the nucleotide sequence of SEQ ID NO:17, or a nucleotide sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide deletions, insertions, or substitutions relative to SEQ ID NO:17.

[0167] In some embodiments, the first RNA fragment, the second RNA fragment, and the third RNA fragment are each selected from nucleotide sequences including:

[0168] (a)(i)N 15-30 GUUUUAGAGCUAG(SEQ ID NO:56), where N 15-30 (ii) SEQ ID NO:3; (iii) SEQ ID NO:4;

[0169] (b)(i)N 15-30 GUUUUAGAGCUAGA(SEQ ID NO:57), where N 15-30 (ii) SEQ ID NO:40; (iii) SEQ ID NO:42;

[0170] (c)(i)N15-30 GUUUUAGAGCUAG(SEQ ID NO:56), where N 15-30 (ii) a spacer region sequence corresponding to a target site in a target nucleic acid (e.g., a genomic DNA molecule); (iii) SEQ ID NO:58; or

[0171] (d)(i)N 15-30 GUUUUAGAGCUAGA(SEQ ID NO:57), where N 15-30 (ii) SEQ ID NO:59; (iii) SEQ ID NO:4.

[0172] In some embodiments, the first splint oligonucleotide comprises the sequence listed in SEQ ID NO:60; SEQ ID NO:44; or SEQ ID NO:61. In some embodiments, the first portion (d)(i) of the first splint oligonucleotide comprises a sequence complementary to the terminal region (a)(i) of the first RNA fragment, wherein the terminal region extends from the 3' end of the first RNA fragment to include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the 3' end of the spacer region sequence of the sgRNA, wherein the 5' end of the spacer region sequence is aligned with the 5' end of the first RNA fragment. In some embodiments, the first portion (d)(i) of the first splint oligonucleotide comprises a sequence complementary to the terminal region (a)(i) of the first RNA fragment, wherein the terminal region is immediately adjacent to or downstream of the 3' end of the spacer region sequence of the sgRNA at a distance of 1, 2, or 3 nt, wherein the 5' end of the spacer region sequence aligns with the 5' end of the first RNA fragment. In some embodiments, the second splint oligonucleotide comprises the nucleotide sequences listed in SEQ ID NO:6; SEQ ID NO:45; or SEQ ID NO:53.

[0173] In some embodiments, the lengths of the first RNA fragment, the second RNA fragment, and the third RNA fragment are each independently about 10 to about 90 nucleotides, about 10 to about 60 nucleotides, about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 20 to about 40 nucleotides, and about 30 to about 40 nucleotides.

[0174] In some embodiments, the first splint oligonucleotide is a DNA oligonucleotide. In some embodiments, the first splint oligonucleotide is an RNA oligonucleotide. In some embodiments, the second splint oligonucleotide is a DNA oligonucleotide. In some embodiments, the second splint oligonucleotide is an RNA oligonucleotide. In some embodiments, the lengths of the first and second splint oligonucleotides are each independently about 20 to about 100 nucleotides, about 20 to about 90 nucleotides, about 20 to about 80 nucleotides, about 20 to about 70 nucleotides, about 20 to about 60 nucleotides, about 30 to about 60 nucleotides, or about 30 to about 50 nucleotides.

[0175] In some embodiments, the first, second, and / or third RNA fragments are synthesized according to the methods described herein, for example, in vitro using RNA polymerase or using solid-phase synthesis employing phosphoramide chemistry. In some embodiments, the RNA fragments are synthesized using phosphoramide chemistry, wherein (i) the synthesis of the first RNA fragment, the second RNA fragment, and / or the third RNA fragment is each performed in a 5' to 3' or 3' to 5' orientation; or (ii) the synthesis of the first RNA fragment is performed in a 5' to 3' or 3' to 5' orientation, and the synthesis of the second RNA fragment and / or the third RNA fragment is each performed in a 3' to 5' orientation. In some embodiments, the RNA fragments are purified after synthesis.

[0176] In some embodiments, the first and / or second splint oligonucleotides are synthesized according to the methods described herein, for example, in vitro using RNA polymerase or using solid-phase synthesis employing phosphoramide chemistry. In some embodiments, the splint oligonucleotides are purified after synthesis.

[0177] In some embodiments, the first, second, and / or third RNA fragments contain one or more modifications to the RNA backbone as described herein, such as backbone bond or nucleoside modifications. In some embodiments, the modification is a phosphate thioester bond. In some embodiments, the modification is 2'-O-methylation of a nucleoside.

[0178] In some embodiments, hybridization is performed according to the methods described herein. In some embodiments, hybridization is performed in solution. In some embodiments, hybridization is performed with or without an annealing step. In some embodiments, the annealing step includes (i) heating the solution to about 80°C to about 95°C for a period of less than about 10 minutes (e.g., 1, 2, 3, 4, or 5 minutes); and (ii) cooling the solution to a temperature for bonding (e.g., about 30°C to about 40°C) at a rate of about 0.1°C / second to about 2°C / second.

[0179] In some embodiments, the ligation reaction is performed according to the methods described herein. In some embodiments, ligation is performed at about 15°C to about 45°C, or about 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C. In some embodiments, ligation is performed for about 0.1 to about 48 hours, or about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 hours. In some embodiments, a protease or chelating agent is used for ligation. In some embodiments, a congesting agent is used for ligation. In some embodiments, ligation is performed to at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% completion. In some embodiments, the sgRNA is purified after synthesis, for example, using chromatographic methods.

[0180] Example

[0181] Unless otherwise stated, the present invention will be practiced using conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry and immunology known to those skilled in the art.Non-limiting examples include Sambrook & Russell (2012) Molecular Cloning: A Laboratory Manual (4th ed.); Ausubel (1987) Current Protocols in Molecular Biology, New York, NY: Wiley (including supplements through 2014); Bollag et al. (1996) Protein Methods. New York, NY: Wiley-Liss; Huang et al. (2005) Nonviral Vectors for Gene Therapy. San Diego: Academic Press; Kaplitt et al. (1995) Viral Vectors: Gene Therapy and Neuroscience Applications. San Diego, CA: Academic Press; Lefkovits (1997) The Immunology Methods Manual: The Comprehensive Sourcebook of Techniques. San Diego, CA: Academic Press; Doyle et al. (1998) Cell and Tissue Culture: Laboratory Procedures in Biotechnology. New York, NY: Wiley; Mullis, Ferré & Gibbs (1994) PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher; Greenfield (2014) Antibodies: A Laboratory Manual (2nd ed.). New York, NY: Cold Spring Harbor Laboratory Press; Beaucage et al. (2000) Current Protocols in Nucleic Acid Chemistry. New York, NY: Wiley, (including the 2014 supplement); and Makrides (2003) Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences BV, the contents of which are incorporated herein by reference.

[0182] Further embodiments are disclosed in more detail in the following examples, which are provided by way of illustration and are not in any way intended to limit the scope of this disclosure or the claims.

[0183] Example 1: Splint-mediated ligation of an exemplary gRNA

[0184] To demonstrate that chemically synthesized RNA fragments can be used to construct full-length gRNA products using splint-mediated ligation, an exemplary gRNA molecule targeting a single locus in the mouse genome was synthesized using this method. The gRNA was split into three RNA fragments, each less than 40 nucleotides in length. Figure 4 The sequences of the three RNA fragments and two DNA clip oligonucleotides are shown in Table 1. The sites for splitting the gRNA were selected to remove internal hairpins that would interfere with hybridization with the DNA clip oligonucleotides. Because these sites for splitting the gRNA are located downstream of the invariant region (i.e., the spacer or variable region) of the gRNA, these sites can be used for any gRNA used by Cas enzymes derived from Streptococcus pyogenes (SpyCas or SpCas), such as SpyFi, and only the RNA at the 5' end of the gRNA construct (such as RNA1 in Table 1) and its corresponding clip (such as clip 1->2 in Table 1) require unique synthesis for different genomic targets.

[0185] Table 1: Exemplary sequences of RNA fragments and DNA clipping oligonucleotides

[0186]

[0187] Selection criteria for gRNA segmentation

[0188] Under biological conditions, RNAs associated with the Cas protein family possess internal structures that can interfere with their hybridization with complementary splint oligonucleotides. For example, when assembled with Cas proteins, gRNAs associated with spCas9 have four stem-loops (hairpins), while gRNAs associated with saCas9 have three. When gRNAs are not associated with Cas proteins, these stem-loops, specifically two tetraloops, are presumed to be present. To minimize intramolecular association of RNA fragments that could hinder splint hybridization, sites for splitting the gRNA are selected within these stem-loop motifs to disrupt these energy-favorable secondary structures. The stability of the stem-loop depends on the stability of the stem / helix (i.e., the number of nucleotides forming the helix) and loop (i.e., the base type and the number of nucleotides forming the loop) regions. Splitting within the loop region removes the stem-loop and is therefore generally preferred. Splitting within the helix is ​​also compatible with this approach, and free energy calculations are used to ensure proper binding of the RNA fragment to the splint.

[0189] Selection of DNA clip oligonucleotides

[0190] The length of the DNA splint oligonucleotide is chosen to promote the formation of a double helix between the splint and the RNA fragment, rather than the formation of intramolecular structures within the RNA fragment. This is done computationally by comparing the energy of the internal structure with the energy of the double helix formed between the splint and the pre-ligated RNA fragment. The minimum free energy prediction algorithm (mFold) is used to calculate the free energy (ΔG) of the RNA secondary structure of a single fragment. intra ) and intermolecular hybridization (ΔG) between fragments and splints inter Extend the length of the clamp until the following criteria are met to ensure a good fit between the segment and the clamp during the connection process.

[0191] The free energy of intramolecular structures is set to be greater than that of intermolecular structures, which is related to their melting temperatures (T). m It is inversely proportional to the others.

[0192] ΔG intra >ΔG inter ~T m-intra <T m-inter

[0193] In addition, the temperature at which the connection reaction takes place (T) rxn The T value is set below that of the RNA / DNA intercalation complex. m .

[0194] T rxn <T m-inter

[0195] DNA-sandwich oligonucleotides are used because i) T4 RNA ligase II can be used to ligate DNA / RNA heteroduplexes, and ii) DNA is easier and less expensive to manufacture than RNA. However, the sandwich can also be synthesized from RNA, non-natural nucleic acids, artificial nucleic acids (e.g., peptide nucleic acids), or any nucleic acid mimic. Procedures for determining free energy and stability can be accessed at URL unafold.rna.albany.edu / ?q=mfold.

[0196] Chemical synthesis and purification of RNA fragments

[0197] RNA fragments and DNA clip oligonucleotides were synthesized using standard phosphorylation chemistry and extended in a 3' to 5' orientation. Since one of the substrates of T4 RNA ligase is the 5' phosphorylated RNA oligomer, all RNA fragments except those that would become the 5' end of the gRNA (e.g., RNA 1 in Table 1) were synthesized with terminal 5' phosphate esters. All RNA fragments were purified by reversed-phase HPLC, ion-exchange chromatography, or PAGE prior to use. Because phosphorylation is the final coupling step in the synthesis of these RNAs, no truncated products from their synthesis are incorporated during enzymatic ligation. The only truncated product that can be incorporated will come from the 5' terminal fragment. For this reason, it is advantageous to purify the 5' terminal fragment prior to ligation and to design this fragment to be less than 40 nucleotides. In some cases, the 5' terminal fragment was synthesized in a 5' to 3' orientation, while other fragments were synthesized in a 3' to 5' orientation, which prevents truncated products from the 5' terminal fragment synthesis from being included in the final ligation product.

[0198] Modified oligonucleotides

[0199] RNA fragments with a non-natural (modified) ribose-phosphate backbone can also be used to synthesize gRNA using splint-mediated ligation as described herein. For example, ligation of RNA fragments with methoxy-substituted 2' hydroxyl groups at or one to two nucleotides from the 3' end ligation site has been achieved. These RNA fragments also contain phosphate-thioester bonds 20 or more nucleotides from the ligation site. These modifications may also be permitted at or near the ligation site.

[0200] Connection reaction

[0201] RNA fragments and DNA clips were combined at concentrations of 10 or 20 μM each in T4 RNA ligase II reaction buffer (New England Biolabs (NEB)). The concentrations of RNA fragments and DNA clips were approximately equal. The solution was heated to 90 °C for 3 minutes and then cooled to 37 °C at a rate of 1 °C / s to disrupt the internal structure of the RNA fragment and allow the DNA clips and RNA fragment to anneal. T4 RNA ligase II was then added to the solution, and the mixture was incubated at 37 °C for 0.5 to 24 hours. According to NEB's definition, one unit of T4 RNA ligase II is the amount of enzyme required to ligate 0.4 μg of an equimolar mixture of 23-mer and 17-mer RNA in a total reaction volume of 20 μL within 30 minutes at 37 °C. The ligation reaction can also be performed at other temperatures, such as 25 °C, in which case the reaction time will be increased, and the reaction can be quenched using protease or EDTA. A congestion agent (e.g., PEG) can be added to the ligation reaction.

[0202] Separation of the entire product

[0203] Full-length gRNA products were isolated from DNA splice oligonucleotides and unlinked RNA 1, 2, or 3 fragments using ion-exchange high-performance liquid chromatography (HPLC). Figure 5 This is an HPLC chromatogram showing the presence of oligonucleotides before (top trace) and after (bottom trace) the ligation reaction. The ligation products RNA2-3, RNA1-2, and full-length gRNA (RNA 1-2-3) were detected.

[0204] The full-length gRNA product was tested using a plasmid containing the target DNA sequence and in combination with SpCas9. Figure 6 As shown, the plasmid was cleaved by SpCas9 at the appropriate target site.

[0205] These results indicate that chemically synthesized RNA fragments can be used to construct full-length gRNA products using splint-mediated ligation.

[0206] Example 2: Splint-mediated modified RNA ligation

[0207] To examine whether splint-mediated ligation can be used to ligate modified RNA fragments, exemplary modified RNAs (RNA 2m1 and RNA 2m2) as shown in Table 2 below were ligated using DNA splint oligonucleotides, splint 1, and splint 2. Figure 7A The chemical structure of unmodified RNA and exemplary modifications are shown.

[0208] Table 2. Sequences of modified RNA fragments and splint oligonucleotides

[0209]

[0210] Figure 7B This is a chromatogram showing the HPLC analysis results of the ligation products. The ligation products RNA2m1-RNA3, RNA1-RNA2m1, and RNA2m2-RNA3 were detected.

[0211] Next, use clip 1 and clip 2 to connect RNA1 and RNA2m1 (see Table 3 below). Figure 8 The RNA is shown as RNA2 and RNA3 to produce full-length gRNA.

[0212] Table 3. Sequences of modified RNA fragments and splint oligonucleotides

[0213]

[0214] Full-length gRNA products were isolated from DNA clip oligonucleotides and partial ligation products (RNA2-RNA3 and RNA1-RNA2) using HPLC. Figure 8 This is a chromatogram showing the HPLC analysis results of the ligation products before and after purification. Full-length RNA products (RNA1-2-3gRNA) were detected, and the purified full-length product is shown in the bottom trajectory. These results indicate that RNA fragments with methylation modifications around the ligation site can be ligated using the splint-mediated ligation described in this disclosure.

[0215] While certain alternatives to this disclosure have been presented, it should be understood that various modifications and combinations are possible and are considered within the spirit and scope of the appended claims. Therefore, there is no intention to limit the precise abstract and disclosure given herein.

[0216] Example 3: Evaluation of response conditions for splint-mediated connections

[0217] Experimental conditions for splint-mediated ligation reactions of modified RNA were compared. This included (i) the addition of magnesium salts to the reaction; and (ii) the evaluation of the use of a thermal annealing step prior to the ligation reaction.

[0218] Splint-mediated ligation was used to prepare the final modified sgRNA product containing a spacer region sequence targeting exon 2 of the human G6PC gene and a backbone for use with SpyCas9. The sequences of the modified sgRNA product and their corresponding unmodified versions are shown in Table 4. Splint-mediated ligation used three RNA fragments and two DNA splint oligonucleotides, each as shown in Table 4. The RNA fragments included:

[0219] (i) 33mer RNA 1 (SEQ ID NO:11), which includes, in the 5' to 3' direction: a spacer region sequence, a crRNA repeat sequence of the final sgRNA product and the final sgRNA product;

[0220] (ii) 39mer RNA 2 (SEQ ID NO:12), which includes, in the 5' to 3' direction: a tetracyclic "AAA", and the 5' segment of the tracrRNA of the final sgRNA product; and

[0221] (iii) 28mer RNA 3 (SEQ ID NO:13), which includes the 3' segment of the tracrRNA of the final sgRNA product.

[0222] like Figure 9A As shown, the DNA clip 1 oligonucleotide (SEQ ID NO:5) was designed to be complementary to the 3' region of RNA 1 and the 5' region of RNA 2. Specifically, the sequence 5'-CTAGCTCTAAAACTC-3' (SEQ ID NO:22) of DNA clip 1 is complementary to the sequence 5'-GUGUUUUAGAGCUAG-3' (SEQ ID NO:23) of RNA 1; and the sequence 5'-CCTTATTTTAACTTGCTATTT-3' (SEQ ID NO:24) of DNA clip 1 is complementary to the sequence 5'-AAAUAGCAAGUUAAAAUAAGG-3' (SEQ ID NO:25) of RNA 2.

[0223] Furthermore, the DNA clip 2 oligonucleotide (SEQ ID NO:6) was designed to be complementary to the 3' region of RNA 2 and the 5' region of RNA 3. Specifically, the sequence 5'-AAGTTGATAACGGACTAG-3' (SEQ ID NO:26) of DNA clip 2 is complementary to the sequence 5'-CUAGUCCGUUAUCAACUU-3' (SEQ ID NO:27) of RNA 2; and the sequence 5'-AAAAGCACCGACTCGGTGCCACTTTTTC-3' (SEQ ID NO:28) of DNA clip 2 is complementary to the sequence 5'-GAAAAAGUGGCACCGAGUCGGUGCUUUU-3' (SEQ ID NO:29) of RNA 3.

[0224] like Figure 9BAs shown, the ligation of RNA fragments occurs at the first ligation site between RNA fragment 1 and RNA fragment 2, located within the GAAA quadruple loop of the repeat-anti-repeat stem loop formed between crRNA and tracrRNA; and at the second ligation site between RNA fragment 2 and RNA fragment 3, located near the GAAA quadruple loop of the second stem loop in tracrRNA. The DNA clip 1 oligonucleotide listed in SEQ ID NO:5 is complementary to a segment of RNA 1, but with one mismatch. The DNA clip 1 oligonucleotide listed in SEQ ID NO:14 is complementary to the same portion of RNA 1, but without a mismatch, and is also used in the ligation reaction.

[0225] Table 4. Unmodified and modified sequences of RNA fragments and splint oligonucleotides used to generate sgRNA targeting G6PC.

[0226]

[0227] Each oligonucleotide was dissolved in water at a concentration of 1 mM. An equimolar mixture of RNA fragments and DNA clips was prepared. The RNA / DNA mixture was then heated to 90 °C for 3 minutes and cooled to 37 °C at a rate of 1 °C / s to disrupt the internal structure of the RNA fragments and allow annealing to form a DNA / RNA hybrid structure (“annealing”). Alternatively, this step can be skipped (“non-annealing step”).

[0228] The annealed or unannealed RNA / DNA mixture was then diluted with 50 U of T4 RNA ligase II in T4 RNA ligase II reaction buffer (New England Biolabs (NEB)) at a final concentration of 10 μM for each RNA and DNA splice oligonucleotide. The ligation reaction was prepared by adding MgCl2 to provide a final concentration of 12.5 mM or without adding MgCl2. The solution was incubated at 37 °C for 16 h. The reaction was terminated by adding proteinase K or by quenching with EDTA. The reaction mixture was then analyzed by ion-exchange HPLC to determine the presence of full-length gRNA product and partial ligation products (RNA2-RNA3 and RNA1-RNA2).

[0229] like Figure 10 As shown, full-length RNA products (RNA1-2-3 gRNA; retention time 30.58 min) were detected in the ligation reactions under each evaluation condition. The results indicate that the clip-mediated ligation reaction of the modified RNA fragments can be achieved without an initial annealing step and can proceed successfully with or without the addition of magnesium salts.

[0230] Example 4: Design variant for achieving clamp-mediated connection response

[0231] Alternative designs were developed to prepare the final sgRNA product (the modified sequence shown in SEQ ID NO: 20) described in Example 3 using a splint-mediated ligation reaction. These designs are based on using two splint oligonucleotides to ligate three RNA fragments. One difference between the designs is the degree of complementarity between the first splint oligonucleotide and the first RNA fragment. As further described below, the first RNA fragment includes a variable spacer region sequence and a partially invariant sequence in the final sgRNA product. The first design has a first splint oligonucleotide complementary only to the invariant region of the first RNA fragment, and the second design has a first splint oligonucleotide complementary to both the invariant region and a portion of the variable region. Therefore, the first design provides a set of components in which only the first RNA fragment is modified to prepare sgRNAs with different target specificities. Based on the second design, both the first RNA fragment and the first splint oligonucleotide are modified to prepare sgRNAs with different target specificities. However, one advantage of this design is the increased overlap between the first splice oligonucleotide and the first RNA fragment, which is expected to increase the stability (i.e., melting temperature) of the RNA / DNA heteroduplex formed between these components, thereby promoting the formation of the heteroduplex at the temperature used for the ligation reaction (e.g., 37°C).

[0232] The first design includes the RNA fragments shown in Table 5, displayed as modified or unmodified versions. Specifically, the RNA fragments include:

[0233] (i) 34mer RNA 1, whose 5' to 3' contains a spacer sequence (SEQ ID NO:20), a crRNA repeat sequence, and part of a quadruple loop of the final sgRNA repeat-anti-repeat stem-loop;

[0234] (ii) 34mer RNA 2, comprising the remainder of the four-loop repeat-anti-repeat stem-loop, the anti-repeat sequence of tracrRNA, and a portion of the tracrRNA containing the bases of the second stem-loop extended into the tracrRNA; and

[0235] (iii) 32mer RNA 3, including the remaining 3' portion of tracrRNA.

[0236] DNA clip 1 oligonucleotide (SEQ ID NO:44) is designed with segments complementary to the 3' region of RNA 1 and the 5' region of RNA 2. Specifically, the 5'-TCTAGCTCTAAAAC-3' sequence of DNA clip 1 (SEQ ID NO:30) is complementary to the 5'-GUUUUAGAGCUAGA-3' sequence of RNA 1 (SEQ ID NO:31); the 5'-TTATTTTAACTTGCTATT-3' sequence of DNA clip 1 (SEQ ID NO:32) is complementary to the 5'-AAUAGCAAGUUAAAAUAA-3' sequence of RNA 2 (SEQ ID NO:33).

[0237] Furthermore, the DNA clip 2 oligonucleotide (SEQ ID NO:45) was designed with segments complementary to the 3' region of RNA 2 and the 5' region of RNA 3. Specifically, the 5'-TGATAACGGACTAGCC-3' (SEQ ID NO:34) sequence of DNA clip 2 is complementary to the 5'-GGCUAGUCCGUUAUCA-3' (SEQ ID NO:35) sequence of RNA 2; and the 5'-TCGGTGCCACTTTTTCAAGT-3' (SEQ ID NO:36) sequence of DNA clip 2 is complementary to the 5'-ACUUGAAAAAGUGGCACCGA-3' (SEQ ID NO:37) sequence of RNA 3.

[0238] One advantage of this design is that the first clip oligonucleotide (DNA clip 1) is complementary to the segment of the first RNA fragment (RNA 1) that does not include the spacer region sequence. Therefore, the remaining first and second clip oligonucleotides (DNA clip 1 and DNA clip 2), as well as the second and third RNA fragments (RNA 2 and RNA 3), are "universal" because they are used to prepare sgRNAs conjugated with SpyCas9 with the invariant main strand sequence listed in SEQ ID NO:17. The first RNA fragment containing only the spacer region sequence most strongly associated with the sgRNA is customized according to the desired targeting specificity of the sgRNA.

[0239] Table 5. Unmodified and modified sequences of RNA fragments and splint oligonucleotides used to generate sgRNA targeting G6PC.

[0240]

[0241] The second design includes the RNA fragments shown in Table 6, displaying modified or unmodified versions, the same as those used in the first design described above. A schematic diagram of the second design is shown below. Figure 11The sequence alignment of the first, second, and third RNA fragments with respect to the first and second DNA splices is provided. The DNA version of the nucleotide sequence of the final sgRNA product is shown in a 5' to 3' orientation (the RNA version of the nucleotide sequence is listed in SEQ ID NO:19), and the regions corresponding to the nucleotide sequences of the first, second, and third RNA fragments are indicated (the RNA versions of the first, second, and third RNA fragments are listed in SEQ ID NO:38, 40, and 42, respectively). The alignment of the first and second DNA splices with the first, second, and third RNA fragments to form RNA / DNA duplexes is also shown, with the nucleotide sequences of the first and second DNA splices shown in a 3' to 5' orientation (the nucleotide sequences are listed in SEQ ID NO:52 and 53, respectively).

[0242] The second design of the DNA clip 1 oligonucleotide (SEQ ID NO:52) has segments complementary to the 3' region of RNA 1 and the 5' region of RNA 2. Specifically, the 5'-TCTAGCTCTAAAACACCAGTATG-3' (SEQ ID NO:46) sequence of DNA clip 1 is complementary to the 5'-CAUACUGGUGUUUUAGAGCUAGA-3' (SEQ ID NO:47) sequence of RNA 1; the 5'-TATTTTAACTTGCTATT-3' (SEQ ID NO:48) sequence of DNA clip 1 is complementary to the 5'-AAUAGCAAGUUAAAAUA-3' (SEQ ID NO:49) sequence of RNA 2. According to this design, DNA clip 1 overlaps with 9 nucleotides present at the 3' end of the spacer region sequence in RNA 1. The expected overlap with the expansion of the first RNA fragment increases the stability of the heteroduplex formed between the first splice oligonucleotide and the first RNA fragment, in particular increasing the melting temperature of the heteroduplex and promoting the formation of the heteroduplex under the conditions used for the ligation reaction.

[0243] Furthermore, the DNA clip 2 oligonucleotide (SEQ ID NO:53) is designed with regions complementary to the 3' portion of RNA 2 and the 5' portion of RNA 3. Specifically, the 5'-TGATAACGGACTAGCCT-3' (SEQ ID NO:50) sequence of DNA clip 2 is complementary to the 5'-AGGCUAGUCCGUUAUCA-3' (SEQ ID NO:51) sequence of RNA 2; and the 5'-GACTCGGTGCCACTTTTTCAAGT-3' (SEQ ID NO:54) sequence of DNA clip 2 is complementary to the 5'-ACUUGAAAAAGUGGCACCGAGUC-3' (SEQ ID NO:55) sequence of RNA 3.

[0244] Table 6. Unmodified and modified sequences of RNA fragments and splint oligonucleotides used to generate sgRNA targeting G6PC.

[0245]

[0246] sequence list

[0247]

[0248]

[0249]

Claims

1. A method for synthesizing guide RNA (gRNA), the method comprising: Provide a first RNA fragment comprising a terminal region containing a 5' phosphate moiety, and a second RNA fragment comprising a terminal region containing a 3' hydroxyl group, wherein the first RNA fragment, the second RNA fragment, or both comprise at least a portion of a sequence capable of binding to an RNA-guided endonuclease; A splice oligonucleotide is provided, comprising a first portion of a terminal region complementary to a first RNA fragment, the terminal region comprising a 5' phosphate moiety and a second portion of a terminal region complementary to a second RNA fragment, the terminal region comprising a 3' hydroxyl group; The first RNA fragment, the second RNA fragment, and the splint oligonucleotide are hybridized together to form a complex; and gRNA is synthesized by using RNA ligase to ligate the first and second RNA fragments at the ligation sites present between the RNA complexes.

2. The method according to claim 1, wherein, The lengths of the first and second RNA fragments are 10 to 90 nucleotides, respectively.

3. The method according to claim 2, wherein, The length of the second RNA fragment is 40 nucleotides or less.

4. The method according to any one of claims 1-3, wherein, The 5' phosphate ester portion is a 5'-phosphate ester or a 5'-thiophosphate ester.

5. The method according to any one of claims 1-3, wherein, The RNA ligase is either T4 RNA ligase I or T4 RNA ligase II.

6. The method according to any one of claims 1-3, wherein, The splint oligonucleotide is a DNA or RNA oligonucleotide.

7. The method according to any one of claims 1-3, wherein, The length of the splint oligonucleotide is 20 to 100 nucleotides.

8. The method according to any one of claims 1-3, wherein, The splice oligonucleotides are attached to a solid carrier.

9. The method according to any one of claims 1-3, wherein, The gRNA is 30 to 160 nucleotides in length.

10. The method according to any one of claims 1-3, wherein, The gRNA contains a sequence complementary to the sequence in the target DNA.

11. The method according to claim 10, wherein, The target DNA is mammalian DNA.

12. The method according to claim 11, wherein, The target DNA is human DNA.

13. The method according to any one of claims 1-3, wherein, The connection site corresponds to a site in the four-loop portion of the stem-loop structure in the synthesized gRNA.

14. The method according to any one of claims 1-3, wherein, The connection site corresponds to a site in the helical portion of the stem-loop structure in the synthesized gRNA.

15. The method according to any one of claims 1-3, wherein, The first RNA fragment, the second RNA fragment, or both contain at least one secondary structure, and wherein hybridization of the first RNA fragment, the second RNA fragment, and the splice oligonucleotide produces a free energy lower than the free energy of the secondary structure having the lowest free energy.

16. The method according to any one of claims 1-3, comprising linking three or more RNA fragments.

17. The method according to any one of claims 1-3, wherein, Providing the first and second RNA fragments includes synthesizing the first and second RNA fragments by enzymatic synthesis or phosphoramide chemistry.

18. The method according to claim 17, wherein, The second RNA fragment is synthesized in a 5' to 3' or 3' to 5' orientation.

19. The method of claim 17, wherein, Providing the first and second RNA fragments includes purifying the first and second fragments after synthesis.

20. The method according to any one of claims 1-3, wherein, The provision of the splint oligonucleotide includes the synthesis of the splint oligonucleotide by enzymatic synthesis or phosphoramide chemistry.

21. The method according to claim 20, wherein, Providing splint oligonucleotides includes purifying the splint oligonucleotides after synthesis.

22. The method according to claim 19, wherein, Purification includes purification using chromatographic methods.

23. The method according to claim 22, wherein, The chromatographic method is reversed-phase HPLC, ion exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, affinity chromatography, or polyacrylamide gel purification or any combination thereof.

24. The method according to any one of claims 1-3, wherein, The first RNA fragment, the second RNA fragment, or both contain at least one modification in the RNA backbone.

25. The method according to claim 24, wherein, The modification is selected from the group consisting of: 2'methoxy (2'OMe), 2'fluorine (2'fluorine), 2'-O-methoxy-ethyl (MOE), locked nucleic acid (LNA), unlocked nucleic acid (UNA), bridging nucleic acid, 2'deoxynucleic acid (DNA), and peptide nucleic acid (PNA).

26. The method according to any one of claims 1-3, wherein, The first RNA fragment, the second RNA fragment, or both contain at least one base modification.

27. The method according to claim 26, wherein, The base modification is selected from the group consisting of: 2-aminopurine, inosine, thymine, 2,6-diaminopurine, 2-pyrimidinone, and 5-methylcytosine.

28. The method according to any one of claims 1-3, wherein, The first RNA fragment, the second RNA fragment, or both contain at least one phosphate thioester bond.

29. The method according to any one of claims 1-3, wherein, Hybridization is carried out in solution.

30. The method according to claim 29, wherein, The concentrations of the splint oligonucleotide, the first RNA fragment, and the second RNA fragment are equal in the solution.

31. The method according to any one of claims 1-3, wherein, The ligation of the first and second RNA fragments was performed at 15°C–45°C.

32. The method according to claim 31, wherein, The ligation of the first and second RNA fragments was performed at 37°C.

33. The method according to any one of claims 1-3, wherein, The ligation of the first and second RNA fragments takes 0.1 to 48 hours.

34. The method according to any one of claims 1-3, wherein, Connecting the first and second RNA fragments also includes using a protease or chelating agent.

35. The method according to claim 34, wherein, The chelating agent is EDTA, EGTA, or a combination of both.

36. The method according to any one of claims 1-3, wherein, Connecting the first and second RNA fragments also includes using one or more congestion agents.

37. The method of claim 36, wherein, The one or more crowding agents include polyethylene glycol (PEG). Ethylene glycol, dextran, or any combination thereof.

38. The method according to any one of claims 1-3, wherein, The ligation of the first and second RNA fragments was completed to a degree of at least 10%.

39. The method according to claim 38, wherein, The ligation of the first and second RNA fragments was completed to a degree of at least 90%.

40. A method for synthesizing guide RNA (gRNA), the method comprising providing: (a) The first RNA fragment, which contains a terminal region containing a 3' hydroxyl group; (b) A second RNA fragment comprising a first terminal region containing a 5' phosphate moiety and a second terminal region containing a 3' hydroxyl group; (c) The third RNA fragment, which contains a terminal region containing a 5' phosphate ester portion; (d) A first splice oligonucleotide comprising (i) a first portion complementary to the terminal region containing the 3' hydroxyl group of the first RNA fragment; and (ii) a second portion complementary to the first terminal region containing the 5' phosphate portion of the second RNA fragment. (e) a second splice oligonucleotide comprising (i) a first portion complementary to the second terminal region containing the 3' hydroxyl group of the second RNA fragment; and (ii) a second portion complementary to the terminal region containing the 5' phosphate moiety of the third RNA fragment; and (f) RNA ligase, in, Hybridization of the first, second, and third RNA fragments with the first and second splice oligonucleotides results in the formation of a complex having a first linker site between the 3' hydroxyl group of the first RNA fragment and the 5' phosphate group of the second RNA fragment, and a second linker site between the 3' hydroxyl group of the second RNA fragment and the 5' phosphate group of the third RNA fragment. and The RNA ligase causes the first and second RNA fragments to be ligated at the first ligation site, and the second and third RNA fragments to be ligated at the second ligation site, thereby synthesizing gRNA.

41. The method according to claim 40, wherein, The gRNA comprises a first RNA fragment connected to the second RNA fragment via a first phosphodiester bond at 5' to 3', and a second RNA fragment connected to the third RNA fragment via a second phosphodiester bond.

42. The method according to claim 41, wherein, The first phosphodiester bond is formed between the 3' hydroxyl group of the first RNA fragment and the 5' phosphate group of the second RNA fragment, and wherein the second phosphodiester bond is formed between the 3' hydroxyl group of the second RNA fragment and the 5' phosphate group of the third RNA fragment.

43. The method according to any one of claims 40-42, wherein, The gRNA is a single-molecule gRNA (sgRNA).

44. The method according to any one of claims 40-42, wherein, The gRNA is 30 to 160 nucleotides in length.

45. The method according to any one of claims 40-42, wherein, The first connection site corresponds to a site in a first stem-loop structure, wherein the first stem-loop structure is formed by the hybridization of the minimum CRISPR repeat sequence and the minimum tracrRNA sequence in the gRNA.

46. ​​The method according to claim 45, wherein, The site in the first stem-ring structure is in the four-ring portion or the spiral portion.

47. The method according to any one of claims 40-42, wherein, The second connection site corresponds to a site in the second stem-loop structure, wherein the second stem-loop structure is present in the tracrRNA sequence of the gRNA.

48. The method according to claim 47, wherein, The site in the second stem-ring structure is in the four-ring portion or the spiral portion.

49. The method according to any one of claims 40-42, wherein, The first RNA fragment, the second RNA fragment, and / or the third RNA fragment contain at least one secondary structure, and the free energy of the complex formed by the hybridization of the first, second, and third RNA fragments with the first and second splice oligonucleotides is lower than the free energy of the secondary structure having the lowest free energy.

50. A method for synthesizing a single-molecule guide RNA (sgRNA) conjugated with an RNA-guided endonuclease, the method comprising: Provides a complex formed between a first RNA fragment, a second RNA fragment, a third RNA fragment, a first splice oligonucleotide, and a second splice oligonucleotide; and RNA ligase, among which (a) The first RNA fragment includes (i) a terminal region containing a 3' hydroxyl group; (b) The second RNA fragment comprises (i) a first terminal region containing a 5' phosphate moiety, and (ii) a second terminal region containing a 3' hydroxyl group; (c) The third RNA fragment includes (i) a terminal region containing a 5' phosphate moiety; (d) The first splice oligonucleotide comprises (i) a first portion complementary to the terminal region containing the 3' hydroxyl group of the first RNA fragment, and (ii) a second portion complementary to the first terminal region containing the 5' phosphate moiety of the second RNA fragment; and (e) The second splice oligonucleotide comprises (i) a first portion complementary to the second terminal region containing the 3' hydroxyl group of the second RNA fragment, and (ii) a second portion complementary to the terminal region containing the 5' phosphate moiety of the third RNA fragment. The complex is formed by hybridization of (a)(i) and (d)(i), (b)(i) and (d)(ii), (b)(ii) and (e)(i), and (c)(i) and (e)(ii). The complex has a first linker site between the 3' hydroxyl group of the first RNA fragment and the 5' phosphate group of the second RNA fragment, and a second linker site between the 3' hydroxyl group of the second RNA fragment and the 5' phosphate group of the third RNA fragment. The RNA ligase induces ligation at the first ligation site and ligation at the second ligation site to form sgRNA, which comprises from 5' to 3': a spacer sequence and an invariant sequence for binding to an RNA-guided endonuclease; the invariant sequence comprises a stem loop formed between a crRNA repeat sequence and a tracrRNA anti-repetition sequence, and a 3' tracrRNA sequence comprising at least one stem loop, thereby synthesizing sgRNA conjugated to an RNA-guided endonuclease.

51. The method according to claim 50, wherein, The first connection site corresponds to a site in the stem-loop formed between the crRNA repeat sequence and the tracrRNA anti-repetition sequence.

52. The method according to claim 51, wherein, The first connection site corresponds to a site in the 5' stem of the stem ring, in the fourth ring of the stem ring, or in the 3' stem of the stem ring.

53. The method according to any one of claims 50-52, wherein, The 3' tracrRNA sequence contains a first stem-loop, a second stem-loop, and a third stem-loop.

54. The method according to claim 53, wherein, The second connection site corresponds to a site in the first stem ring, in the second stem ring, or in the third stem ring.

55. The method according to claim 53, wherein, The second connection site corresponds to a site in the second stem ring, wherein the site is a site in the 5' stem of the second stem ring, a site in the fourth ring of the second stem ring, or a site in the 3' stem of the second stem ring.

56. The method according to claim 53, wherein, The second connection site corresponds to a site adjacent to the 5' base of the second stem-loop or to the 3' base of the second stem-loop.

57. The method according to any one of claims 50-52, wherein, The first RNA fragment contains a nucleotide sequence that is the 5' first linkage site.

58. The method according to any one of claims 50-52, wherein, The second RNA fragment contains a nucleotide sequence located between the first linker site and the second linker site.

59. The method according to any one of claims 50-52, wherein, The third RNA fragment contains a nucleotide sequence from 3' to the second linkage site.

60. The method according to any one of claims 50-52, wherein, The terminal region of (a)(i) comprises a nucleotide sequence of 10 to 30 nucleotides located at the 3' end of the first RNA fragment.

61. The method according to claim 60, wherein, The terminal region of (a)(i) contains the spacer region sequence of the sgRNA.

62. The method according to claim 60, wherein, The terminal region of (a)(i) does not contain the spacer region sequence of the sgRNA.

63. The method according to any one of claims 50-52, wherein, The first portion of (d)(i) is completely complementary to the terminal region of (a)(i), or has 1, 2 or 3 mismatches relative to the terminal region of (a)(i).

64. The method according to any one of claims 50-52, wherein, (b)(i) The first terminal region contains a nucleotide sequence of 10 to 30 nucleotides located at the 5' end of the second RNA fragment.

65. The method according to any one of claims 50-52, wherein, The second portion of (d)(ii) is completely complementary to the first terminal region of (b)(i), or has 1, 2 or 3 mismatches relative to the terminal region of (d)(ii).

66. The method according to any one of claims 50-52, wherein, (b)(ii) The second terminal region contains a nucleotide sequence of 10 to 30 nucleotides located at the 3' end of the second RNA fragment.

67. The method according to any one of claims 50-52, wherein, The first portion of (e)(i) is completely complementary to the second terminal region of (b)(ii), or has one, two, or three mismatches relative to the terminal region of (b)(ii).

68. The method according to any one of claims 50-52, wherein, The terminal region of (c)(i) comprises a nucleotide sequence of 10 to 40 nucleotides located at the 5' end of the third RNA fragment.

69. The method according to any one of claims 50-52, wherein, The second portion of (e)(ii) is completely complementary to the terminal region of (c)(i), or has 1, 2 or 3 mismatches relative to the terminal region of (c)(i).

70. The method according to claim 40 or 50, wherein, The lengths of the first RNA fragment, the second RNA fragment, and the third RNA fragment are each independently 10 to 90 nucleotides, 10 to 60 nucleotides, 10 to 50 nucleotides, 10 to 40 nucleotides, 20 to 40 nucleotides, and 30 to 40 nucleotides, respectively.

71. The method according to claim 40 or 50, wherein, The RNA ligase is either T4 RNA ligase I or T4 RNA ligase II.

72. The method according to claim 40 or 50, wherein, The first splint oligonucleotide is a DNA or RNA oligonucleotide, and the second splint oligonucleotide is a DNA or RNA oligonucleotide.

73. The method according to claim 40 or 50, wherein, The lengths of the first and second splint oligonucleotides are independently 20 to 100 nucleotides, 20 to 90 nucleotides, 20 to 80 nucleotides, 20 to 70 nucleotides, 20 to 60 nucleotides, 30 to 60 nucleotides, or 30 to 50 nucleotides.

74. The method according to claim 40 or 50, wherein, The gRNA or sgRNA contains a spacer region sequence that is complementary to the sequence in the target DNA.

75. The method according to claim 74, wherein, The target DNA is mammalian DNA or human DNA.

76. The method according to claim 1, 40, or 50, wherein, The RNA-guided endonuclease is a small Cas nuclease or a small RNA-guided endonuclease.

77. The method according to claim 1, 40, or 50, wherein, The RNA-guided endonuclease is selected from the group consisting of Cas9, Cas12, aCas13 and their variants.

78. The method according to claim 77, wherein, The RNA-guided endonuclease is either Streptococcus pyogenes Cas9 (SpyCas9) or Staphylococcus aureus (SaCas9).

79. The method according to claim 1, 40, or 50, wherein, The RNA-guided endonuclease is a variant of Cas9, and the variant of Cas9 is selected from the group consisting of: small Cas9, dead Cas9 (dCas9), and Cas9 cleavage enzyme.

80. The method according to any one of claims 50-52, wherein, The RNA-guided endonuclease is Streptococcus pyogenes Cas9 (SpyCas9).

81. The method according to any one of claims 80, wherein, The invariant sequence comprises the nucleotide sequence of SEQ ID NO:

17.

82. The method according to claim 80, wherein, The first RNA fragment, the second RNA fragment, and the third RNA fragment are each selected from the following nucleotide sequences: (a)(i)N 15-30 GUUUUAGAGCUAG(SEQ ID NO:56), where N 15-30 Corresponding to the interval region sequence; (ii) SEQ ID NO:3; and (iii)SEQ ID NO:4; (b)(i)N 15-30 GUUUUAGAGCUAGA(SEQ ID NO:57), where N 15-30 Corresponding to the interval region sequence; (ii)SEQ ID NO:40; and (iii)SEQ ID NO:42; (c)(i)N 15-30 GUUUUAGAGCUAG(SEQ ID NO:56), where N 15-30 Corresponding to the aforementioned interval sequence; (ii)SEQ ID NO:58; and (iii) SEQ ID NO:42; or (d)(i)N 15-30 GUUUUAGAGCUAGA(SEQ ID NO:57), where N 15-30 Corresponding to the interval region sequence; (ii)SEQ ID NO:59; and (iii)SEQ ID NO:

4.

83. The method according to claim 80, wherein, The first splint oligonucleotide contains the nucleotide sequence listed in SEQ ID NO:60; SEQ ID NO:44; or SEQ ID NO:

61.

84. The method according to claim 83, wherein, No part of the first splint oligonucleotide is complementary to the spacer region sequence.

85. The method according to claim 83, wherein, The first splint oligonucleotide further comprises a 3' segment having a nucleotide sequence complementary to the spacer sequence or to one or more of the 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides present at the 3' end of the spacer sequence.

86. The method according to claim 81, wherein, The second splint oligonucleotide contains the nucleotide sequence listed in SEQ ID NO:6; SEQ ID NO:45; or SEQ ID NO:

53.

87. The method according to claim 40 or 50, wherein, Providing the first RNA fragment, the second RNA fragment, and the third RNA fragment includes synthesizing the RNA fragments using enzymatic synthesis or phosphoramide chemistry.

88. The method according to claim 87, wherein, The synthesis of the RNA fragment using the phosphoramidite chemical method includes: (i) Synthesizing the first RNA fragment, the second RNA fragment, and the third RNA fragment in a 5' to 3' or 3' to 5' orientation; or (ii) Synthesize the first RNA fragment in a 5' to 3' or 3' to 5' orientation, and synthesize the second RNA fragment and the third RNA fragment in a 3' to 5' orientation.

89. The method according to claim 40 or 50, wherein, Providing the first and second splint oligonucleotides includes synthesizing the oligonucleotides using enzymatic synthesis or phosphoramide chemistry, optionally including purifying the oligonucleotides after synthesis.

90. The method according to claim 40 or 50, wherein, The first RNA fragment, the second RNA fragment, and / or the third RNA fragment contain at least one modification in the RNA backbone.

91. The method of claim 90, wherein the modification is selected from the group consisting of: 2'-methoxy (2'OMe), 2'-fluorine (2'-fluorine), 2'-O-methoxy-ethyl (MOE), locked nucleic acid (LNA), unlocked nucleic acid (UNA), bridging nucleic acid, 2'-deoxynucleic acid (DNA), and peptide nucleic acid (PNA).

92. The method according to claim 40 or 50, wherein, The first RNA fragment, the second RNA fragment, and / or the third RNA fragment contain at least one base modification.

93. The method according to claim 92, wherein, The base modification is selected from the group consisting of: 2-aminopurine, inosine, thymine, 2,6-diaminopurine, 2-pyrimidinone, and 5-methylcytosine.

94. The method according to claim 40 or 50, wherein, The first RNA fragment, the second RNA fragment, and / or the third RNA fragment contain at least one phosphate thioester bond.

95. The method according to claim 40 or 50, wherein, Hybridization is carried out in solution, with or without an annealing step.

96. The method according to claim 95, wherein, The annealing step includes (i) heating the solution to 80°C to 95°C for a period of less than 10 minutes; and (ii) cooling the solution to the temperature for connection at a rate of 0.1°C to 2°C per second.

97. The method according to claim 95, wherein, The concentrations of the first splint oligonucleotide, the second splint oligonucleotide, the first RNA fragment, the second RNA fragment, and the third RNA fragment are equal in solution.

98. The method according to claim 40 or 50, wherein, The connection is made at 15°C to 45°C, or at 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C.

99. The method according to claim 40 or 50, wherein, The connection lasts for 0.1 to 48 hours, or 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 hours.

100. The method according to claim 40 or 50, wherein, The connection also includes the use of proteases or chelating agents.

101. The method according to claim 100, wherein, The chelating agent is EDTA, EGTA, or a combination of both.

102. The method according to claim 40 or 50, wherein, The connection also includes the use of one or more congestion agents.

103. The method according to claim 102, wherein, The one or more crowding agents include polyethylene glycol (PEG). Ethylene glycol, dextran, or any combination thereof.

104. The method according to claim 40 or 50, wherein, The connection is completed to a degree of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

105. The method of claim 87, wherein providing the first RNA fragment, the second RNA fragment, and the third RNA fragment further comprises purifying the RNA fragments after synthesis.

106. The method according to claim 1, 40 or 50, further comprising purifying the gRNA or sgRNA after synthesis.

107. The method according to claim 106, wherein, Purifying the gRNA or sgRNA includes purification using chromatographic methods.

108. The method according to claim 107, wherein, The chromatographic method is reversed-phase HPLC, ion exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, affinity chromatography, or polyacrylamide gel purification or any combination thereof.

109. A method for generating a bimolecular gRNA comprising crRNA and tracrRNA, the method comprising: Provide a first RNA fragment comprising a terminal region containing a 5' phosphate moiety, and a second RNA fragment comprising a terminal region containing a 3' hydroxyl group, wherein the first RNA fragment, the second RNA fragment, or both comprise at least a portion of a sequence capable of binding to an RNA-guided endonuclease; A splice oligonucleotide is provided, comprising a first portion of the terminal region complementary to the first RNA fragment, the terminal region comprising a 5' phosphate moiety and a second portion of the terminal region complementary to the second RNA fragment, the terminal region comprising a 3' hydroxyl group; The first RNA fragment, the second RNA fragment, and the splint oligonucleotide are hybridized together to form a complex; The first and second RNA fragments are ligated at the ligation sites present between the RNA complexes using RNA ligase, thereby synthesizing tracrRNA; Provide crRNA containing a sequence complementary to the sequence in the target DNA; and The tracrRNA and crRNA are hybridized to produce bimolecular gRNA.

110. The method according to claim 109, wherein, The crRNA is provided by means of enzymatic synthesis or phosphoramide chemical synthesis.

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