Method for synthesizing RNA molecules

Abstract

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

Description

Technical Field

[0001] Cross - reference to related applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 941,174, filed on November 27, 2019. The entire content thereof is incorporated herein by reference.

[0002] The present disclosure generally relates to the fields of molecular biology and biotechnology, including methods for synthesizing nucleic acids and RNA molecules related to endonucleases also known as guide RNAs.

Background Art

[0003] Targeting of DNA using the RNA - guided, DNA - targeting principle of the CRISPR (clustered regularly interspaced short palindromic repeats) - Cas (CRISPR - associated) system is widely used in the art. The CRISPR - Cas system can be classified into two classes: Class 1 systems that utilize complexes of multiple Cas proteins (such as type I, type III, and type IV CRISPR - Cas systems), and Class 2 systems that utilize a single Cas protein (such as type II, type V, and type VI CRISPR - Cas systems). Type II CRISPR - Cas - based systems are used for genome editing and require a Cas polypeptide or a variant thereof, which is guided by a customizable guide RNA (gRNA) for programmable DNA targeting. The guide RNA of type II CRISPR - Cas - based systems typically ranges in length from 30 to 130 nucleotides (Chylinski et al. (2013) RNA Biology, 10(5):726 - 737).

[0004] Methods for the synthesis of gRNA include, for example, intracellular transcription of exogenous plasmids or solid-phase synthesis using phosphoramidite chemistry. Direct chemical synthesis of gRNA allows incorporation of chemical modifications that enhance the chemical stability of the RNA, reduce immunogenicity, and reduce the likelihood of off-target effects (i.e., cleaving genomic DNA at unwanted sites). One limitation of chemical synthesis of some sequences, such as gRNA, is the length of the desired single-stranded RNA, which is typically about 60 to 100 nucleotides (nt) in the case of gRNA. For example, if the coupling efficiency of the phosphoramidite chemistry being used is about 0.99 X (where X is the number of nucleotides), when synthesizing a gRNA of about 100 nucleotides in length, it is expected that about 30-40% full-length product (FLP) will be obtained in the overall synthesis process. Complete separation of the FLP from incomplete couplings (truncation products) and the remaining by-products formed from deprotection is currently not achievable by standard purification methods (e.g., chromatography) for RNA molecules of about 100 nucleotides in length. Due to these limitations, it is desirable to design a more efficient method for synthesizing gRNA.

Summary of the Invention

[0005] The applicant has discovered an improvement in a method for synthesizing RNA, particularly medium-length RNA (mlRNA) such as guide RNA used in gene editing. Accordingly, the present disclosure provides a method for synthesizing mlRNA using splint-mediated ligation of two or more RNA fragments. In some embodiments, the present disclosure provides a method for synthesizing mlRNA using splint-mediated ligation of two RNA fragments or three RNA fragments. Such methods include, for example, providing a first RNA fragment comprising a terminal region comprising a 5' phosphate moiety and a second RNA fragment comprising a terminal region comprising 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, for example, an RNA-guided endonuclease; providing a splint oligonucleotide comprising a first portion complementary to the first RNA fragment at a terminal region comprising a 5' phosphate moiety and a second portion complementary to the second RNA fragment at a terminal region comprising a 3' hydroxyl group; hybridizing the first RNA fragment, the second RNA fragment, and the splint oligonucleotide together to form a complex; and ligating the first and second RNA fragments using a ligase at a ligation site present within the complex, thereby synthesizing an mlRNA or a portion of an mlRNA.

[0006] In some embodiments, the method comprises: (a) a first RNA fragment comprising a terminal region comprising a 3'-hydroxyl group; (b) a second RNA fragment comprising (i) a terminal region comprising a 5'-phosphate moiety and (ii) a terminal region comprising a 3'-hydroxyl group; (c) a third RNA fragment comprising a terminal region comprising a 5'-phosphate group; (d) a first splint oligonucleotide comprising (i) a first portion complementary to the terminal region of the first RNA fragment comprising the 3'-hydroxyl group and (ii) a second portion complementary to the first terminal region of the second RNA fragment comprising the 5'-phosphate moiety; (e) a second splint oligonucleotide comprising (i) a first portion complementary to the second terminal region of the second RNA fragment comprising the 3'-hydroxyl group and (ii) a second portion complementary to the terminal region of the third RNA fragment comprising the 5'-phosphate moiety; (f) a ligase, wherein the first RNA fragment, the second RNA fragment, the third RNA fragment, the first splint oligonucleotide, and the second splint oligonucleotide hybridize together to form a complex, the complex comprising a first ligation site between the 3'-hydroxyl group of the first RNA fragment and the 5'-phosphate group of the second RNA fragment and a second ligation site between the 3'-hydroxyl group of the second RNA fragment and the 5'-phosphate group of the third RNA fragment, and the ligase effects ligation of the first and second RNA fragments at the first ligation site and the second and third RNA fragments at the second ligation site, thereby synthesizing an mlRNA or a portion of an mlRNA. In some embodiments, the first RNA fragment, the second RNA fragment, the third RNA fragment, or a combination thereof comprises at least a portion of a sequence that binds to an RNA-guided endonuclease (e.g., Cas9), for example. In some embodiments, the first RNA fragment, the second RNA fragment, the third RNA fragment, or a combination thereof comprises a spacer sequence that targets a target sequence in a target DNA (e.g., a genomic DNA molecule).

[0007] In one aspect, a method for synthesizing a guide RNA (gRNA) is provided herein. The method includes providing a first RNA fragment comprising a terminal region comprising a 5'-phosphate moiety and a second RNA fragment comprising a terminal region comprising 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; providing a splint oligonucleotide comprising a first portion complementary to the first RNA fragment at the terminal region comprising the 5'-phosphate moiety and a second portion complementary to the second RNA fragment at the terminal region comprising the 3'-hydroxyl group; hybridizing the first RNA fragment, the second RNA fragment, and the splint oligonucleotide together to form a complex; and ligating the first and second RNA fragments using a ligase at a ligation site present between the RNA complexes, thereby synthesizing the gRNA. In some embodiments, the lengths of the first and second RNA fragments are each 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 5'-phosphate or 5'-phosphorothioate. In some embodiments, the ligase is T4 DNA ligase, T4 RNA ligase I, or T4 RNA ligase II. In some embodiments, the splint oligonucleotide is a DNA or RNA oligonucleotide. In some embodiments, the length of the splint oligonucleotide is 20 to 100 nucleotides. In some embodiments, the splint oligonucleotide is bound to a solid support. In some embodiments, the length of the gRNA is 30 to 160 nucleotides. In some embodiments, the gRNA comprises a sequence complementary to a sequence in a 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 of the synthesized gRNA.In some embodiments, the ligation site corresponds to a site in the helical portion of the stem-loop structure of 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 sprint oligonucleotide results in a lower free energy than that of the secondary structure with the lowest free energy of the sprint oligonucleotide. 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 via enzymatic synthesis or phosphoramidite chemistry. In some embodiments, the second RNA fragment is synthesized in the 5' to 3' or 3' to 5' direction. In some embodiments, providing the first and second RNA fragments includes purifying the first and second fragments after synthesis. In some embodiments, providing the sprint oligonucleotide includes synthesizing the sprint oligonucleotide via enzymatic synthesis or phosphoramidite chemistry. In some embodiments, providing the sprint oligonucleotide includes purifying the sprint oligonucleotide after synthesis. In some embodiments, purifying includes purifying by chromatography methods. In some embodiments, the chromatography methods are reverse 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), bridged nucleic acid, 2'-deoxy nucleic 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 phosphorothioate bond. In some embodiments, hybridizing comprises hybridizing in solution. In some embodiments, the concentration of the splint oligonucleotide, the concentration of the first RNA fragment, and the concentration of the second RNA fragment in solution are substantially equal. In some embodiments, ligating the first and second RNA fragments is performed at 15°C to 45°C. In some embodiments, ligating the first and second RNA fragments is performed at about 37°C. In some embodiments, ligating the first and second RNA fragments is performed for about 0.1 to about 48 hours. In some embodiments, ligating the first and second RNA fragments further comprises using a protease or a chelating agent. In some embodiments, the chelating agent is EDTA, EGTA, or a combination of both. In some embodiments, ligating the first and second RNA fragments further comprises using one or more crowding agents. In some embodiments, the one or more crowding agents include polyethylene glycol (PEG), Ficoll®, ethylene glycol, dextran, or any combination thereof. In some embodiments, ligating the first and second RNA fragments proceeds until at least 10% is complete.In some embodiments, ligating the first and second RNA fragments proceeds to at least 90% completion. In some embodiments, the method further comprises purifying the gRNA after synthesis. In some embodiments, purifying the gRNA comprises purifying using a chromatography method. In some embodiments, the chromatography method is reverse 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, Cas13, 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, inactive Cas9 (dCas9), and Cas9 nickase.

[0008] In another aspect, the present disclosure provides a method of synthesizing a gRNA, the method comprising: (a) a first RNA fragment comprising a terminal region comprising a 3'-hydroxyl group; (b) a second RNA fragment comprising a first terminal region comprising a 5'-phosphate moiety and a second terminal region comprising a 3'-hydroxyl group; (c) a third RNA fragment comprising a terminal region comprising a 5'-phosphate moiety; (d) a first splint oligonucleotide comprising: (i) a first portion complementary to the terminal region of the first RNA fragment comprising the 3'-hydroxyl group and (ii) a second portion complementary to the first terminal region of the second RNA fragment comprising the 5'-phosphate moiety; (e) a second splint oligonucleotide comprising: (i) a first portion complementary to the second terminal region of the second RNA fragment comprising the 3'-hydroxyl group and (ii) a second portion complementary to the terminal region of the third RNA fragment comprising the 5'-phosphate moiety; and (f) a ligase, wherein hybridizing the first, second, and third RNA fragments and the first and second splint oligonucleotides results in the formation of a complex having a first ligation site between the 3'-hydroxyl group of the first RNA fragment and the 5'-phosphate group of the second RNA fragment and a second ligation site between the 3'-hydroxyl group of the second RNA fragment and the 5'-phosphate group of the third RNA fragment, and the ligase effects ligation of the first and second RNA fragments at the first ligation site and the second and third RNA fragments at the second ligation site, thereby synthesizing the gRNA. In some embodiments, the gRNA comprises, from 5' to 3', a first RNA fragment linked to a second RNA fragment by a first phosphodiester bond and a second RNA fragment linked to a third RNA fragment by 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 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 has a length of about 30 to about 160 nucleotides, or a length of about 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or 160 nucleotides. In some embodiments, the first ligation site corresponds to a site of the first stem-loop structure, and the first stem-loop structure is formed by hybridization of the minimal CRISPR repeat sequence and the minimal tracrRNA sequence in the synthesized gRNA. In some embodiments, the site of the first stem-loop structure is in the tetraloop portion or the helix portion. In some embodiments, the second ligation site corresponds to a site of the second stem-loop structure. In some embodiments, the second stem-loop is present in the tracrRNA sequence of the gRNA. In some embodiments, the site of the second stem-loop structure is in the tetraloop portion or the helix portion.

[0009] In some aspects, the present disclosure provides a method for synthesizing a single-guide RNA (sgRNA) for use with an RNA-guided endonuclease, the method comprising providing a first RNA fragment, a second RNA fragment, a third RNA fragment, a first splint oligonucleotide, and a second splint oligonucleotide, and a ligase, wherein (a) the first RNA fragment comprises (i) a terminal region comprising a 3'-hydroxyl group, (b) the second RNA fragment comprises (i) a first terminal region comprising a 5'-phosphate moiety and (ii) a second terminal region comprising a 3'-hydroxyl group, (c) the third RNA fragment comprises (i) a terminal region comprising a 5'-phosphate moiety, (d) the first splint oligonucleotide comprises (i) a first portion complementary to a terminal region of the first RNA fragment comprising the 3'-hydroxyl group and (ii) a second portion complementary to a first terminal region of the second RNA fragment comprising the 5'-phosphate moiety, (e) the second splint oligonucleotide comprises (i) a first portion complementary to a second terminal region of the second RNA fragment comprising the 3'-hydroxyl group and (ii) a second portion complementary to a terminal region of the third RNA fragment comprising the 5'-phosphate, the complex being 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 having a first ligation 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 ligation site present between the 3'-hydroxyl group of the second RNA fragment and the 5'-phosphate group of the third RNA fragment, the ligase effecting ligation at the first ligation site and ligation at the second ligation site to form, 5' to 3', an sgRNA comprising a spacer sequence and a constant sequence that binds to an RNA-guided endonuclease, the constant sequence comprising a stem-loop formed between a crRNA repeat sequence and a tracrRNA reverse repeat sequence and a 3'tracrRNA sequence comprising at least one stem-loop,Thereby, synthesize a sgRNA for use with an RNA-guided endonuclease.

[0010] In any of the foregoing or related aspects, the first ligation site corresponds to a site within a stem-loop formed between a crRNA repeat sequence and a tracrRNA reverse repeat sequence. In some embodiments, the first ligation site corresponds to a site in the 5' stem of the stem-loop, the tetraloop of the stem-loop, or the 3' stem of the stem-loop. In some embodiments, the 3'tracrRNA sequence includes 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 ligation site corresponds to a site in the first stem-loop, the second stem-loop, or the third stem-loop. In some embodiments, the second ligation site corresponds to a site within the second stem-loop. In some embodiments, the site is within the 5' stem of the second stem-loop, a site within the tetraloop of the second stem-loop, or a site within the 3' stem of the second stem-loop. In some embodiments, the second ligation 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 a site adjacent 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 includes a nucleotide sequence that is 5' of the first ligation site. In some embodiments, the second RNA fragment includes a nucleotide sequence that is between the first ligation site and the second ligation site. In some embodiments, the third RNA fragment includes a nucleotide sequence that is 3' of the second ligation site.

[0011] 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 the spacer sequence of the sgRNA. In some embodiments, the terminal region of (a)(i) does not comprise the spacer sequence of the sgRNA. In some embodiments, the 5' end of the spacer sequence is aligned with the 5' end of the first RNA fragment, and the terminal region of (a)(i) comprises the spacer sequence of the sgRNA. In some embodiments, the terminal region of (a)(i) extends from the 3' end of the first RNA fragment and includes 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 sequence. In some embodiments, the terminal region of (a)(i) extends from the 3' end of the first RNA fragment and is directly adjacent to the 3' end of the spacer 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 1, 2, or 3 mismatches with respect 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 1, 2, or 3 mismatches with respect 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 1, 2, or 3 mismatches with respect 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 fully complementary to the terminal region of (c)(i). In some embodiments, the second portion of (e)(ii) has 1, 2, or 3 mismatches with respect to the terminal region of (c)(i).

[0012] In any of the foregoing or related aspects, 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, or about 30 to about 40 nucleotides in length. In some embodiments, the first sprint oligonucleotide is a DNA or RNA oligonucleotide, and the second sprint oligonucleotide is a DNA or RNA oligonucleotide. In some embodiments, the first sprint oligonucleotide and the second sprint 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 in length.

[0013] In any of the foregoing or related aspects, the gRNA or sgRNA comprises a spacer sequence that is complementary to the sequence of 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, Cas13, 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, inactive Cas9 (dCas9), and Cas9 nickase.

[0014] 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 each comprise, (a) (i) N 15-30 GUUUUAGAGCUAG (SEQ ID NO: 56) (N 15-30 corresponds 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) (N 15-30 corresponds to the spacer sequence), (ii) SEQ ID NO: 40, and (iii) SEQ ID NO: 42, (c) (i) N 15-30 GUUUUAGAGCUAG (SEQ ID NO: 56) (N 15-30(corresponding to the spacer sequence), (ii) SEQ ID NO: 58, and (iii) SEQ ID NO: 42, or (d) (i) N 15-30 GUUUUAGAGCUAGA (SEQ ID NO: 57) (N 15-30 is selected from nucleotide sequences comprising (i) (corresponding to the spacer sequence), (ii) SEQ ID NO: 59, and (iii) SEQ ID NO: 4. In some embodiments, the spacer sequence targets a target site of a target nucleic acid molecule (e.g., genomic DNA). In some embodiments, the spacer sequence is about 10 to about 30 nucleotides in length. In some embodiments, the spacer sequence is 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the spacer sequence is 19 nucleotides in length. In some embodiments, the spacer sequence is 20 nucleotides in length. In some embodiments, the spacer sequence is 21 nucleotides in length. In some embodiments, the spacer sequence is 22 nucleotides in length. In some embodiments, the first split oligonucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 60, SEQ ID NO: 44, or SEQ ID NO: 61. In some embodiments, no portion of the first split oligonucleotide is complementary to the spacer sequence. In some embodiments, the first split oligonucleotide further comprises a 3' end having a nucleotide sequence that is complementary to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides present at the 3' end of the spacer sequence or the spacer sequence.

[0015] 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, and the complex formed by hybridizing the first, second, and third RNA fragments, and the first and second splint oligonucleotides results in a free energy lower than the free energy of the secondary structure with the lowest free energy. In some embodiments, providing the first RNA fragment, the second RNA fragment, and the third RNA fragment includes the enzymatic synthesis or the synthesis of the RNA fragments using phosphoramidite chemistry. In some embodiments, providing the RNA fragments further includes purifying the RNA fragments after synthesis. In some embodiments, the synthesis of the RNA fragments using phosphoramidite chemistry includes the synthesis of the first RNA fragment, the second RNA fragment, and the third RNA fragment in the 5' to 3' or 3' to 5' direction, respectively. In some embodiments, the synthesis of the RNA fragments using phosphoramidite chemistry includes the synthesis of the first RNA fragment in the 5' to 3' or 3' to 5' direction, and the synthesis of the second RNA fragment and the third RNA fragment in the 3' to 5' direction, respectively. In some embodiments, providing the first and second splint oligonucleotides includes the enzymatic synthesis or the synthesis of the oligonucleotides using phosphoramidite chemistry. In some embodiments, providing the splint oligonucleotides further includes purifying the oligonucleotides after synthesis. In some embodiments, the first RNA fragment, the second RNA fragment, and / or the third RNA fragment comprises at least one modification to 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), bridged nucleic acid, 2'-deoxy nucleic 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 RNA fragment, the second RNA fragment, and / or the third RNA fragment comprises 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, and / or the third RNA fragment comprises at least one phosphorothioate bond. In some embodiments, hybridizing is performed in solution. In some embodiments, hybridizing is performed without an annealing step. In some embodiments, hybridizing is performed in an annealing step. In some embodiments, the annealing step comprises (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 at a rate of about 0.1°C to about 2°C / second (e.g., 1°C / second) to the temperature used 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). In some embodiments, the concentration of the first splint oligonucleotide, the concentration of the second splint oligonucleotide, the concentration of the first RNA fragment, the concentration of the second RNA fragment, and the concentration of the third RNA fragment in the solution are substantially 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, ligating the first and second RNA fragments further comprises using a protease or a chelating agent. In some embodiments, the chelating agent is EDTA, EGTA, or a combination of both. In some embodiments, ligating the first and second RNA fragments further comprises using one or more crowding agents. In some embodiments, the one or more crowding agents include polyethylene glycol (PEG), Ficoll®, ethylene glycol, dextran, or any combination thereof. In some embodiments, the ligation proceeds to at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% completion. In some embodiments, the method further comprises purifying the gRNA or sgRNA after synthesis. In some embodiments, purifying the gRNA or sgRNA comprises purifying using a chromatography method. In some embodiments, the chromatography method is reverse-phase HPLC, ion-exchange chromatography, size-exclusion chromatography, hydrophobic interaction chromatography, affinity chromatography, or polyacrylamide gel purification, or any combination thereof.

[0016] In another aspect, provided herein is a method for generating a dual molecule gRNA comprising a crRNA and a tracrRNA, the method comprising: providing a first RNA fragment comprising a terminal region comprising a 5' phosphate moiety, and a second RNA fragment comprising a terminal region comprising 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; providing a splint oligonucleotide comprising a first portion complementary to the first RNA fragment at a terminal region comprising a 5' phosphate moiety, and a second portion complementary to the second RNA fragment at a terminal region comprising a 3' hydroxyl group; hybridizing the first RNA fragment, the second RNA fragment, and the splint oligonucleotide together to form a complex; ligating the first and second RNA fragments using a ligase at a ligation site present between the RNA complexes, thereby synthesizing a tracrRNA; providing a crRNA comprising a sequence complementary to a sequence of a target DNA; and enabling the tracrRNA and the crRNA to hybridize, thereby generating a dual molecule gRNA. In some embodiments, providing the crRNA comprises synthesizing the crRNA via enzymatic synthesis or phosphoramidite chemistry.

[0017] Unless defined otherwise, 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 belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0018] Other features and advantages of the present invention will become apparent from the following detailed description and the claims.

Brief Description of the Drawings

[0019]

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Mode for Carrying Out the Invention

[0020] The present disclosure provides a method for synthesizing RNA, particularly medium-length RNA (mlRNA) such as guide RNA (gRNA), by ligating RNA fragments using one or more splinted oligonucleotides and ligase. Optionally, one or more of the RNA fragments comprise at least a portion of a sequence capable of binding to an RNA-guided endonuclease. In some embodiments, one or more of the RNA fragments comprise a spacer sequence for targeting a target sequence in a target DNA (e.g., a genomic DNA molecule).

[0021] Current methods for the synthesis of mlRNA include, for example, intracellular transcription of exogenous plasmids or solid-phase synthesis using phosphoramidite chemistry. One limitation of the chemical synthesis of mlRNA is the length of the resulting single-stranded RNA. For example, when synthesizing an RNA of about 100 nucleotides in length where the coupling efficiency of the phosphoramidite chemistry used is about 0.99 X (X is the number of nucleotides), it is expected that about 30-40% full-length product (FLP) will be obtained in the overall synthesis process. Complete separation of the FLP from incomplete coupling (truncation products) and the remaining by-products formed from deprotection is not achievable at present using standard purification methods (e.g., chromatography) for RNA molecules of about 100 nucleotides in length. The present disclosure provides a more efficient method for synthesizing mlRNA that improves the yield of full-length product and reduces the number of truncation products generated. Furthermore, the methods of the present disclosure are demonstrated herein to provide for the synthesis of both unmodified mlRNA (e.g., gRNA or sgRNA) and mlRNA comprising one or more chemical modifications, such as backbone modifications (e.g., phosphorothioate linkages) and / or nucleoside modifications (e.g., 2'-O-methylation).

[0022] The present disclosure also relates, at least in part, to the discovery that single molecule guide RNAs (sgRNAs) for use with RNA-guided endonucleases (e.g., Cas9) can be effectively synthesized using the splint-mediated ligation techniques described herein, such as the ligation of two or three RNA fragments using one or more splint oligonucleotides and a ligase. In some embodiments, the ligation includes two RNA fragments, one splint oligonucleotide, and a ligase, where the two RNA fragments hybridize with the splint oligonucleotide to form a complex comprising a ligation site, and the ligase effects ligation at the ligation site, thereby forming an sgRNA for use with an RNA-guided endonuclease. In some embodiments, the sgRNA includes a nucleotide sequence that is 5' of the ligation site and a nucleotide sequence that is 3' of the ligation site, the first RNA fragment corresponds to the nucleotide sequence that is 5' of the ligation site, the second RNA fragment corresponds to the nucleotide sequence that is 3' of the ligation site, and ligation at the ligation site enables the joining of the first and second RNA fragments to form the nucleotide sequence of the sgRNA.

[0023] In some embodiments, the ligation involves three RNA fragments, two splint oligonucleotides, and a ligase. The three RNA fragments hybridize the two splint oligonucleotides to form a complex comprising first and second ligation sites, and the ligase effects ligation at the first and second ligation sites, thereby forming an sgRNA for use with an RNA-guided endonuclease. In some embodiments, the sgRNA comprises a nucleotide sequence that is 5' of the first ligation site, a nucleotide sequence that is 3' of the first ligation site and 5' of the second ligation site, and a nucleotide sequence that is 3' of the second ligation site. The first RNA fragment corresponds to the nucleotide sequence that is 5' of the first ligation site, the second RNA fragment corresponds to the nucleotide sequence that is 3' of the first ligation site and 5' of the second ligation site, and the third RNA fragment corresponds to the nucleotide sequence that is 3' of the second ligation site. Ligation at the first and second ligation sites enables the joining of the first, second, and third RNA fragments to form the nucleotide sequence of the sgRNA.

[0024] In some embodiments, the nucleotide sequence of the sgRNA comprises, from 5' to 3', a spacer sequence for targeting a target site of a nucleic acid molecule (e.g., a genomic DNA molecule) and a constant sequence that binds to an RNA-guided endonuclease. The constant sequence comprises, from 5' to 3', a stem-loop formed between a CRISPR repeat sequence and a tracrRNA reverse repeat sequence, and a tracrRNA comprising at least one stem-loop. In some embodiments, the splint-mediated ligation approach provides at least one RNA fragment, or combination of RNA fragments, comprising the spacer sequence, and at least one RNA fragment, or combination of RNA fragments, comprising the constant sequence.

[0025] In some embodiments, the splint-mediated ligation approach includes the placement of ligation sites in the sgRNA, within or proximal to a stem loop in the invariant sequence of the sgRNA (e.g., within or proximal to a stem loop formed between the CRISPR repeat and the tracrRNA reverse repeat, e.g., within or proximal to a stem loop of the tracrRNA). As described herein, the placement of the ligation site in the stem loop prevents the formation of secondary structure in an RNA fragment (i.e., an RNA fragment ligated at the ligation site) that would prevent or disfavor hybridization of the RNA fragment with the splint oligonucleotide. For example, disruption of the stem loop by the ligation site provides an RNA fragment (i.e., an RNA fragment ligated at the ligation site) having (i) minimal secondary structure and / or (ii) a secondary structure having a free energy higher than the free energy resulting from hybridization of the RNA fragment and the splint oligonucleotide.

[0026] In some embodiments, the splint-mediated ligation approach involves the placement of first and second ligation sites in the sgRNA, where the placement of the first ligation site disrupts the stem-loop formation of an RNA fragment comprising a nucleotide sequence that is 5' of the first ligation site and an RNA fragment comprising a nucleotide sequence that is 3' of the first ligation site, the first and second ligation sites are each within or proximal to a stem-loop (e.g., a stem-loop formed between a CRISPR repeat sequence and a tracrRNA reverse repeat sequence, e.g., a stem-loop of tracrRNA) in the invariant sequence of the sgRNA, the placement of the second ligation site disrupts the stem-loop formation in an RNA fragment comprising a nucleotide sequence that is 5' of the second ligation site and an RNA fragment comprising a nucleotide sequence that is 3' of the second ligation site, and the formation of secondary structure in each or all of the RNA fragments used in the splint-mediated ligation reaction is disadvantageous compared to hybridization with the splint oligonucleotide.

[0027] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof, and in which are shown by way of illustration specific embodiments in which the invention may be practiced. In the drawings, like numerals describe substantially identical components throughout the several views. The exemplary embodiments described in the detailed description, drawings, and claims are not intended to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects generally described herein and illustrated in the figures can be arranged, substituted, combined, and designed in a wide variety of different configurations, all of which are explicitly contemplated and form a part of this application.

[0028] Unless otherwise defined, all technical terms, notations, and other scientific terms or expressions used in this specification are intended to have the meanings commonly understood by those skilled in the art to which this application pertains. In some cases, terms with commonly understood meanings are defined in this specification for clarity and / or ready reference, and including such definitions in this specification should not necessarily be construed as representing a substantial difference from what is commonly understood in the art.

[0029] "Medium-length RNA (mlRNA)" means an RNA molecule having a length of about 30 to about 160 nucleotides (e.g., about 30 to about 150, about 30 to about 140, about 30 to about 130, about 30 to about 120, about 30 to about 110, about 30 to about 100, about 30 to about 90, about 30 to about 80, about 30 to about 70, about 30 to about 60, about 30 to about 50, about 30 to about 40, about 40 to about 160, about 40 to about 150, about 40 to about 140, about 40 to about 130, about 40 to about 120, about 40 to about 110, about 40 to about 100, about 40 to about 90, about 40 to about 80, about 40 to about 70, about 40 to about 60, about 40 to about 50, about 50 to about 160, about 50 to about 150, about 50 to about 140, about 50 to about 130, about 50 to about 120, about 50 to about 110, about 50 to about 100, about 50 to about 90, about 50 to about 80, about 50 to about 70, about 50 to about 60, about 60 to about 160, about 60 to about 150, about 60 to about 140, about 60 to about 130, about 60 to about 120, about 60 to about 110, about 60 to about 100, about 60 to about 90, about 60 to about 80, about 60 to about 70, about 70 to about 160, about 70 to about 150, about 70 to about 140, about 70 to about 130, about 70 to about 120, about 70 to about 110, about 70 to about 100, about 70 to about 90, about 70 to about 80, about 80 to about 160, about 80 to about 150, about 80 to about 140, about 80 to about 130, about 80 to about 120, about 80 to about 110, about 80 to about 100, about 80 to about 90, about 90 to about 160, about 90 to about 150, about 90 to about 140, about 90 to about 130, about 90 to about 120, about 90 to about 110, about 90 to about 100, about 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).

[0030] As used herein, the term "RNA-guided endonuclease" refers to a polypeptide that can bind to an RNA (e.g., gRNA) to form a complex that targets a specific DNA sequence (e.g., within a target DNA). Exemplary RNA-guided endonucleases are Cas polypeptides (e.g., Cas endonucleases such as Cas9 endonuclease). Thus, in some embodiments, an RNA-guided endonuclease as described herein is targeted to a specific DNA sequence in a target DNA by the RNA molecule to which it binds. The RNA molecule includes a sequence that is complementary to and can hybridize with the target sequence within the target DNA, thus enabling the bound polypeptide to be targeted to a specific location within the target DNA.

[0031] As used herein, "guide RNA" or "gRNA" is a site-specific targeting RNA that can bind to an RNA-guided endonuclease to form a complex and direct the activity of the bound RNA-guided endonuclease (such as a Cas endonuclease) to a specific target sequence within a target nucleic acid. A guide RNA can comprise one or more RNA molecules.

[0032] As used herein, the "secondary structure" of a nucleic acid molecule (e.g., an RNA fragment, or a gRNA) refers to base-pairing interactions within the nucleic acid molecule.

[0033] As used herein, "target DNA" is DNA that includes a "target site" or "target sequence." The term "target sequence" is used herein to refer to a nucleic acid sequence present in a target DNA to which a DNA targeting sequence or segment of a gRNA (also referred to herein as a "spacer") can hybridize when sufficient conditions for hybridization exist. For example, a target sequence 5’GAGCATATC-3’ within a target DNA is targeted (or can hybridize or is complementary) by an RNA sequence 5’-GAUAUGCUC-3’. Hybridization between a DNA targeting sequence or segment of a gRNA and a target sequence can be based on, for example, Watson-Crick type base pairing rules that allow programmability in the DNA targeting sequence or segment. A DNA targeting sequence or segment of a gRNA can be designed, for example, to hybridize to any target sequence.

[0034] As used herein, the term "Cas endonuclease" or "Cas nuclease" includes, but is not limited to, for example, RNA-guided DNA endonucleases associated with the CRISPR adaptive immune system.

[0035] Unless otherwise specified, "nuclease" and "endonuclease" are used interchangeably herein and mean an enzyme having endonuclease catalytic activity for polynucleotide cleavage.

[0036] As used herein, "cleavage" means the cleavage of the covalent backbone of a DNA molecule. Both single-strand cleavage and double-strand cleavage are possible, and double-strand cleavage can occur as a result of two different single-strand cleavage events.

[0037] The term "domain" is used herein to describe a segment of a protein or nucleic acid. Unless otherwise specified, a domain need not have a particular functional property.

[0038] A "splice oligonucleotide" is an oligonucleotide that, when hybridized to other polynucleotides (e.g., RNA fragments), acts as a "splice" to bring the ends of the polynucleotides adjacent to each other so that they can be ligated together. A splice oligonucleotide can be any oligomer that can hybridize to a polynucleotide via Watson-Crick base pair interactions. A splice oligonucleotide can be DNA, RNA, a non-natural or artificial nucleic acid (e.g., a peptide nucleic acid). A splice oligonucleotide can contain a nucleotide sequence that is partially complementary to the nucleotide sequences from two or more different oligonucleotides. Generally, RNA ligase, DNA ligase, or another type of ligase can be used to ligate two nucleotide sequences together.

[0039] The "spacer" or "variable region" of a gRNA contains a nucleotide sequence that is complementary to a specific sequence within the target DNA (the complementary strand of the target DNA). In some embodiments, the spacer confers target specificity to the gRNA when combined with an RNA-guided endonuclease, enabling the RNA-guided endonuclease to cleave at the target site targeted by the spacer of the target DNA. As used herein, the term "spacer" is used interchangeably with the term "spacer sequence".

[0040] The term "constant region" of a gRNA refers to the nucleotide sequence of the gRNA that binds to an RNA-guided endonuclease. In some embodiments, the gRNA comprises a crRNA and a trans-activating crRNA (tracrRNA), and the crRNA and tracrRNA hybridize to each other to form a double strand. In some embodiments, the crRNA comprises, from 5' to 3': a spacer sequence and a minimal CRISPR repeat (also referred to herein as a "crRNA repeat sequence"), and the tracrRNA comprises a minimal tracrRNA sequence complementary to the minimal CRISPR repeat sequence (also referred to herein as a "tracrRNA reverse repeat sequence") and a 3' tracrRNA sequence. In some embodiments, the constant region of the gRNA refers to the minimal CRISPR repeat sequence and the portion of the crRNA that is the tracrRNA.

[0041] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein and refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, these terms include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids / triplexes, or polymers having purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0042] As used herein, "binding" refers to non-covalent interactions between macromolecules (e.g., between a protein and a nucleic acid). While in a state of non-covalent interaction, the macromolecules are said to be "bound" or "interacting" or "associated" (e.g., when molecule X is said to interact with molecule Y, it means that molecule X is non-covalently bound to molecule Y). Binding interactions generally have a dissociation constant (Kd) of 10 -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, 10 -12Less than M, 10 -13 Less than M, 10 -14 Less than M, or 10 -15 Characterized by less than M. Kd depends on environmental conditions such as pH and temperature as known to those skilled in the art. "Affinity" refers to the strength of binding, and an increase in binding affinity correlates with a decrease in Kd.

[0043] The terms "hybridizing" or "hybridize" refer to pairs of nucleic acid sequences that are substantially complementary or complementary within two different molecules. The pair can be achieved by any process by which a nucleic acid sequence binds to a substantially or completely complementary sequence via base pairs to form a hybridization complex. In some embodiments, "hybridizing" or "hybridize" includes denaturing the molecule to disrupt the intramolecular structure (e.g., secondary structure) within the molecule. In some embodiments, denaturing the molecule includes heating the solution containing the molecule to a temperature sufficient to disrupt the intramolecular structure of the molecule. In some cases, denaturing the molecule includes adjusting the pH of the solution containing the molecule to a pH sufficient to disrupt the intramolecular structure of the molecule. For the purpose of hybridization, two nucleic acid sequences or segments of sequences are "substantially complementary" if at least 80% of their individual bases are complementary to each other. For example, a primer oligonucleotide sequence is generally less than about 50% identical to one of two polynucleotides (e.g., an RNA fragment) that it is designed to be complementary to. However, the complementary portions of each sequence can be referred to herein as "segments", and the segments are substantially complementary if they have 80% or more identity.

[0044] Where a range of values is provided, unless the context clearly dictates otherwise, each intervening value between the upper and lower limits of that range, in increments of one tenth of the lower limit unit, and any other stated or intervening value in the stated range are understood to be included within the scope of the present invention. The upper and lower limits of these smaller ranges may independently be included within the smaller range, and are also included within the present disclosure in accordance with any specifically excluded limit values within the stated range. If the stated range includes one or both of these limit values, ranges excluding either or both of these included limit values are also included within the present disclosure.

[0045] Certain ranges are presented herein with numerical values preceded by the term "about." The term "about" is used herein to provide literal support for the exact numerical value that it precedes, as well as for a value that is near to or approximately the numerical value that the term precedes. When determining whether a number is near to or approximates a specifically recited number, a non-recited number that is near to or approximates the specifically recited number may be a number that provides substantially the same as the specifically recited number in the context in which it is presented.

[0046] It is understood that for clarity, certain features of the present disclosure that are described in the context of separate embodiments may also be provided in combination in a single embodiment. The various features of the present disclosure may be described in the context of a single embodiment, but the features may also be provided separately, or in any suitable sub-combination. All combinations of embodiments related to the present disclosure are specifically included by the present disclosure and are disclosed herein as if each combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and their elements are also specifically included by the present disclosure and are disclosed herein as if each such sub-combination was individually and explicitly disclosed herein.

[0047] I. Sprint Oligonucleotides The present disclosure provides methods for synthesizing mlRNAs, such as gRNAs, that use splint-mediated ligation of RNA, particularly 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. Essential to these methods are splint oligonucleotides. These hybridize with a first RNA fragment and a second RNA fragment to form a complex, which facilitates 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 splint-mediated ligation of two RNA fragments using one splint oligonucleotide. In some embodiments, the method includes the use of splint-mediated ligation of three RNA fragments using two splint oligonucleotides. In some embodiments, the method includes the use of splint-mediated ligation of more than three RNA fragments (e.g., four, five, six, seven, or eight RNA fragments) using an appropriate number of splint oligonucleotides necessary to ligate the RNA fragments.

[0048] A splint oligonucleotide includes a first portion complementary to a first RNA fragment in a terminal region that includes, for example, a 5' phosphate moiety. It further includes a second portion complementary to a second RNA fragment in a terminal region that includes a 3' hydroxyl group. The splint oligonucleotide can hybridize with the first RNA fragment and the second RNA fragment to form a complex. In the complex, the RNA fragments are favorably positioned for ligation at a ligation site present between the RNA fragments.

[0049] The split oligonucleotide may contain a sequence complementary to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides, either continuously or discontinuously, in the first RNA fragment in the terminal region containing the 5'-phosphate moiety. The split oligonucleotide may contain a sequence complementary to the 1-20 nucleotides, 21-40 nucleotides, 41-60 nucleotides, or 61-80 nucleotides of the first RNA fragment, and the nucleotides may be continuous or discontinuous. The split oligonucleotide may contain a sequence complementary to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides, either continuously or discontinuously, in the second RNA fragment in the terminal region containing the 3'-hydroxyl group. The split oligonucleotide may contain a sequence complementary to the 1-20 nucleotides, 21-40 nucleotides, 41-60 nucleotides, or 61-80 nucleotides of the second RNA fragment, and the nucleotides may be continuous or discontinuous.

[0050] The length of the sequence of the split oligonucleotide complementary to the first RNA fragment and the length of the sequence complementary to the second RNA fragment may be the same or different.

[0051] The split oligonucleotide can be designed to preferentially facilitate complex formation between the RNA fragment and the split oligonucleotide over the intramolecular structures (e.g., secondary structures) present in the RNA fragment and / or the split oligonucleotide. Minimum free energy prediction algorithms can be used to design suitable split oligonucleotides provided by the methods of the present disclosure. Theoretically, the lower the free energy, the higher the likelihood of forming a complex between the RNA fragment and the split oligonucleotide. The minimum free energy structure of a sequence is the secondary structure calculated to have the lowest free energy value (and thus the highest theoretical likelihood of formation). As an example, using a minimum free energy prediction algorithm, ΔGintra The free energy of the secondary structure of the RNA fragment represented by, and ΔG inter The free energy of intermolecular hybridization between the RNA fragment represented by and the sprint oligonucleotide can be calculated. In some cases, nearest neighbor approximation is used. The melting temperature (Tm) of the secondary structure of the RNA fragment is T m-intra It is represented by. The melting temperature of the RNA fragment and the sprint oligonucleotide hybrid is T m-inter It is represented by. The length of the sprint oligonucleotide is such that ΔG intra is greater than ΔG inter and / or T m-inter is greater than T m-intra It can be designed to ensure that. Exemplary free energy prediction algorithms are accessible from those available at URL:: / / unafold.rna.albany.edu / ?q=mfold.

[0052] The first RNA fragment, the second RNA fragment, or both contain at least one secondary structure, and when hybridizing the first RNA fragment, the second RNA fragment, and the sprint oligonucleotide, it results in a free energy lower than the free energy associated with one of the at least one secondary structure. Hybridizing the first RNA fragment, the second RNA fragment, and the sprint oligonucleotide can also result in a free energy lower than, for example, the free energy of the secondary structure having the lowest free energy (or minimum free energy) of the first RNA fragment, the second RNA fragment, or both.

[0053] One or more splint oligonucleotides can be used to hybridize with RNA fragments to mediate ligation of the RNA fragments. The number of splint oligonucleotides used to mediate ligation can be less than the number of RNA fragments to be ligated. For example, the number of splint 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, the number of splint oligonucleotides can be n - 1.

[0054] The length of the splinted 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 100, 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 60, 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 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).

[0055] Sprint oligonucleotides can be bound to a support. Sprint oligonucleotides can be bound to a support using various techniques. For example, sprint oligonucleotides can be bound directly to the support or immobilized on the support by chemical immobilization. For example, chemical immobilization can occur between a functional group on the support and a corresponding functional group of the sprint oligonucleotide. Such a corresponding functional element of the sprint oligonucleotide is either an inherent chemical group of the sprint oligonucleotide, such as a hydroxyl group, or can be additionally introduced. An example of such a functional group is an amine group. Typically, the sprint oligonucleotides to be immobilized contain a functional amine group or are chemically modified to contain a functional amine group. Means and methods for such chemical modification are known in the art.

[0056] Using the localization of functional groups within the sprint oligonucleotides to be immobilized, the binding behavior and / or orientation of the sprint oligonucleotides can be controlled and formed. For example, the functional groups can be located at the 5' or 3' end of the sprint oligonucleotide or within the sequence of the sprint oligonucleotide. A typical support for the sprint oligonucleotides to be immobilized contains a moiety that can bind to such sprint oligonucleotides, such as an amine-functionalized nucleic acid. Non-limiting examples of such supports include carboxy, aldehyde, and epoxy supports.

[0057] The support to which the sprint oligonucleotides can be immobilized can be chemically activated, for example, by activation of the functional groups available on the support. The term "activated substrate" relates to a material in which interacting or reactive chemical functional groups have been established or made possible by chemical modification procedures. For example, a support containing a carboxyl group can be activated before use. Additionally, certain supports contain functional groups that can react with a specific moiety already present in the sprint oligonucleotide.

[0058] The covalent bonds used to attach the splint oligonucleotide to the support can be considered as direct and indirect bonds in the sense that, although the splint oligonucleotide is attached by a "direct" covalent bond, there may be a chemical moiety or linker that separates the "first" nucleotide of the splint oligonucleotide from the support (i.e., an indirect bond). In some cases, the splint oligonucleotide immobilized on the support by a covalent bond and / or a chemical linker generally appears to be directly immobilized or attached to the support. The splint oligonucleotide may not be directly attached to the support, but may interact indirectly, for example, by binding to a molecule that itself is directly or indirectly attached to the support. The splint oligonucleotide can also be indirectly attached to the support (e.g., via a solution containing a polymer).

[0059] When the splint oligonucleotide is indirectly immobilized on the support, for example, via hybridization to a surface oligonucleotide to which the splint oligonucleotide can bind, the splint oligonucleotide may further contain an upstream sequence that can hybridize to the 5'-end of the surface oligonucleotide (the 5' of the sequence that hybridizes to two or more RNA fragments as described herein).

[0060] The splint oligonucleotide can be attached to the support via its 5'-end or its 3'-end. The splint oligonucleotide attached to the support can be synthesized in situ on the support.

[0061] II. Methods for synthesizing RNA The methods disclosed herein for synthesizing mlRNA generally involve providing a first RNA fragment, a second RNA fragment, and a splint oligonucleotide. The first RNA fragment, the second RNA fragment, and the splint oligonucleotide are hybridized together to form a complex. When such a complex is formed, the first and second RNA fragments are positioned in proximity to facilitate ligation. A ligase is then used to ligate the first and second RNA fragments across the ligation site, thereby synthesizing mlRNA.

[0062] In some embodiments, the method involves providing a first RNA fragment, a second RNA fragment, a third RNA fragment, a first splint oligonucleotide, and a second oligonucleotide. The first RNA fragment, the second RNA fragment, and the first splint oligonucleotide are hybridized together, and the second RNA fragment, the third RNA fragment, and the second splint oligonucleotide are hybridized together, thereby forming a complex comprising the first, second, and third RNA fragments, and the first and second splint oligonucleotides. Formation of the complex positions (i) the 3'-hydroxyl group of the first RNA fragment and the 5'-phosphate moiety of the second RNA fragment in proximity to provide a first ligation site, and (ii) the 3'-hydroxyl group of the second RNA fragment and the 5'-phosphate moiety of the third RNA fragment in proximity to provide a second ligation site. The method further provides a ligase that ligates the first and second RNA fragments at the first ligation site and the second and third RNA fragments at the second ligation site, thereby synthesizing mlRNA.

[0063] Hybridization of the first RNA fragment, the second RNA fragment, and the sprint oligonucleotide can be performed in solution. In some embodiments, the hybridization further comprises a third RNA fragment and a second sprint oligonucleotide that are performed in solution. When hybridizing in solution, the concentration of the first RNA fragment can be, for example, approximately equal to the concentration of the second RNA fragment. In some embodiments, when the hybridization further comprises a third RNA fragment, the concentrations of the first RNA fragment and the second RNA fragment are each approximately equal to the concentration of the third RNA fragment. Depending on the method, fragment, and sprint oligonucleotide used, the concentration of the sprint oligonucleotide in solution or the concentration of the second RNA fragment in solution can be approximately equal to, greater than, or less than the concentration of the first RNA fragment in solution. For example, the concentrations of the sprint oligonucleotide, the first RNA fragment, and the second RNA fragment can be approximately equal. In some embodiments, the method comprises the first, second, and third RNA fragments, and the first and second sprint oligonucleotides, and the concentrations of the first sprint oligonucleotide, the second sprint oligonucleotide, the first RNA fragment, the second RNA fragment in solution, and the third RNA fragment are each approximately equal.

[0064] In some cases, for hybridization, the RNA fragment and / or the splint oligonucleotide are denatured, i.e., the intramolecular structure of the RNA fragment and / or the splint oligonucleotide is disrupted, enabling annealing between the RNA fragment and the splint oligonucleotide. Denaturation can be achieved, for example, by heating a solution containing the RNA fragment and the splint oligonucleotide 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 can involve heating the solution to a temperature of about 80°C to about 100°C, e.g., about 82°C to about 98°C, about 84°C to about 96°C, about 86°C to about 94°C, or about 88°C to about 92°C (e.g., 81°C, about 82°C, about 83°C, about 84°C, about 85°C, about 86°C, about 87°C, about 88°C, about 89°C, about 90°C, about 91°C, about 92°C, about 93°C, about 94°C, about 95°C, about 96°C, about 97°C, about 98°C, or about 99°C). In other examples, hybridization does not involve heating the solution.

[0065] In some cases, hybridizing may involve cooling the solution to a temperature of about 20°C to about 45°C, such as about 22°C to about 43°C, about 25°C to about 40°C, or about 27°C to about 38°C (e.g., about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41°C, about 42°C, about 43°C, or about 44°C) after heating. For example, in some cases, hybridizing may involve cooling the solution to about 37°C after heating. Hybridizing may include cooling the solution to a temperature that retains sufficient ligase activity for the ligase used in the methods described herein to ligate the first and second RNA fragments, and / or a temperature lower than the melting temperature of the complex formed by the RNA fragments and the splint oligonucleotides during hybridization. If hybridizing does not include heating of the solution, hybridizing can be performed at a temperature lower than the melting temperature of the complex formed by the RNA fragments and the splint oligonucleotides during hybridization. Depending on the particular method being performed, cooling the solution after heating can include decreasing the temperature of the solution at a constant rate or an uncontrolled rate.

[0066] The methods described in this disclosure include ligating a first and a second RNA fragment using a ligase at a ligation site. Ligation can include ligating the 5' phosphate group of the terminal region of the first RNA fragment to the 3' hydroxyl group of the terminal region of the second RNA fragment. Catalyzed by the ligase, the 5' phosphate group and the 3' hydroxyl group react to form a phosphodiester bond. The ligation site can be the site where a phosphodiester bond is formed between the 5' phosphate group and the 3' hydroxyl group.

[0067] In some embodiments, the method comprises ligating a first RNA fragment and a second RNA fragment using a ligase at a first ligation site, and ligating the second RNA fragment and a third RNA fragment using a ligase at a second ligation site. In some embodiments, the ligation comprises ligation of a 3’ hydroxyl group at the end of the first RNA fragment and a 5’ phosphate at the end of the second RNA fragment, and ligation of a 3’ hydroxyl group at the end of the second RNA fragment and a 5’ phosphate at the end of the third RNA fragment, each ligation resulting in the formation of a phosphodiester bond.

[0068] Generally, ligating the first and second RNA fragments can be performed at a temperature of about 15°C to about 45°C, such as about 17°C to about 43°C, about 20°C to about 40°C, or about 22°C to about 38°C (e.g., about 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 about 45°C). For example, ligating the first and second RNA fragments can be performed at 37°C. Ligating the first and second RNA fragments can be performed for various periods depending on the method being implemented, such as about 0.1 to about 48 hours, such as 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, 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 used in the splint-mediated ligation reaction is independent of the number of RNA fragments used in the reaction. For example, the reaction temperature and / or reaction time suitable for splint-mediated ligation involving two RNA fragments is suitable for splint-mediated ligation reactions involving three or more RNA fragments.

[0069] In some cases, it may be useful to quench the ligation reaction following synthesis of the gRNA. For example, the ligation reaction can be quenched using a protease or a chelating agent. Non-limiting examples of proteases include Proteinase K. Non-limiting examples of chelating agents include EDTA and EGTA, or combinations of both.

[0070] In some cases, ligating the first and second RNA fragments further comprises using one or more crowding agents. Non-limiting examples of crowding agents include polyethylene glycol (PEG), Ficoll®, ethylene glycol, and dextran, or any combination thereof. In some embodiments, the use of one or more crowding agents is suitable for a splint-mediated ligation reaction involving two, three, or more RNA fragments.

[0071] In the methods described herein, various ligases can be used. 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, a thermostable DNA ligase (e.g., 5’AppDNA / RNA ligase), or an ATP-dependent DNA ligase. In some cases, combinations of any two or more such ligases can be used.

[0072] 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 cleaved and / or can contain mutations. For example, T4 RNA ligase II can contain the K227Q mutation and / or the R55K mutation. In some cases, T4 RNA ligase II can be cleaved and can have the K227Q and / or R55K mutations. Also useful in the methods described herein is PBCV-1 DNA ligase (i.e., Chlorella virus DNA ligase, SplintR® ligase). In some cases, the ligase can be a DNA ligase (e.g., 9°N® DNA ligase).

[0073] In some of the methods described herein, three or more (e.g., three, four, or five) RNA fragments can be ligated to synthesize an mlRNA. Ligation of three or more RNA fragments can be performed in the same step or in separate steps (such as a stepwise method).

[0074] The methods described herein can further include purifying the mlRNA after synthesis. Purification can separate the full-length RNA product from unreacted RNA fragments and / or splint oligonucleotides. Purification can include, for example, enzymatically degrading unreacted RNA fragments using an exonuclease, such as one specific for 5'-phosphate-containing RNA. An exemplary exonuclease is XRN-1.

[0075] Purification of full-length RNA products from unreacted RNA fragments and / or splint oligonucleotides can also be carried out using ultrafiltration or chromatography methods. Non-limiting examples of chromatography methods include reverse-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 chromatography (CE), capillary gel chromatography (CGE), and polyacrylamide gel purification.

[0076] Also provided herein are methods of synthesizing an RNA molecule comprising all or a portion of a tracrRNA sequence using any of the methods described herein. The RNA molecule is optionally purified and can be used to generate a full-length gRNA (e.g., sgRNA). To generate a full-length sgRNA, a splint oligonucleotide is used to ligate an additional RNA fragment comprising a spacer and a minimal CRISPR repeat (such as a crRNA) to a previously synthesized RNA molecule. These methods can generate a full-length gRNA specific to any target DNA sequence, for example, by ligating an RNA fragment comprising the corresponding spacer sequence to a previously synthesized RNA molecule comprising all or a portion of a tracrRNA sequence. Figure 3 is a representative schematic showing a 3-fragment system in which two of the RNA fragments (Fragment II and III) are first ligated using a splint oligonucleotide to form an invariant RNA construct comprising a portion of the tracrRNA sequence. This RNA product can be purified and stored, for example, for later use. Next, an RNA fragment I' comprising a sequence complementary to a specific target DNA can be combined with the previously synthesized RNA construct to form a full-length gRNA.

[0077] III. RNA Fragments As described in the present disclosure, a method for synthesizing mlRNA involves 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, and the RNA is synthesized by ligating the first and second RNA fragments. When synthesizing gRNA, the first RNA fragment, the second RNA fragment, or both can comprise at least a portion of a sequence capable of binding to an RNA-guided endonuclease. Exemplary mlRNA synthesized by the described method can comprise, from 5' to 3', the second RNA fragment followed by the first RNA fragment. The second RNA fragment may not contain a 5' phosphate moiety. The 5' phosphate moiety can be, for example, 5'-phosphate or 5'-phosphorothioate. The first fragment, the second RNA fragment, or both can comprise a sequence or a portion of a sequence that is complementary to a sequence in the target DNA. In some cases, the second RNA fragment comprises a sequence that is complementary to the sequence of the target DNA.

[0078] mlRNA can be synthesized by ligating three or more (e.g., 3, 4, 5, or 6) RNA fragments. Figure 2 is a schematic diagram showing the ligation of three RNA fragments using two splint oligonucleotides. In some cases, RNA is synthesized by ligating less than 6 RNA fragments. As an example, in the case of RNA synthesized by ligating RNA fragments A, B, and C (enumerated in the order from 5' to 3'), before ligation, RNA fragment A can contain a 3'-hydroxyl group and may contain a 5'-phosphate moiety, RNA fragment B can contain both a 3'-hydroxyl group and a 5'-phosphate moiety, RNA fragment C can contain a 5'-phosphate moiety and may or may not contain a 3'-hydroxyl group. Ligation of RNA fragments A, B, and C can include the formation of phosphodiester bonds 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 of C.

[0079] The length of any of the RNA fragments can be from 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, 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 50, 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 85, 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, 80 to 90, 80 to 85, or 85 to 90 nucleotides). The lengths of the first and second RNA fragments can be, for example, 10 to 90 nucleotides each. The length of the second RNA fragment can be about 40 nucleotides or less (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.

[0080] The RNA fragment can include one or more secondary structures. The secondary structure of an RNA molecule (e.g., an RNA fragment or mlRNA) can include stems and loops, or combinations thereof. Non-limiting examples of the secondary structure of an RNA molecule include stem-loops, hairpins, hairpin loops, tetraloops, internal loops, bulges, pseudoknots, and cloverleafs. In some cases, the RNA fragment does not include a secondary structure (e.g., a stem-loop). The RNA synthesized by the methods provided herein can include, but is not limited to, one or more secondary structures such as one or more stem-loop structures formed upon ligation of the RNA fragments. In some cases, the ligation site present between the RNA fragments corresponds to a site of the secondary structure (e.g., a stem-loop structure) of the synthesized RNA. The ligation site can correspond to a site of a portion of the secondary structure, including but not limited to, the tetraloop portion or the helix portion of the stem-loop structure.

[0081] The method of the present disclosure can include predicting the secondary structure of an RNA fragment and / or the free energy associated with the secondary structure based on the sequence of the RNA fragment. Methods for predicting the secondary structure of RNA are known in the art and include 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. The secondary structure of RNA can be predicted from the RNA sequence by minimization of free energy, such as that described in Mathews and Turner (2006) Current Opinion in Structural Biology, 16:270-278. Non-limiting examples of software for RNA secondary structure prediction can be found by referring to the URLs available at en.wikipedia.org / wiki / List_of_RNA_structure_prediction_software.

[0082] Modification The RNA fragment can include one or more modifications. For example, the RNA fragment can include at least one modification to 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), bridged nucleic acid, 2'-deoxy nucleic acid (DNA), and peptide nucleic acid (PNA). Alternatively or additionally, the RNA fragment can 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 includes at least one phosphorothioate bond.

[0083] The modification of RNA fragments can be used, for example, to enhance stability, reduce the likelihood or extent of the innate immune response, and / or enhance other attributes, and new types of modifications are being developed regularly. Examples of various types of modifications include, but are not limited to, one or more nucleotides modified at the 2'-position of the sugar, such as 2'-O-alkyl, 2'-O-alkyl-O-alkyl, or 2'-fluoro modified nucleotides. DNA (2'-deoxy-) nucleotide substitutions are also contemplated. Non-limiting examples of RNA modifications also include 2'-fluoro, 2'-amino, 2'O-methyl modifications on the ribose of pyrimidines, and base residues or inverted bases at the 3'-end of RNA. Such modifications are incorporated into oligonucleotides, and these oligonucleotides have been shown to have a higher T m (i.e., higher target binding affinity) than 2'-deoxyoligonucleotides for a given target. In some embodiments, the modification of the RNA fragments disclosed herein includes 2'O-methyl modification of one or more nucleosides in the RNA fragment.

[0084] An RNA fragment according to any of the embodiments described herein can include, for example, a modification that increases resistance to nuclease digestion compared to natural nucleic acids. In some cases, the modified nucleic acid includes a modified backbone selected from, for example, phosphorothioate, phosphotriester, methylphosphonate, short chain alkyl or cycloalkyl sugar linkages, and short chain heteroatom or heterocyclic sugar linkages. The nucleic acid can have a phosphorothioate backbone or a heteroatom backbone, such as CH 2 -NH-O-CH 2 , CH-N(CH 3 )-O-CH 2 (known as the methylene(methylimino) or MMI backbone), CH 2 -O-N(CH 3 )-CH 2 , CH 2 -N(CH 3 )-N(CH 3 )-CH 2and O-N(CH 3 )-CH 2 -CH 2It can have a backbone, an amide backbone (see De Mesmaeker et al. (1995) Acc. Chem. Res., 28(9):366-374), a morpholino backbone structure (see Summerton and Weller, U.S. Patent No. 5,034,506), a peptide nucleic acid (PNA) backbone (wherein the phosphodiester backbone of an oligonucleotide is replaced by a polyamide backbone and the nucleotides are directly or indirectly attached to the aza nitrogen atoms of the polyamide backbone, see Nielsen et al. (1991) Science, 254(5037):1497-1500). Phosphorus-containing linkages include phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl, and other alkylphosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having a normal 3'-5' linkage, their 2'-5' linked analogs, and those having inverted polarity, but are not limited thereto, and adjacent pairs of nucleoside units are linked 3'-5' and 5'-3' or 2'-5' and 5'-2', see U.S. Patent Nos. 3,687,808, 4,469,863, 4,476,301, 5,023,243, 5,177,196, 5,188,897, 5,264,423, 5,276,019, 5,278,302, 5,286,717, 5,321,131, 5,399,676, 5,405,939, 5,453,496, 5,455,233, 5,466,677, 5,476,925, 5,519,126, 5,536,821, 5,541,306, 5,550,111, 5,563,253, 5,571,799, 5,587,361, and 5,625,050.In some embodiments, the modification of the RNA fragments disclosed herein includes one or more phosphorothioate bonds in the backbone of the RNA fragment.

[0085] Morpholino-based oligomeric compounds are 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, Lacerra 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 are described in Wang et al. (2000) J. Am. Chem. Soc., 122(36):8595-8602.

[0086] An RNA fragment according to any of the embodiments described herein can include a backbone that does not contain phosphorus atoms internally, for example, a short-chain alkyl or cycloalkyl nucleoside internucleoside linkage, a mixed heteroatom and alkyl or cycloalkyl nucleoside internucleoside linkage, or a backbone formed by one or more short-chain heteroatom or heterocyclic nucleoside internucleoside linkages. These include those having a morpholino linkage (partially formed from the sugar moiety of the nucleoside), a siloxane backbone, a sulfide, a sulfoxide and a sulfone backbone, a formacetyl and a thioformacetyl backbone, a methyleneformacetyl and a thioformacetyl backbone, an alkene-containing backbone, a sulfamate backbone, a methyleneimino and a methylenehydrazino backbone, a sulfonic acid and a sulfonamide backbone, an amide backbone, and N, O, S, CH 2including those in which components are mixed, see, for example, U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 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.

[0087] An RNA fragment according to any of the embodiments described herein may have, for example, at the 2'-position one of the following: OH, SH, SCH 3 , F, OCN, OCH 3 , OCH 3 O(CH 2 )nCH 3 , O(CH 2 )nNH 2 , or O(CH 2 )nCH 3 (where n is from 1 to 10), C1-C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl, Cl, Br, CN, CF 3 , OCF 3 , O-, S-, or N-alkyl, O-, S-, or N-alkenyl, SOCH 3 , SO2CH 3 , ONO 2 , NO 2 , N 3 , NH 2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. For example, the modification can include 2'-methoxyethoxy (2'-O-CH 2 CH 2 OCH 3 , 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-CH 3 ), 2'-propoxy (2'-OCH 2 CH 2 CH 3) and include 2'-fluoro (2'-F). Similar modifications can also be made at other positions of the oligonucleotide, particularly at the 3'-position of the sugar on the 3'-terminal nucleotide and the 5'-position of the 5'-terminal nucleotide. The oligonucleotide may also have a sugar mimic such as cyclobutyl instead of a pentofuranosyl group. In some embodiments, both the sugar of the nucleotide unit and the internucleoside linkage, e.g., the backbone, are replaced with novel groups. The base units are maintained for hybridization with a suitable nucleic acid target compound. An oligonucleotide mimic, which is one such oligomeric compound and has been shown to have excellent hybridization properties, is called a peptide nucleic acid (PNA). In a PNA compound, the sugar backbone of the oligonucleotide is replaced with an amide containing a backbone such as an aminoethylglycine backbone. The nucleobases are retained and are attached directly or indirectly to the azanitrogen atom of the amide portion of the backbone. Representative U.S. patents that teach 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. Further teachings of PNA compounds can be found in Nielsen et al. (1991) Science, 254(5037):1497-1500. The RNA fragments described herein can include 2'-O-thionocarbamate MP (2'-O-methyl-3'-phosphonoacetate) and MSP (O-methyl-3'-thiophosphonoacetate) (Ryan et al., (2017) Nuc. Acids Res. 46(2):792-803).

[0088] The RNA fragments described herein can include one or more modifications selected from the group consisting of pseudouridine, N 1 -methylpseudouridine, and 5-methoxyuridine. For example, incorporating one or more N 1 -methylpseudouridine into an RNA fragment can enhance RNA stability and reduce immunogenicity in animal cells such as mammalian cells (e.g., human and mouse cells). N 1The N1-methylpseudouridine modification can also be incorporated in combination with one or more 5-methylcytidines.

[0089] For example, there are a number of commercial vendors of modified RNAs, including Trilink Biotech, Axolabs, Bio-Synthesis Inc., and Dharmacon. As described by Trilink, for example, 5-methyl-CTP can be used to confer desirable properties such as increased nuclease stability or reduced interaction of innate immune receptors with in vitro transcribed RNA. 5’-methylcytidine-5’-triphosphate (5-methyl-CTP), N6-methyl-ATP, and pseudouridine triphosphate 2-thio-UTP have also been shown to reduce innate immune stimulation in culture and in vivo, as shown by Kormann et al. (2011) Nat. Biotechnol., 29:154-157 and Warren et al. (2010) Cell Stem Cell, 7(5):618-630.

[0090] RNA fragments can incorporate modifications designed to bypass the innate antiviral response. 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 anti-reverse cap analog (ARCA). See, for example, Warren et al. (2010) Cell Stem Cell, 7(5):618-630.

[0091] A variety of modifications have been developed and applied to enhance RNA stability, reduce innate immune responses, and / or achieve other advantages. See, for example, reviews by 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, Bremsen et al. (2012) Front. Genet., 3:154.

[0092] Mimetic RNA fragments can be nucleic acid mimics. The term "mimic" as applied to polynucleotides is intended to include polynucleotides in which only the furanose ring, or both the furanose ring and the internucleotide linkage, are replaced by non-furanose groups. Replacement 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 maintained for hybridization with a suitable target nucleic acid. A polynucleotide mimic, which is one such nucleic acid and has been shown to have excellent hybridization properties, is called a peptide nucleic acid (PNA). In PNA, the sugar backbone of the polynucleotide is replaced by an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleotides are retained and are attached directly or indirectly to the aza nitrogen atoms of the amide portions of the backbone. Representative U.S. patents that teach 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.

[0093] RNA fragments can be polynucleotide mimics based on linked morpholino units (morpholino nucleic acids) having a heterocyclic base attached to a morpholino ring. A number of linking groups that link the morpholino monomer units in morpholino nucleic acids have been reported. One class of linking groups has been selected to give nonionic oligomeric compounds. Morpholino-based polynucleotides are nonionic mimics of oligonucleotides and 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. A variety of compounds within the morpholino classification of polynucleotides have been prepared and have various different linking groups that join the monomer subunits.

[0094] RNA fragments can be polynucleotide mimics called cyclohexenyl nucleic acids (GeNA), in which the furanose ring normally present in DNA / RNA molecules is replaced by a cyclohexenyl ring. Phosphoramidite monomers protected with GeNA DMT are prepared and used in the synthesis of oligomeric compounds following classical phosphoramidite chemistry. Completely modified GeNA oligomeric compounds and oligonucleotides having specific positions modified with GeNA have been prepared and studied (see Wang et al. (2000) J. Am. Chem. Soc., 122(36):8595-8602). Studies incorporating the GeNA structure into natural nucleic acid structures have shown facile conformational adaptation by NMR and circular dichroism.

[0095] RNA fragments are locked nucleic acids (LNA), in which the 2'-hydroxyl group is linked to the 4'-carbon atom of the sugar ring, forming a 2'-C,4'-C-oxymethylene bond, thereby forming a bicyclic sugar moiety. In certain embodiments, the linkage is a methylene (-CH2) n group, where n is 1 or 2 (Singh et al. (1998) Chem. Commun., 4:455-456). LNAs and LNA analogs exhibit very high duplex thermal stability, with complementary DNA and RNA (T m = +3 to +10 °C), stability against 3'-exonuclease degradation, and good solubility properties. Potent and non-toxic antisense oligonucleotides containing LNA have been described (Wahlestedt et al. (2000) Proc. Natl. Acad. Sci. U.S.A, 97(10):5633-5638). The synthesis and preparation of the LNA monomers adenine, cytosine, guanine, 5-methylcytosine, thymine, and uracil, and 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 WO99 / 14226.

[0096] Modified sugar moiety The RNA fragment can include one or more substituted sugar moieties containing a sugar substituent selected from, for example, OH, F, O—, S—, or N-alkyl, O—S—, or N-alkenyl, O—, S— or N-alkynyl, or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl can be substituted or unsubstituted C1-C10 alkyl or C2-C10 alkenyl and alkynyl. Particularly suitable are O((CH 2 ) n O) m CH 3 、O(CH 2 ) n OCH 3 、O(CH z ) n NH 2 、O(CH 2 )CH 3 、O(CH 2 ) n ONH 2 and O(CH 2 ) n ON((CH 2 ) n CH 3 ) 2 wherein n and m are from 1 to about 10. Other RNA fragments are C1-C10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 、OCN、Cl、Br、CN、CF 3 、OCF 3 、SOCH 3 、SO 2 CH 3 、ONO 2 、NO 2 、N 3 、NH 2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, and RNA cleavage groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of oligonucleotides, or groups for improving the pharmacodynamic properties of oligonucleotides, and other suitable sugar substituents selected from other substituents having similar properties. Suitable modifications include 2'-methoxyethoxy 2'-O-CH 2 -CH 2 OCH 3 (also known as -2'-O-(2-methoxyethyl) or 2'-MOE) For example, an alkoxyalkoxy group is included (Martin et al. (1995) Helv. Chim. Acta, 78(2):486-504). Further suitable modifications include 2'-dimethylaminooxyethoxy, for example, also known as 2'-DMAOE described in the examples hereinbelow O(CH 2 ) 2 ON(CH 3 ) 2 group, and 2'dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethyl-aminoethoxyethyl or 2'DMAEOE), for example 2'-O-CH 2 -O-CH 2 -N(CH 3 ) 2 is included.

[0097] Other suitable sugar substituents include methoxy (-O-CH 3 ), aminopropoxy (-O-CH 2 CH 2 CH 2 NH 2 ), allyl (-CH 2 -CH=CH 2 ), -O-allyl (-O-CH 2 -CH=CH 2) and Fluoro (F). The 2'-modification may be in the arabino (up) or ribo (down) position. A preferred 2'-arabino modification is 2'-F. Similar modifications may also be made at other positions of the oligomeric compound, for example, the 3'-position of the sugar on the 3'-terminal nucleotide, or in a 2'-5' linked oligonucleotide, and at the 5'-position of the 5'-terminal nucleotide. The oligonucleotide may also have a sugar mimic such as a cyclobutyl moiety instead of a pentofuranosyl sugar.

[0098] Base Modifications and Substitutions An RNA fragment according to any of the embodiments described herein can additionally or alternatively include nucleic acid base (often simply referred to as "base" in the art) modifications or substitutions. As used herein, "unmodified" or "natural" nucleic acid bases include adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U). Modified nucleic acid bases include nucleic acid bases that are rarely or only transiently found in natural nucleic acids, such as hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (also called 5-methyl-2'-deoxycytosine and often referred to as 5-Me-C in the art), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, and synthetic nucleic acid bases such as 2-aminoadenine, 2-(methylamino)adenine, 2-(imidazolylalkyl)adenine, 2-(aminoalkylamino)adenine, or other hetero-substituted alkyladenines, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6(6-aminohexyl)adenine, and 2,6-diaminopurine (Kornberg et al. (1980) DNA Replication (2 nd(see Gebeyehu et al. (1987) Nucl. Acids Res., 15(11):4513-4534; ed.)(pp.75-77). San Francisco, CA: W.H. Freeman & Co.). "Universal" bases known in the art, such as inosine, can also be included. 5-Me-C substitution has been shown to improve the stability of nucleic acid duplexes by 0.6 - 1.2 °C (Sanghvi (1993). Antisense Research and Applications, (pp.276-278). Crooke, S.T. and Lebleu, B., (Eds.), Boca Raton, FL: CRC Press), and is an embodiment of base substitution.

[0099] 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-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azauracil, cytosine and thymine, 5-uracil (pseudo-uracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, as well as 3-deazaguanine and 3-deazaadenine.

[0100] Furthermore, nucleobases include those disclosed in U.S. Patent No. 3,687,808, those disclosed by Kroschwitz (Ed.) (1990). The Concise Encyclopedia of Polymer Science and Engineering, (pp. 858 - 859). Hoboken, N.J.: John Wiley & Sons, those disclosed by Englisch et al. (1991) Angewandte Chemie International Edition, 30(6):613 - 722, and those disclosed by Sanghvi (1993) Chapter 15, Antisense Research and Applications, (pp. 289 - 302), Crooke, S.T. and Lebleu, B. (Eds), Boca Raton, FL: CRC Press. Certain of these nucleobases are particularly useful for enhancing the binding affinity of the oligomeric compounds of the present disclosure. These include 5 - substituted pyrimidines, 6 - azapyrimidines, and N - 2, N - 6, and - O - 6 substituted purines, including 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 °C (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, particularly when combined with 2’ - O - methoxyethyl sugar modification.Modified nucleobases are described, for example, in U.S. Patent Nos. 3,687,808, 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, 5,502,177, 5,525,711, 5,552,540, 5,587,469, 5,596,091, 5,614,617, 5,681,941, 5,750,692, 5,763,588, 5,830,653, 6,005,096, as well as U.S. Patent Application Publication No. 2003 / 0158403.

[0101] RNA fragments according to any of the embodiments described herein that include nucleobase modifications or substitutions may not be uniformly modified at all positions. For example, an RNA fragment may have a modification incorporated into a single nucleoside.

[0102] IV. Synthesis of RNA Fragments and Sprint Oligonucleotides The RNA fragments and sprint oligonucleotides provided by the present disclosure can be synthesized by any method suitable for oligonucleotide synthesis described herein or known in the art. Non-limiting examples include enzymatic synthesis and chemical synthesis (e.g., phosphoramidite chemistry).

[0103] Methods for synthesizing RNA from a DNA template are known in the art. For example, RNA fragments and sprint oligonucleotides can be synthesized in vitro using an RNA polymerase enzyme (e.g., T7 polymerase, T3 polymerase, SP6 polymerase, etc.). Solid-phase synthesis using phosphoramidite chemistry involves the assembly of protected 2'-deoxynucleosides (dA, dC, dG, and T), ribonucleosides (A, C, G, and U), or chemically modified nucleosides, such as LNA or BNA monomers. The monomers are sequentially coupled to the growing oligonucleotide chain in the order required for the sequence of the product. Once the chain assembly is complete, the product is released from the solid phase into solution, deprotected, and recovered.

[0104] RNA fragments and sprint oligonucleotides can be synthesized in the 5' to 3' direction or the 3' to 5' direction. In some cases, a second RNA fragment is synthesized in the 5' to 3' direction. The synthesized RNA fragments and sprint oligonucleotides can be purified prior to ligation according to known methods in the art, including but not limited to high-performance liquid chromatography (HPLC), reverse-phase HPLC, ion-exchange chromatography, size-exclusion chromatography, hydrophobic interaction chromatography, affinity chromatography, and polyacrylamide gel purification.

[0105] V. Medium-length RNA Exemplary mlRNAs synthesized using any of the methods described herein are guide RNAs (gRNAs) (e.g., any of the gRNAs described herein). The gRNAs synthesized by the methods of the present disclosure can be single molecule gRNAs (sgRNAs) or double molecule gRNAs. The gRNA provides target specificity by binding to an RNA-guided endonuclease and induces the activity of the RNA-guided endonuclease. The RNAs of the present disclosure can be synthesized from two or more RNA molecules (referred to as RNA fragments) using one or more splints. An exemplary double molecule gRNA includes a crRNA and a trans-activating crRNA (tracrRNA), and the crRNA and tracrRNA hybridize to each other to form a double strand. The double molecule gRNA can also be a duplex of two crRNAs. The gRNA duplex can bind an RNA-guided endonuclease such that the gRNA and the RNA-guided endonuclease form a complex. The crRNA includes both a spacer sequence capable of hybridizing to a target nucleic acid sequence of interest and a crRNA repeat sequence. The tracrRNA can be in any form (e.g., full-length tracrRNA or an active partial tracrRNA) and can be of various lengths. For example, the tracrRNA can include all or a portion of a wild-type tracrRNA sequence (e.g., about or at least 20, 26, 32, 45, 48, 54, 63, 67, 85 or more nucleotides of the wild-type tracrRNA sequence) or can consist thereof. Examples of wild-type tracrRNA sequences from S. pyogenes include 171-nucleotide, 89-nucleotide, 75-nucleotide and 65-nucleotide versions. See, e.g., Deltcheva et al. (2011) Nature 471:602-607 and WO2014 / 093661. As an example, the crRNA has, in the 5' to 3' direction, an optional spacer extension sequence, a spacer sequence, and a minimal CRISPR repeat sequence. The tracrRNA can have a minimal tracrRNA sequence (complementary to the minimal CRISPR repeat sequence), a 3' tracrRNA sequence, and an optional tracrRNA extension sequence.Any tracrRNA extension can have one or more hairpin structures that can have elements conferring additional functionality to the gRNA. The crRNA and tracrRNA hybridize via the minimal CRISPR repeat and the minimal tracrRNA sequence to form the gRNA.

[0106] An exemplary sgRNA comprises a nucleotide sequence complementary to the sequence of a target DNA and a nucleotide sequence capable of binding to an RNA-guided endonuclease. As an example, the sgRNA can have, in the 5' to 3' direction, an optional spacer extension sequence, a spacer sequence, a minimal CRISPR repeat sequence, a single molecule guide linker, a minimal tracrRNA sequence, a 3' tracrRNA sequence, and an optional tracrRNA extension sequence. Optionally, the sgRNA can have, in the 5' to 3' direction, a minimal CRISPR repeat and a spacer sequence. The single molecule guide linker binds the minimal CRISPR repeat and the minimal tracrRNA sequence to form a hairpin structure. In some embodiments, the single molecule guide linker is a tetraloop. Exemplary gRNAs are described, for example, in WO2018 / 002719.

[0107] Generally, CRISPR repeat sequences include one or more of the following: (1) excision of a DNA target segment adjacent to an intracellular CRISPR repeat sequence that includes a corresponding tracr sequence, and (2) formation of a CRISPR complex at the target sequence, and include any sequence having sufficient complementarity to the tracr sequence to facilitate these, where the CRISPR complex includes a CRISPR repeat sequence hybridized to the tracr sequence. Generally, the degree of complementarity is related to the optimal alignment of the CRISPR repeat sequence and the tracr sequence along the length of the shorter of the two sequences. The optimal alignment is determined by any suitable alignment algorithm and may further account for secondary structures such as self-complementarity within either the tracr sequence or the CRISPR repeat sequence. In some cases, the degree of complementarity between the tracr sequence and the CRISPR repeat sequence along the 30 nucleotide length of the shorter of the two when optimally aligned is about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or more. The tracr sequence can be, for example, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50 or more nucleotides in length.

[0108] The spacer of the gRNA includes a nucleotide sequence that is complementary to the sequence of the target DNA. In other words, the spacer of the gRNA interacts sequence-specifically with the target DNA via hybridization (e.g., base pairing). Thus, the nucleotide sequence of the spacer can vary and determines the position within the target DNA where the gRNA and the target DNA interact. The spacer of the gRNA can be selected to hybridize to any desired sequence within the target DNA.

[0109] The spacer can have a length of, for example, 10 to 30 nucleotides (such as any length from 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 can have a length of 13 to 25 nucleotides, 15 to 23 nucleotides, 18 to 22 nucleotides, or 20 to 22 nucleotides.

[0110] The percent sequence complementarity between the spacer of the gRNA and the target sequence of the target DNA can be, for example, at least about 60% (such as any of at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%).

[0111] The length of the gRNA synthesized by the method described herein can be, for example, 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 (such as any length from 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 method described herein can include a spacer. In some embodiments, the gRNA synthesized by the method described herein includes a sequence complementary to a sequence in the target DNA, including but not limited to, target mammalian DNA. For example, the target DNA can be human DNA.

[0112] Modification of mlRNA The mlRNA described herein can include one or more modifications useful, for example, for tracking, increasing stability, targeting the RNA to a specific intracellular location, or reducing immunogenicity. Modifications of the gRNA can be used to enhance the formation or stability of a DNA editing complex comprising the gRNA and an RNA-guided endonuclease (e.g., a Cas endonuclease such as Cas9 endonuclease). The modification of the gRNA can also or alternatively be used to enhance the initiation, stability, or kinetics of the interaction between the DNA editing complex and the target sequence within the target DNA, for example, to enhance the on-target activity. The modification of the gRNA can also or alternatively be used to increase specificity, e.g., the relative rate of DNA editing at the on-target site, as compared to the effect at other (off-target) sites.

[0113] The modification can also or alternatively be used to increase the stability of the guide RNA, for example, by increasing resistance to degradation by ribonucleases (RNases) present within the cell, thereby increasing the half-life within the cell.

[0114] An mlRNA according to any of the embodiments described herein can include a segment at either the 5' or 3' end that provides any of the above features. For example, suitable segments include riboswitch sequences (e.g., to enable regulated stability and / or regulated accessibility by proteins and protein complexes), stability control sequences, sequences that form dsRNA duplexes (i.e., hairpins), sequences that target the RNA to an intracellular location (e.g., nucleus, mitochondria, chloroplasts, etc.), modifications or sequences that provide for tracking (e.g., direct conjugation to a fluorescent molecule, conjugation to a moiety that facilitates fluorescence detection, sequences that enable fluorescence detection, etc.), modifications or sequences that provide a response to light or radiation (e.g., UV, vis, IR optogenetic elements), modifications or sequences that provide a binding site for a protein (e.g., a protein that acts on DNA, including transcriptional activators, transcriptional repressors, DNA methyltransferases, DNA demethylases, histone acetyltransferases, histone deacetylases, etc.), and combinations thereof.

[0115] An mlRNA according to any of the embodiments described herein can also include a modification that reduces the likelihood or extent of inducing an innate immune response when the RNA is introduced into a cell. As described below and in the art, such responses, which are well-characterized in the context of RNA interference (RNAi) including small interfering RNAs (siRNAs), tend to be associated with a reduction in the half-life of the RNA and / or the induction of cytokines or other factors related to the immune response.

[0116] An mlRNA according to any of the embodiments described herein can also include one or more modifications selected from modifications that enhance the stability of the RNA (e.g., by reducing its degradation by RNases in the cellular context) and modifications that reduce the likelihood or extent of inducing an innate immune response when the RNA is introduced into a cell. Combinations of modifications such as those described above and others can likewise be used.

[0117] Stability control sequence An mlRNA according to any of the embodiments described herein can include a stability control sequence that affects the stability of the RNA. Non-limiting examples of suitable stability control sequences are transcription terminator segments (e.g., transcription termination sequences). The transcription terminator segment of an RNA can have a total length of about 10 nucleotides to about 100 nucleotides, such as about 10 nucleotides (nt) to about 20 nt, about 20 nt to about 30 nt, about 30 nt to about 40 nt, about 40 nt to about 50 nt, about 50 nt to about 60 nt, about 60 nt to about 70 nt, about 70 nt to about 80 nt, about 80 nt to about 90 nt, or about 90 nt to about 100 nt. For example, the transcription terminator segment can have a length of about 15 nucleotides (nt) to about 80 nt, about 15 nt to about 50 nt, about 15 nt to about 40 nt, about 15 nt to about 30 nt, or about 15 nt to about 25 nt.

[0118] The transcription termination sequence can function in eukaryotic cells and / or prokaryotic cells.

[0119] Nucleotide sequences that can be included in a stability control sequence (e.g., a transcription termination segment, or any segment of an RNA that provides for improved stability) include, for example, the Rho-independent trp termination site.

[0120] Conjugate An mlRNA according to any of the embodiments described herein can include modifications that include chemically linking to the gRNA one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the RNA. These targeting moieties or conjugates can include conjugate groups covalently bound to a functional group such as a primary or secondary hydroxyl group. Conjugate groups include intercalating agents, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of the oligomer, and groups that enhance the pharmacokinetic properties of the oligomer. Suitable conjugate groups include, but are not limited to, cholesterol, lipids, phospholipids, biotin, phenazine, folic acid, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dyes. Groups that enhance the pharmacodynamic properties include groups that improve uptake, enhance resistance to degradation, and / or enhance sequence-specific hybridization to the target nucleic acid. Groups that enhance the pharmacokinetic properties include groups that improve the uptake, distribution, metabolism, or excretion of the nucleic acid.

[0121] The mlRNA according to any of the embodiments described herein may include a cholesterol moiety (Letsinger et al. (1989) Proc. Natl. Acad. Sci. U.S.A., 86(17):6553-6556), cholic acid (Manoharan et al. (1994) Bioorg. Med. Chem. Let., 4(8):1053-1060), thioethers such as hexyl-S-tritylthiol (Manoharan et al. (1992). Ann. N.Y. Acad. Sci., 660(1):306-309, and Manoharan et al. (1993) Bioorg. Med. Chem. Let., 3(12):2765-2770), thiocolesterol (Oberhauser et al. (1992) Nucl. Acids Res., 20(3):533-538), aliphatic chains such as 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 such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al. (1995) Tetrahedron Lett., 36(21):3651-3654; Shea et al. (1990) Nucl. Acids Res., 18(13):3777-3783)), polyamines or polyethylene glycol chains (Manohoran et al. (1995) Nucleos. Nucleot. Nucl., 14(3-5):969-973), adamantaneacetic acid (Manoharan et al. (1995) Tetrahedron Lett., 36(21):3651-3654), palmitoyl moiety (Mishra et al. (1995) Biochim. Biophys.Acta, 1264(2):229-237), or can include a chemically linked conjugate moiety including, but not limited to, a lipid moiety such as octadecylamine or hexylaminocarbonyl-t-oxy cholesterol moiety (Crooke et al. (1996) J. Pharmacol. Exp. Ther., 277(2):923-937).

[0122] An mlRNA according to any of the embodiments described herein may include a chemically linked conjugate that includes a "protein transduction domain" or PTD (also known as a cell-penetrating peptide, or CPP), and may refer to an organic or inorganic compound that facilitates passage through a polypeptide, polynucleotide, carbohydrate, or lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. A PTD conjugated to another molecule ranges from small polar molecules to large macromolecules and / or nanoparticles, and for example, facilitates the passage of molecules through membranes, moving from the extracellular space into the intracellular space, or from the cytosol into an organelle. The PTD can covalently bind to the gRNA. Exemplary PTDs include the minimal undecapeptide protein transduction domain (corresponding to residues 47-57 of HIV-1 TAT containing YGRKKRRQRRR (SEQ ID NO: 1)), polyarginine sequences containing a sufficient number of arginines to direct entry into cells (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines), the VP22 domain (Zender et al. (2002) Cancer Gene Ther., 9(6):489-496), the Drosophila antennapedia protein transduction domain (Noguchi et al. (2003) Diabetes, 52(7):1732-1737), the truncated human calcitonin peptide (Trehin et al. (2004) Pharm. Research, 21(7):1248-1256), polylysine (Wender et al. (2000) Proc. Natl. Acad. Sci. USA, 97(24):13003-13008), but are not limited thereto. The PTD can be an activatable CPP (ACPP) (Aguilera et al. (2009) Integr. Biol. (Camb), 1(5-6):371-381). ACPPs include a polycationic CPP (e.g., Arg9 or "R9") connected via a cleavable linker to a corresponding polyanion (e.g., Glu9 or "E9"), reducing the net charge to near zero, thereby inhibiting adhesion and uptake by cells.When the linker is cleaved, the polyanion is released, locally unmasking the polyarginine and its inherent adhesiveness and "activating" the ACPP to cross the membrane. The PTD can be chemically modified to enhance its bioavailability. Exemplary modifications are described in Mae et al. (2009) Expert Opin. Drug Deliv., 6(11):1195-1205.

[0123] The mlRNA according to any of the embodiments described herein can 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.

[0124] VI. RNA-Induced Endonuclease The methods disclosed herein for synthesizing gRNA generally include providing a first RNA fragment, a second RNA fragment, wherein the first RNA fragment, the second RNA fragment, or both include at least a portion of a sequence capable of binding to an RNA-induced endonuclease.

[0125] The RNA-guided endonuclease can be either naturally occurring or non-naturally occurring. 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 functional derivatives thereof. In some embodiments, the RNA-guided endonuclease is a Cas9 endonuclease. The Cas9 endonuclease can be derived from, for example, Streptococcus pyogenes (SpyCas9), Staphylococcus lugdunensis (SluCas9), or Staphylococcus aureus (SaCas9). In some cases, the RNA-guided endonuclease is a variant of Cas9, and the variant of Cas9 is selected from the group consisting of mini-Cas9, inactive Cas9 (dCas9), and Cas9 nickase.

[0126] The RNA-guided endonuclease can be a small RNA-guided endonuclease. The small RNA-guided endonuclease can be engineered from a portion of an RNA-guided endonuclease described herein or known in the art. The small RNA-guided endonuclease can be, for example, a small Cas endonuclease (e.g., PCT / US2018 / 065863, PCT / US2019 / 023044). In some cases, the small RNA-guided nuclease is, for example, smaller than about 1,100 amino acids in length.

[0127] The RNA-guided endonuclease can be a mutant RNA-guided endonuclease. For example, the RNA-guided endonuclease can be a variant of a naturally occurring RNA-guided endonuclease. The mutant RNA-guided endonuclease can also be a mutant RNA-guided endonuclease having altered activity compared to a naturally occurring RNA-guided endonuclease, such as altered endonuclease activity (e.g., altered or inhibited DNA endonuclease activity without substantially reducing the binding affinity to DNA). Such modifications enable sequence-specific DNA targeting of the mutant RNA-guided endonuclease for transcriptional regulation (e.g., activation or repression), epigenetic or chromatin modification by methylation, demethylation, acetylation or deacetylation, or other modifications of DNA-binding and / or DNA-modifying proteins known in the art. In some cases, the mutant RNA-guided endonuclease does not have DNA endonuclease activity.

[0128] The RNA-guided endonuclease may be a nickase that cleaves the complementary strand of the target DNA but has reduced ability to cleave the non-complementary strand of the target DNA, or that cleaves the non-complementary strand of the target DNA but has reduced ability to cleave the complementary strand of the target DNA. In some cases, the RNA-guided endonuclease has reduced ability to cleave both the complementary and non-complementary strands of the target DNA.

[0129] VII. Methods for synthesizing sgRNA In some embodiments, the present disclosure provides a method for synthesizing an sgRNA for use with an RNA-guided endonuclease. 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 variant. In some embodiments, the RNA-guided endonuclease is a small RNA-guided endonuclease. In some embodiments, the RNA-guided endonuclease is a small Cas endonuclease.

[0130] In some embodiments, the sgRNA comprises, from 5' to 3', a crRNA and a tracrRNA, and the crRNA and tracrRNA hybridize to form a double strand. In some embodiments, the crRNA comprises a spacer 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 inverted repeat 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 inverted repeat sequence, for example, by a tetraloop, and the crRNA repeat sequence and the tracrRNA inverted repeat sequence hybridize to form the sgRNA. In some embodiments, the sgRNA comprises, from 5' to 3', a spacer sequence, a crRNA repeat sequence, a tetraloop, a tracrRNA inverted repeat sequence, and a 3'tracrRNA sequence. In some embodiments, the sgRNA further comprises a 5' spacer 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. In some embodiments, the 3'tracrRNA consists of one, two, or three stem loops.

[0131] In some embodiments, the method comprises synthesizing the sgRNA using a splint-mediated ligation approach that includes two RNA fragments and one splint oligonucleotide. In some embodiments, the method comprises providing a complex formed between a first RNA fragment, a second RNA, and the splint oligonucleotide, and a ligase, wherein (a) the first RNA fragment includes a terminal region that includes a 3'-hydroxyl group, (b) the second RNA fragment includes a terminal region that includes a 5'-phosphate moiety, (c) the splint oligonucleotide includes (i) a first portion that is complementary to the terminal region of the first RNA fragment that includes the 3'-hydroxyl group, and (ii) a second portion that is complementary to the first terminal region of the second RNA fragment that includes the 5'-phosphate moiety, the complex is formed by hybridization of (a) and (c)(i) and hybridization of (b) and (c)(ii), the complex includes a ligation site that exists between the 3'-hydroxyl group of the first RNA fragment and the 5'-phosphate group of the second RNA fragment, the ligase effects ligation at the ligation 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 ligation forms an sgRNA that includes a spacer sequence and a constant sequence from 5' to 3', the constant sequence includes a double-strand formed between a crRNA repeat sequence and a tracrRNA reverse repeat sequence, and a 3'tracrRNA sequence that includes at least one stem-loop, thereby synthesizing the sgRNA. In some embodiments, the ligation site corresponds to a double-stranded site formed between the crRNA repeat sequence and the tracrRNA reverse repeat sequence. In some embodiments, the ligation site is in the crRNA repeat sequence, a tetraloop that binds the crRNA repeat sequence and the tracrRNA reverse repeat sequence, or the tracrRNA reverse repeat sequence. In some embodiments, the ligation site is within a stem-loop of the 3'tracrRNA sequence.In some embodiments, the first RNA fragment comprises the nucleotide sequence of the sgRNA that is 5' of the ligation site, and the second RNA fragment comprises the nucleotide sequence of the sgRNA that is 3' of the ligation site.

[0132] In some embodiments, the method includes synthesizing the sgRNA using a splint-mediated ligation approach that includes three RNA fragments and two splint oligonucleotides. In some aspects, the method includes providing a complex formed between a first RNA fragment, a second RNA fragment, a third RNA fragment, a first splint oligonucleotide, and a second splint oligonucleotide, and a ligase, wherein (a) the first RNA fragment includes a terminal region that includes a 3'-hydroxyl group, (b) the second RNA fragment includes (i) a first terminal region that includes a 5'-phosphate moiety and (ii) a second terminal region that includes a 3'-hydroxyl group, (c) the third RNA fragment includes a terminal region that includes a 5'-phosphate moiety, (d) the first splint oligonucleotide includes (i) a first portion complementary to the terminal region of the first RNA fragment that includes the 3'-hydroxyl group and (ii) a second portion complementary to the first terminal region of the second RNA fragment that includes the 5'-phosphate moiety, (e) the second splint oligonucleotide includes (i) a first portion complementary to the second terminal region of the second RNA fragment that includes the 3'-hydroxyl group and (ii) a second portion complementary to the terminal region of the third RNA fragment that includes the 5'-phosphate, 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 ligation site that exists between the 3'-hydroxyl group of the first RNA fragment and the 5'-phosphate group of the second RNA fragment, and a second ligation site that exists between the 3'-hydroxyl group of the second RNA fragment and the 5'-phosphate group of the third RNA fragment, and the ligase results in ligation at the first ligation 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 results in ligation at the second ligation site to,A phosphodiester bond is formed between the 3'-hydroxyl group of the second RNA fragment and the 5'-phosphate group of the third RNA fragment, and ligation forms an sgRNA containing a spacer sequence and a constant sequence from 5' to 3'. The constant sequence includes a double-stranded region formed between the crRNA repeat sequence and the tracrRNA reverse repeat sequence, and a 3'tracrRNA sequence containing at least one stem-loop, thereby synthesizing the sgRNA.

[0133] In some embodiments, the first ligation site corresponds to a double-stranded site formed between the crRNA repeat sequence and the tracrRNA reverse repeat sequence. In some embodiments, the first ligation site is in the crRNA repeat sequence, a tetraloop that binds the crRNA repeat sequence, the crRNA repeat sequence, and the tracrRNA reverse repeat sequence, or the tracrRNA reverse repeat sequence.

[0134] In some embodiments, the 3'tracrRNA sequence includes first, second, and third stem-loops. In some embodiments, the second ligation site corresponds to a site in the first stem-loop, the second stem-loop, or the third stem-loop. In some embodiments, the second ligation site corresponds to a site within the second stem-loop. In some embodiments, the second ligation site corresponds to a site in the 5'stem of the second stem-loop, a tetraloop of the second stem-loop, or a 3'stem of the second stem-loop. In some embodiments, the second ligation site is (i) directly adjacent to the base of the 5'stem of the second stem-loop, (ii) proximal to the base of the 5'stem of the second stem-loop (e.g., ±1 nt, ±2 nt, or ±3 nt from the base of the 5'stem), (iii) directly adjacent to the base of the 3'stem of the second stem-loop, or (iv) proximal to the base of the 3'stem of the second stem-loop (e.g., ±1 nt, ±2 nt, or ±3 nt from the base of the 3'stem).

[0135] In some embodiments, the first RNA fragment comprises the nucleotide sequence of the sgRNA that is 5' of the first ligation site, the second RNA fragment comprises the nucleotide sequence of the sgRNA that is 3' of the first ligation site and 5' of the second ligation site, and the third RNA fragment comprises the nucleotide sequence of the sgRNA that is 3' of the second ligation site.

[0136] In some embodiments, the first RNA fragment comprises, from 5' to 3', a spacer sequence and a portion of the crRNA repeat sequence of the sgRNA. In some embodiments, the first RNA fragment comprises, from 5' to 3', a spacer sequence and the crRNA repeat sequence of the sgRNA. In some embodiments, the first RNA fragment comprises, from 5' to 3', a spacer sequence, the crRNA repeat sequence, and a portion of the tetraloop of the sgRNA. In some embodiments, the first RNA fragment comprises, from 5' to 3', a spacer sequence, the crRNA repeat sequence, and the tetraloop of the sgRNA. In some embodiments, the first RNA fragment comprises, from 5' to 3', a spacer sequence, the crRNA repeat sequence, the tetraloop, and a portion of the tracrRNA repeat sequence of the sgRNA. In some embodiments, the first RNA fragment comprises, from 5' to 3', a spacer sequence, the crRNA repeat sequence, the tetraloop, and the tracrRNA repeat sequence of the sgRNA. In some embodiments, the terminal region of (a)(i) that is complementary to (d)(i) of the first splint oligonucleotide comprises a nucleotide sequence that is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 nucleotides in length, and the 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., the 5' end of the spacer sequence is aligned 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 and includes 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 sequence. In some embodiments, the first portion of (d)(i) of the first splint oligonucleotide is completely complementary to the terminal region of (a)(i).In some embodiments, the first portion of (d)(1) of the first sprint oligonucleotide has 1, 2, or 3 mismatches with respect to the terminal region of (a)(i).

[0137] In some embodiments, the second RNA fragment comprises, from 5' to 3', a crRNA repeat sequence, a tetraloop, a tracrRNA repeat sequence, and a portion of the 3'tracrRNA sequence of the sgRNA (i.e., the portion that is 5' relative to the second ligation). In some embodiments, the second RNA fragment comprises, from 5' to 3', a portion of the crRNA repeat sequence, a tetraloop, a tracrRNA repeat sequence, and a portion of the 3'tracrRNA sequence of the sgRNA (i.e., the portion that is 5' relative to the second ligation). In some embodiments, the second RNA fragment comprises, from 5' to 3', a tetraloop, a tracrRNA repeat sequence, and a portion of the 3'tracrRNA sequence of the sgRNA (i.e., the portion that is 5' relative to the second ligation). In some embodiments, the second RNA fragment comprises, from 5' to 3', a portion of the tetraloop, a tracrRNA repeat sequence, and a portion of the 3'tracrRNA sequence of the sgRNA (i.e., the portion that is 5' relative to the second ligation). In some embodiments, the second RNA fragment comprises, from 5' to 3', a tracrRNA repeat sequence, and a portion of the 3'tracrRNA of the sgRNA (i.e., the portion that is 5' relative to the second ligation). In some embodiments, the second RNA fragment comprises, from 5' to 3', a portion of the tracrRNA repeat sequence, and a portion of the 3'tracrRNA of the sgRNA (i.e., the portion that is 5' relative to the second ligation). In some embodiments, the terminal region (b)(i) that is complementary to the second part (d)(ii) of the first sprint oligonucleotide has a length 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 comprises a nucleotide sequence located at the 5' end of the second RNA fragment. In some embodiments, the second part (d)(ii) of the first sprint oligonucleotide is completely complementary to the terminal region of (b)(i) or has 1, 2, or 3 mismatches relative to the terminal region of (d)(ii).In some embodiments, the terminal region (b)(ii) of the second RNA fragment that is complementary to the first portion (e)(i) of the second sprint oligonucleotide has a length 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 includes a nucleotide sequence located at the 3' end of the second RNA fragment. In some embodiments, the first portion (e)(i) of the second sprint oligonucleotide is completely complementary to the terminal region of (b)(ii) or has 1, 2, or 3 mismatches with respect to the terminal region of (b)(ii).

[0138] In some embodiments, the third RNA fragment includes a portion of the 3'tracrRNA sequence of the sgRNA (i.e., 3' to the second ligation site). In some embodiments, the terminal region (c)(i) of the third RNA fragment that is complementary to the second portion (e)(ii) of the second sprint oligonucleotide has a length 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 includes a nucleotide sequence located at the 5' end of the third RNA fragment. In some embodiments, the second portion (e)(ii) of the second sprint oligonucleotide is completely complementary to the terminal region of (c)(i) or has 1, 2, or 3 mismatches with respect to the terminal region of (c)(i).

[0139] In some embodiments, the invariant sequence of the sgRNA comprises the nucleotide sequence of SEQ ID NO: 17, or a nucleotide sequence having a deletion, insertion, or substitution of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides relative to SEQ ID NO: 17. In some embodiments, the sgRNA is for use with the SpyCas9 endonuclease, and the invariant sequence of the sgRNA comprises the nucleotide sequence of SEQ ID NO: 17, or a nucleotide sequence having a deletion, insertion, or substitution of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides relative to SEQ ID NO: 17.

[0140] In some embodiments, the first RNA fragment, the second RNA fragment, and the third RNA fragment are each (a)(i)N 15-30 GUUUUAGAGCUAG (SEQ ID NO: 56) (where N 15-30 corresponds to a spacer sequence targeting a target site of a target nucleic acid (e.g., a genomic DNA molecule), (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 corresponds to a spacer sequence targeting a target site of a target nucleic acid (e.g., a genomic DNA molecule), (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 corresponds to a spacer sequence targeting a target site of a target nucleic acid (e.g., a genomic DNA molecule), (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 corresponds to a spacer sequence targeting a target site of a target nucleic acid (e.g., a genomic DNA molecule), (ii) SEQ ID NO: 59, and (iii) SEQ ID NO: 4, and is selected from the nucleotide sequences comprising

[0141] In some embodiments, the first split oligonucleotide comprises the sequence set forth 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 split oligonucleotide comprises a sequence complementary to the terminal region (a)(i) of the first RNA fragment, the terminal region extending from the 3' end of the first RNA fragment and comprising 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 sequence of the sgRNA, with the 5' end of the spacer sequence aligned with the 5' end of the first RNA fragment. In some embodiments, the first portion (d)(i) of the first split oligonucleotide comprises a sequence complementary to the terminal region (a)(i) of the first RNA fragment, the terminal region being directly adjacent to or 1, 2, or 3 nt downstream of the 3' end of the spacer sequence of the sgRNA, with the 5' end of the spacer sequence aligned with the 5' end of the first RNA fragment. In some embodiments, the second split oligonucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 6, SEQ ID NO: 45, or SEQ ID NO: 53.

[0142] In some embodiments, 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, about 30 to about 40 nucleotides in length.

[0143] 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 first splint oligonucleotide and the second splint 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 in length.

[0144] In some embodiments, the first, second, and / or third RNA fragments are synthesized in vitro according to the methods described herein, for example, using an RNA polymerase enzyme, or using solid-phase synthesis using phosphoramidite chemistry. In some embodiments, the RNA fragment is synthesized using phosphoramidite chemistry and includes (i) synthesis of the first RNA fragment, synthesis of the second RNA fragment, and synthesis of the third RNA fragment in the 5' to 3' or 3' to 5' direction, or (ii) synthesis of the first RNA fragment in the 5' to 3' or 3' to 5' direction, and synthesis of the second RNA fragment and the third RNA fragment in the 3' to 5' direction. In some embodiments, the RNA fragment is purified after synthesis.

[0145] In some embodiments, the first and / or second splint oligonucleotides are synthesized in vitro according to the methods described herein, for example, using an RNA polymerase enzyme, or using solid-phase synthesis using phosphoramidite chemistry. In some embodiments, the splint oligonucleotide is purified after synthesis.

[0146] In some embodiments, the first, second, and / or third RNA fragments comprise one or more modifications of the RNA backbone described herein, such as backbone linkages or nucleoside modifications. In some embodiments, the modification is a phosphorothioate linkage. In some embodiments, the modification is a 2'-O-methylation of the nucleoside.

[0147] 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, annealing comprises (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 used for ligation (e.g., about 30°C to about 40°C) at a rate of about 0.1°C to about 2°C per second.

[0148] 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, ligation is performed using a protease or a chelating agent. In some embodiments, ligation is performed using a crowding agent. In some embodiments, ligation proceeds 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 chromatography methods. The present disclosure relates to, for example, the following. [1] A method of synthesizing a guide RNA (gRNA), the method comprising To 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 at least a part of the first RNA fragment, the second RNA fragment, or both contains a sequence capable of binding to an RNA-induced endonuclease, and To provide a splint oligonucleotide comprising a first part complementary to the first RNA fragment in the terminal region containing the 5'-phosphate moiety and a second part complementary to the second RNA fragment in the terminal region containing the 3'-hydroxyl group, and To hybridize the first RNA fragment, the second RNA fragment, and the splint oligonucleotide together to form a complex, and To ligate the first and second RNA fragments using a ligase at a ligation site present between the RNA complexes, thereby synthesizing a gRNA, a method. [2] The method according to [1] above, wherein the lengths of the first and second RNA fragments are each 10 to 90 nucleotides. [3] The method according to [2] above, wherein the length of the second RNA fragment is 40 nucleotides or less. [4] The method according to any one of [1] to [3] above, wherein the 5'-phosphate moiety is 5'-phosphate or 5'-phosphorothioate. [5] The method according to any one of [1] to [4] above, wherein the ligase is T4 DNA ligase, T4 RNA ligase I, or T4 RNA ligase II. [6] The method according to any one of [1] to [5] above, wherein the splint oligonucleotide is a DNA or RNA oligonucleotide. [7] The method according to any one of [1] to [6], wherein the length of the split oligonucleotide is 20 to 100 nucleotides. [8] The method according to any one of [1] to [7], wherein the split oligonucleotide is bound to a solid support. [9] The method according to any one of [1] to [8], wherein the length of the gRNA is 30 to 160 nucleotides.

[10] The method according to any one of [1] to [9], wherein the gRNA contains a sequence complementary to a sequence in the target DNA.

[11] The method according to

[10] , wherein the target DNA is mammalian DNA.

[12] The method according to

[11] , wherein the target DNA is human DNA.

[13] The method according to any one of [1] to

[12] , wherein the ligation site corresponds to a site in the tetraloop portion of the stem-loop structure of the synthesized gRNA.

[14] The method according to any one of [1] to

[12] , wherein the ligation site corresponds to a site in the helix portion of the stem-loop structure of the synthesized gRNA.

[15] The method according to any one of [1] to

[14] , wherein 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 split oligonucleotide results in a lower free energy than that of the secondary structure having the lowest free energy.

[16] The method according to any one of [1] to

[15] , comprising ligating three or more RNA fragments.

[17] The method according to any one of [1] to

[16] above, wherein providing the first and second RNA fragments comprises synthesizing the first and second RNA fragments via enzymatic synthesis or phosphoramidite chemistry.

[18] The method according to

[17] above, wherein the second RNA fragment is synthesized in the 5' to 3' or 3' to 5' direction.

[19] The method according to

[17] or

[18] above, wherein providing the first and second RNA fragments comprises purifying the first and second fragments after synthesis.

[20] The method according to any one of [1] to

[19] above, wherein providing the sprint oligonucleotide comprises synthesizing the sprint oligonucleotide via enzymatic synthesis or phosphoramidite chemistry.

[21] The method according to

[20] above, wherein providing the sprint oligonucleotide comprises purifying the sprint oligonucleotide after synthesis.

[22] The method according to

[19] or

[21] above, wherein purifying comprises purifying by a chromatography method.

[23] The method according to

[22] above, wherein the chromatography method is reverse 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 [1] to

[23] above, 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

[24] above, wherein the modification is selected from the group consisting of 2'-methoxy (2'-OMe), 2'-fluorine (2'-fluoro), 2'-O-methoxy-ethyl (MOE), locked nucleic acid (LNA), unlocked nucleic acid (UNA), bridged nucleic acid, 2'-deoxy nucleic acid (DNA), and peptide nucleic acid (PNA).

[26] The method according to any one of [1] to

[25] above, wherein the first RNA fragment, the second RNA fragment, or both contain at least one base modification.

[27] The method according to

[26] above, 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 [1] to

[27] above, wherein the first RNA fragment, the second RNA fragment, or both contain at least one phosphorothioate bond.

[29] The method according to any one of [1] to

[28] above, wherein hybridizing comprises hybridizing in solution.

[30] The method according to

[29] above, wherein the concentration of the splint oligonucleotide, the concentration of the first RNA fragment, and the concentration of the second RNA fragment in the solution are substantially equal.

[31] The method according to any one of [1] to

[30] above, wherein ligating the first and second RNA fragments is carried out at 15°C to 45°C.

[32] The method according to

[31] above, wherein ligating the first and second RNA fragments is carried out at about 37°C.

[33] The method according to any one of [1] to

[32] above, wherein ligating the first and second RNA fragments is carried out for about 0.1 to about 48 hours.

[34] The method according to any one of [1] to

[33] above, wherein ligating the first and second RNA fragments further comprises using a protease or a chelating agent.

[35] The method according to

[34] above, wherein the chelating agent is EDTA, EGTA, or a combination of both.

[36] The method according to any one of [1] to

[35] above, wherein ligating the first and second RNA fragments further comprises using one or more crowding agents.

[37] The method according to

[36] above, wherein the one or more crowding agents comprise polyethylene glycol (PEG), Ficoll®, ethylene glycol, dextran, or any combination thereof.

[38] The method according to any one of [1] to

[37] above, wherein ligating the first and second RNA fragments proceeds to at least 10% completion.

[39] The method according to

[38] above, wherein ligating the first and second RNA fragments proceeds to at least 90% completion.

[40] A method for synthesizing a guide RNA (gRNA), the method comprising: (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 group; (d) (i) a first splint oligonucleotide comprising 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; (e)(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 portion of the third RNA fragment, of a second splint oligonucleotide; (f) a ligase, and comprising providing, Hybridizing the first, second, and third RNA fragments, and the first and second splint oligonucleotides, results in the formation of a complex having a first ligation 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 ligation site present between the 3'-hydroxyl group of the second RNA fragment and the 5'-phosphate group of the third RNA fragment; A method wherein the ligase results in ligation of the first and second RNA fragments at the first ligation site and the second and third RNA fragments at the second ligation site, thereby synthesizing a gRNA.

[41] The method according to

[40] , wherein the gRNA comprises, from 5' to 3', the first RNA fragment linked to the second RNA fragment by a first phosphodiester bond, and the second RNA fragment linked to the third RNA fragment by a second phosphodiester bond.

[42] The method according to

[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 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

[40] to

[42] , wherein the gRNA is a single-guide RNA (sgRNA).

[44] The method according to any one of

[40] to

[43] , wherein the gRNA has a length of about 30 to about 160 nucleotides.

[45] The method according to any one of

[40] to

[44] , wherein the first ligation site corresponds to a site of a first stem-loop structure, and the first stem-loop structure is formed by hybridization of a minimum CRISPR repeat sequence and a minimum tracrRNA sequence in the gRNA.

[46] The method according to

[45] , wherein the site of the first stem-loop structure is in a tetraloop portion or a helix portion.

[47] The method according to any one of

[40] to

[46] , wherein the second ligation site corresponds to a site of a second stem-loop structure, and the second stem-loop structure is present in the tracrRNA sequence of the gRNA.

[48] The method according to

[47] , wherein the site of the second stem-loop structure is in a tetraloop portion or a helix portion.

[49] The method according to any one of

[40] to

[48] , wherein the first RNA fragment, the second RNA fragment, and / or the third RNA fragment includes at least one secondary structure, and the complex formed by hybridizing the first, second, and third RNA fragments and the first and second sprint oligonucleotides has a free energy lower than that of the secondary structure having the lowest free energy.

[50] A method for synthesizing a single molecule guide RNA (sgRNA) for use 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 sprint oligonucleotide, and a second sprint oligonucleotide, and a ligase. (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 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, (e) The second splint 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 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 ligation 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 ligation site present between the 3'-hydroxyl group of the second RNA fragment and the 5'-phosphate group of the third RNA fragment, The method, wherein the ligase effects ligation at the first ligation site and at the second ligation site to form, 5' to 3', an invariant sequence that binds to a spacer sequence and an RNA-guided endonuclease, the invariant sequence comprising a stem-loop formed between a crRNA repeat sequence and a tracrRNA reverse repeat sequence, and a 3' tracrRNA sequence comprising at least one stem-loop, thereby synthesizing the sgRNA for use with the RNA-guided endonuclease.

[51] The method according to

[50] , wherein the first ligation site corresponds to a site within a stem-loop formed between a crRNA repeat sequence and a tracrRNA reverse repeat sequence.

[52] The method according to

[51] , wherein the first ligation site corresponds to a site in the 5' stem of the stem-loop, the tetraloop of the stem-loop, or the 3' stem of the stem-loop.

[53] The method according to any one of

[50] to

[53] , wherein the 3' tracrRNA sequence comprises a first stem-loop, a second stem-loop, and a third stem-loop.

[54] The method according to

[53] , wherein the second ligation site corresponds to a site in the first stem-loop, the second stem-loop, or the third stem-loop.

[55] The method according to

[53] or

[54] , wherein the second ligation site corresponds to a site in the second stem-loop, and the site is in the 5' stem of the second stem-loop, a site within the tetraloop of the second stem-loop, or the 3' stem of the second stem-loop.

[56] The method according to

[53] or

[54] , wherein the second ligation site corresponds to a site adjacent to the 5' base of the second stem-loop or a site adjacent to the 3' base of the second stem-loop.

[57] The method according to any one of

[50] to

[56] , wherein the first RNA fragment comprises a nucleotide sequence that is 5' of the first ligation site.

[58] The method according to any one of

[50] to

[57] , wherein the second RNA fragment comprises a nucleotide sequence that is between the first ligation site and the second ligation site.

[59] The method according to any one of

[50] to

[58] , wherein the third RNA fragment comprises a nucleotide sequence that is 3' to the second ligation site.

[60] The method according to any one of

[50] to

[59] , wherein 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.

[61] The method according to

[60] , wherein the terminal region of (a)(i) comprises the spacer sequence of the sgRNA.

[62] The method according to

[60] , wherein the terminal region of (a)(i) does not comprise the spacer sequence of the sgRNA.

[63] The method according to any one of

[50] to

[62] , wherein the first part of (d)(i) is completely complementary to the terminal region of (a)(i) or has 1, 2, or 3 mismatches to the terminal of (a)(i).

[64] The method according to any one of

[50] to

[63] , wherein the first 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.

[65] The method according to any one of

[50] to

[64] , wherein the second part of (d)(ii) is completely complementary to the first terminal region of (b)(i) or has 1, 2, or 3 mismatches with respect to the terminus of (d)(ii).

[66] The method according to any one of

[50] to

[65] , wherein the second terminal region of (b)(ii) comprises a nucleotide sequence of about 10 to about 30 nucleotides located at the 3' terminus of the second RNA fragment.

[67] The method according to any one of

[50] to

[66] , wherein the first part of (e)(i) is completely complementary to the second terminal region of (b)(ii) or has 1, 2, or 3 mismatches with respect to the terminus of (b)(ii).

[68] The method according to any one of

[50] to

[67] , wherein the terminal region of (c)(i) comprises a nucleotide sequence of about 10 to about 40 nucleotides located at the 5' terminus of the third RNA fragment.

[69] The method according to any one of

[50] to

[68] , wherein the second part of (e)(ii) is completely complementary to the terminal region of (c)(i) or has 1, 2, or 3 mismatches with respect to the terminus of (c)(i).

[70] The method according to any one of

[40] to

[69] , wherein the first RNA fragment, the second RNA fragment, and the third RNA fragment are each independently of a length of 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, about 30 to about 40 nucleotides.

[71] The method according to any one of

[40] to

[70] , wherein the ligase is T4 DNA ligase, T4 RNA ligase I, or T4 RNA ligase II.

[72] The method according to any one of

[40] to

[71] , wherein the first sprint oligonucleotide is a DNA or RNA oligonucleotide, and the second sprint oligonucleotide is a DNA or RNA oligonucleotide.

[73] The method according to any one of

[40] to

[72] , wherein the first sprint oligonucleotide and the second sprint 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 in length.

[74] The method according to any one of

[40] to

[73] , wherein the gRNA or the sgRNA comprises a spacer sequence complementary to the sequence of the target DNA.

[75] The method according to

[74] , wherein the target DNA is mammalian DNA or human DNA.

[76] The method according to any one of [1] to

[75] , wherein the RNA-guided endonuclease is a small Cas nuclease or a small RNA-guided endonuclease.

[77] The method according to any one of [1] to

[75] , wherein the RNA-guided endonuclease is selected from the group consisting of Cas9, Cas12, Cas13, and variants thereof.

[78] The method according to

[77] , wherein the RNA-guided endonuclease is Streptococcus pyogenes Cas9 (SpyCas9) or Staphylococcus aureus (SaCas9).

[79] The RNA-guided endonuclease is a variant of Cas9, and the variant of Cas9 is selected from the group consisting of small Cas9, inactive Cas9 (dCas9), and Cas9 nickase, and the method according to any one of [1] to

[75] above.

[80] The RNA-guided endonuclease is Streptococcus pyogenes Cas9 (SpyCas9), and the method according to any one of

[50] to

[75] above.

[81] The invariant sequence includes the nucleotide sequence of SEQ ID NO: 17, or a nucleotide sequence having a deletion, insertion, or substitution of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides relative to SEQ ID NO: 17, and the method according to

[80] above.

[82] The first RNA fragment, the second RNA fragment, and the third RNA fragment are each, (a)(i)N 15-30 GUUUUAGAGCUAG (SEQ ID NO: 56) (N 15-30 corresponds 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) (N 15-30 corresponds to the spacer sequence), (ii) SEQ ID NO: 40, and (iii) SEQ ID NO: 42, (c)(i)N 15-30 GUUUUAGAGCUAG (SEQ ID NO: 56) (N 15-30 corresponds to the spacer sequence), (ii) SEQ ID NO: 58, and (iii) SEQ ID NO: 42, or (d)(i)N 15-30 GUUUUAGAGCUAGA (SEQ ID NO: 57) (N 15-30 corresponds to the spacer sequence), (ii) SEQ ID NO: 59, and (iii) The method according to

[80] or

[81] , which is selected from the nucleotide sequences including SEQ ID NO: 4.

[83] The method according to any one of

[80] to

[82] , wherein the first split oligonucleotide comprises the nucleotide sequence shown in SEQ ID NO: 60, SEQ ID NO: 44, or SEQ ID NO: 61.

[84] The method according to

[83] , wherein no part of the first split oligonucleotide is complementary to the spacer sequence.

[85] The method according to

[83] , further comprising a 3' end having a nucleotide sequence that is complementary to the spacer sequence or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides present at the 3' end of the spacer sequence, wherein the first split oligonucleotide is complementary thereto.

[86] The method according to any one of

[81] to

[85] , wherein the second split oligonucleotide comprises the nucleotide sequence shown in SEQ ID NO: 6, SEQ ID NO: 45, or SEQ ID NO: 53.

[87] The method according to any one of

[40] to

[86] , wherein providing the first RNA fragment, the second RNA fragment, and the third RNA fragment includes synthesizing the RNA fragments using enzymatic synthesis or phosphoramidite chemistry, and optionally purifying the RNA fragments after synthesis.

[88] The synthesis of the RNA fragments using phosphoramidite chemistry is (i) synthesizing the first RNA fragment, the second RNA fragment, and the third RNA fragment in the 5' to 3' or 3' to 5' direction, or (ii) synthesizing the first RNA fragment in the 5' to 3' or 3' to 5' direction, and synthesizing the second RNA fragment and the third RNA fragment in the 3' to 5' direction, the method according to

[87] .

[89] Providing the first and second splint oligonucleotides includes synthesizing the oligonucleotides using enzymatic synthesis or phosphoramidite chemistry, and optionally purifying the oligonucleotides after synthesis, according to any one of the preceding items

[40] to

[88] .

[90] The method according to any one of the preceding items

[40] to

[89] , wherein the first RNA fragment, the second RNA fragment, and / or the third RNA fragment comprises at least one modification in the RNA backbone.

[91] The method according to item

[90] , wherein 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), bridged nucleic acid, 2'-deoxy nucleic acid (DNA), and peptide nucleic acid (PNA).

[92] The method according to any one of the preceding items

[40] to

[91] , wherein the first RNA fragment, the second RNA fragment, and / or the third RNA fragment comprises at least one base modification.

[93] The method according to item

[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 any one of the preceding items

[40] to

[93] , wherein the first RNA fragment, the second RNA fragment, and / or the third RNA fragment comprises at least one phosphorothioate bond.

[95] Hybridizing is performed in solution, and hybridizing is performed regardless of the presence or absence of an annealing step, according to any one of the preceding items

[40] to

[94] .

[96] 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, and (ii) cooling the solution to the temperature used for the ligation at a rate of about 0.1°C to about 2°C per second, the method according to

[95] .

[97] The method according to

[95] or

[96] , wherein the concentrations of the first split oligonucleotide, the second split oligonucleotide, the first RNA fragment, the second RNA fragment, and the third RNA fragment in the solution are substantially equal.

[98] The method according to any one of

[40] to

[97] , wherein the ligation is carried out 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.

[99] The method according to any one of

[40] to

[98] , wherein the ligation is carried out for about 0.1 to about 48 hours, or about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 hours.

[0100] The method according to any one of

[40] to

[99] , wherein the ligation further comprises using a protease or a chelating agent.

[0101] The method according to

[0100] , wherein the chelating agent is EDTA, EGTA, or a combination of both.

[0102] The method according to any one of

[40] to

[0101] , wherein the ligation further comprises using one or more crowding agents.

[0103] The method according to

[0102] , wherein the one or more crowding agents include polyethylene glycol (PEG), ficoll (registered trademark), ethylene glycol, dextran, or any combination thereof.

[0104] The method according to any one of the above

[40] to

[0103] , wherein the ligation proceeds until at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% is completed.

[0105] The method according to any one of the above [1] to

[0104] , further comprising purifying the gRNA or the sgRNA after synthesis.

[0106] The method according to

[0105] , wherein purifying the gRNA or sgRNA comprises purifying using a chromatography method.

[0107] The method according to

[0106] , wherein the chromatography method is reverse phase HPLC, ion exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, affinity chromatography, or polyacrylamide gel purification, or any combination thereof.

[0108] A method for generating a bimolecular gRNA comprising crRNA and tracrRNA, the method comprising: providing a first RNA fragment comprising a terminal region comprising a 5' phosphate moiety and a second RNA fragment comprising a terminal region comprising 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; providing a splint oligonucleotide comprising a first portion complementary to the first RNA fragment at the terminal region comprising the 5' phosphate moiety and a second portion complementary to the second RNA fragment at the terminal region comprising the 3' hydroxyl group; hybridizing the first RNA fragment, the second RNA fragment, and the splint oligonucleotide together to form a complex; Ligating the first and second RNA fragments using a ligase at a ligation site present between the RNA complexes, thereby synthesizing tracrRNA; Providing a crRNA comprising a sequence complementary to a sequence in a target DNA; Enabling the tracrRNA and the crRNA to hybridize, thereby generating a bimolecular gRNA, the method comprising.

[0109] The method according to

[0108] , wherein providing the crRNA comprises synthesizing the crRNA via enzymatic synthesis or phosphoramidite chemistry.

Example

[0149] The practice of the present invention uses conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology that are known in the art, unless otherwise indicated. 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, Ferre & 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 supplements up to 2014), and Makrides (2003) Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences B.V., the disclosures of which are hereby incorporated by reference.

[0150] Additional embodiments are disclosed in more detail in the following examples, which are provided by way of example and are in no way intended to limit the present disclosure or the claims.

[0151] Example 1: Sprint-Mediated Ligation of Exemplary gRNAs To demonstrate that full-length gRNA products can be created using sprint-mediated ligation with chemically synthesized RNA fragments, this method was used to synthesize an exemplary gRNA molecule targeting a single locus in the mouse genome. The gRNA was divided into three RNA fragments, each having a length of less than 40 nucleotides (Figure 4). The sequences of the three RNA fragments and two DNA sprint oligonucleotides are shown in Table 1. Positions for segmenting the gRNA were chosen to remove internal hairpins that would interfere with hybridization to the DNA sprint oligonucleotides. These positions for segmenting the gRNA were in the invariant region of the gRNA (i.e., downstream of the spacer or variable region), so these positions can be used for any gRNA used by a Cas enzyme derived from Streptococcus pyogenes (SpyCas or SpCas) such as SpyFi, and only the RNA at the 5' end position of the gRNA construct (e.g., RNA1 in Table 1) and its corresponding sprint (e.g., Splint1->2 in Table 1) need to be uniquely synthesized for different genomic targets. [Table 1]

[0152] Selection Criteria for gRNA Segmentation Under biological conditions, RNAs associated with the Cas protein family have internal structures that can prevent hybridization to complementary split oligonucleotides. For example, when assembled with the Cas protein, the gRNA associated with SpCas9 has four stem-loops (hairpins), and the gRNA associated with saCas9 has three stem-loops. These stem-loops, particularly the two tetraloops, are assumed to be present when the gRNA is not bound to the Cas protein. To minimize the intramolecular association of RNA fragments that prevent hybridization to the split, the location of gRNA segmentation was selected within these stem-loop motifs to disrupt these energetically 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 type of bases and the number of nucleotides forming the loop) regions. Cleavage within the loop region is generally preferred as it removes the stem-loop. Cleavage within the helix is also compatible with this approach, and free energy calculations were used to ensure proper binding of the RNA fragment to the split.

[0153] Selection of DNA Split Oligonucleotides The length of the DNA split oligonucleotide was selected to promote duplex formation between the split and the RNA fragment rather than the formation of intramolecular structures within the RNA fragment. This was achieved in silico by comparing the energy of the internal structure to the energy of the duplex formed between the split and the RNA fragment pre-ligated to the split. The minimum free energy prediction algorithm (mFold) was used to calculate the free energy (ΔG intra ) of the RNA secondary structure of the individual fragments and the intermolecular hybridization (ΔG inter) was calculated. The length of the sprint was extended until the criteria described below were met to ensure that the fragments were well - ligated to the sprint during ligation.

[0154] The free energy of the intramolecular structure was set to be greater than that of the intermolecular structure and is inversely proportional to the melting temperature (T m ). ΔG intra >ΔG inter ~T m-intra <T m-inter

[0155] In addition, the temperature (T rxn ) at which the ligation reaction is carried out was set lower than the T m of the RNA / DNA sprint complex. T rxn <T m-inter

[0156] DNA sprint oligonucleotides were used because i) T4 RNA ligase II can use DNA / RNA heteroduplexes for ligation and ii) DNA can be made more easily and inexpensively than RNA. However, the sprint can also be synthesized from RNA, non - natural nucleic acids, artificial nucleic acids (e.g., peptide nucleic acids), or any nucleic acid mimics. The program used to determine free energy and stability is accessible at the URL: unafold.rna.albany.edu / ?q = mfold.

[0157] Chemical synthesis and purification of RNA fragments RNA fragments and DNA splint oligonucleotides were synthesized using standard phosphoramidite chemistry that extends in the 3’ to 5’ direction. Since one of the substrates for T4 RNA ligase is a 5’ phosphorylated RNA oligomer, all RNA fragments except for the fragment that becomes the 5’ end of the gRNA (e.g., RNA1 in Table 1) were synthesized with a terminal 5’ phosphate. All RNA fragments were purified by reverse-phase HPLC, ion-exchange chromatography, or PAGE prior to use. Since phosphorylation is the final coupling step during the synthesis of these RNAs, truncation products from their synthesis are not incorporated during enzymatic ligation. The only truncation products that can be incorporated are those derived from the 5’ end fragment. For this reason, the 5’ end fragment is purified prior to ligation, and it is advantageous to design this fragment to be less than 40 nucleotides. In some cases, the 5’ end fragment is synthesized in the 5’ to 3’ direction and the other fragments are synthesized in the 3’ to 5’ direction so that cleavage products from the synthesis of the 5’ end fragment are not included in the final ligation product.

[0158] Modified oligonucleotide It is also possible to synthesize gRNAs using the splint-mediated ligation described herein using RNA fragments having non-natural (modified) ribophosphate backbones. As an example, ligation of RNA fragments having a methoxy substitution of the 2’ hydroxyl group at the 3’ end ligation site or 1 to 2 nucleotides away from the 3’ end ligation site was achieved. These RNA fragments also included phosphorothioate linkages more than 20 nucleotides away from the ligation site. These modifications may also be tolerated at or near the ligation site.

[0159] Ligation reaction The RNA fragment and DNA splint were mixed in a T4 RNA ligase II reaction buffer (New England Biolabs (NEB)) at concentrations of 10 or 20 μM each. The concentrations of the RNA fragment and DNA splint were substantially equal. The solution was heated at 90 °C for 3 minutes and cooled to 37 °C at a rate of 1 °C / second to disrupt the internal structure of the RNA fragment and enable annealing of the DNA splint and RNA fragment. Next, T4 RNA ligase II was added to the solution and incubated at 37 °C for 0.5 - 24 hours. As defined by NEB, 1 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 RNAs in a total reaction volume of 20 μL at 37 °C for 30 minutes. The ligation reaction can also function at other temperatures, such as 25 °C, the reaction time can be extended, and the reaction can be quenched using protease or EDTA. A crowding agent (e.g., PEG) can be added to the ligation reaction.

[0160] Isolation of the full-length product Ion-exchange high-performance liquid chromatography (HPLC) was used to isolate the full-length gRNA product from the DNA splint oligonucleotide and unligated RNA 1, 2, or 3 fragments. Figure 5 is an HPLC chromatogram showing the presence of oligonucleotides before (upper panel) and after (lower panel) the ligation reaction. The presence of the ligation products RNA2-3, RNA1-2, and full-length gRNA (RNA 1-2-3) was detected.

[0161] The full-length gRNA product was tested in combination with SpCas9 using a plasmid containing the target DNA sequence. As shown in Figure 6, the plasmid was cleaved by SpCas9 at the appropriate target site.

[0162] These results demonstrate that full-length gRNA products can be created using chemically synthesized RNA fragments and splint-mediated ligation.

[0163] Example 2: Split-mediated ligation of modified RNAs To examine whether RNA fragments containing modifications can be ligated using split-mediated ligation, the modified RNAs (RNA2m1 and RNA2m2) shown in Table 2 below were ligated using DNA splint oligonucleotides Splint1 and Splint2. Figure 7A shows the chemical structures of unmodified RNA and exemplary modifications. [Table 2]

[0164] Figure 7B is a chromatogram showing the results from HPLC analysis of the ligation products. The ligation products RNA2m1-RNA3, RNA1-RNA2m1, and RNA2m2-RNA3 were detected.

[0165] Next, RNA1, RNA2m1 (shown as RNA2 in Table 3 and Figure 8 below), and RNA3 were ligated to generate full-length gRNA using Splint1 and Splint2. [Table 3]

[0166] The full-length gRNA product was isolated from the DNA splint oligonucleotides and partial ligation products (RNA2-RNA3 and RNA1-RNA2) using HPLC. Figure 8 is a chromatogram showing the results from HPLC analysis of the ligation products before and after purification. The full-length RNA product (RNA1-2-3 gRNA) was detected, and the purified full-length product is shown in the lower panel. These results demonstrate that RNA fragments having methylation modifications around the ligation site can be ligated using the split-mediated ligation described in the present disclosure.

[0167] While certain alternative embodiments of the present disclosure have been disclosed, various modifications and combinations are possible and should be understood to be within the true spirit and scope of the appended claims. Accordingly, there is no intention to limit the exact summary and disclosure presented herein.

[0168] Example 3: Evaluation of Reaction Conditions for Split-Mediated Ligation Experimental conditions for performing the split-mediated ligation reaction of modified RNAs were compared. This included an evaluation of the use of (i) addition of magnesium salts to the reaction and (ii) a thermal annealing step prior to performing the ligation reaction.

[0169] The split-mediated ligation was designed to prepare a spacer sequence targeting exon 2 of the human G6PC gene and a final modified sgRNA product having a backbone suitable for use with SpyCas9. The sequences of the modified sgRNA products and their corresponding unmodified versions are shown in Table 4. In the split-mediated ligation, three RNA fragments and two DNA split oligonucleotides were used, as shown in Table 4 respectively. The RNA fragments were (i) 33mer RNA1 (SEQ ID NO: 11) containing, in the 5’ to 3’ direction, the spacer sequence, the crRNA repeat sequence of the final sgRNA product, and the first guanine of the tetraloop of the final sgRNA product, and (ii) 39mer RNA2 (SEQ ID NO: 12) containing, in the 5’ to 3’ direction, the “AAA” of the tetraloop of the final sgRNA product and the 5’ segment of the tracrRNA, and (iii) 28mer RNA3 (SEQ ID NO: 13) containing the 3’ segment of the tracrRNA of the final sgRNA product.

[0170] As shown in Fig. 9A, the DNA splint 1 oligonucleotide (SEQ ID NO: 5) was designed using segments complementary to the 3’ segment of RNA1 and the 5’ segment of RNA2. Specifically, the sequence 5’-CTAGCTCTAAAACTC-3’ (SEQ ID NO: 22) of DNA splint 1 is complementary to the sequence 5’-GUGUUUUAGAGCUAG-3’ (SEQ ID NO: 23) of RNA1, and the sequence 5’-CCTTATTTTAACTTGCTATTT-3’ (SEQ ID NO: 24) of DNA splint 1 is complementary to the sequence 5’-AAAUAGCAAGUUAAAAUAAGG-3’ (SEQ ID NO: 25) of RNA2.

[0171] In addition, the DNA splint 2 oligonucleotide (SEQ ID NO: 6) was designed using segments complementary to the 3’ segment of RNA2 and the 5’ segment of RNA3. Specifically, the sequence 5’-AAGTTGATAACGGACTAG-3’ (SEQ ID NO: 26) of DNA splint 2 is complementary to the sequence 5’-CUAGUCCGUUAUCAACUU-3’ (SEQ ID NO: 27) of RNA2, and the sequence 5’-AAAAGCACCGACTCGGTGCCACTTTTTC-3’ (SEQ ID NO: 28) of DNA splint 2 is complementary to the sequence 5’-GAAAAAGUGGCACCGAGUCGGUGCUUUU-3’ (SEQ ID NO: 29) of RNA3.

[0172] As shown in Fig. 9B, the ligation of the RNA fragments occurs at the first ligation site between RNA fragment 1 and RNA fragment 2 located in the GAAA tetraloop of the repetitive inverted repeat stem-loop formed between the crRNA and the tracrRNA, and at the second ligation site between RNA fragment 2 and RNA fragment 3 adjacent to the GAAA tetraloop of the second stem-loop of the tracrRNA. The DNA splint 1 oligonucleotide shown in SEQ ID NO: 5 is complementary to the segment of RNA1 but has one mismatch. The DNA splint 1 oligonucleotide shown in SEQ ID NO: 14 is complementary to the same part of RNA1 but has no mismatch and is also suitable for use in the ligation reaction.

Table 4

[0173] Each oligonucleotide was dissolved in water at a concentration of 1 mM. An equimolar ratio mixture of RNA fragments and DNA splints was prepared. Next, the RNA / DNA mixture was heated at 90 °C for 3 minutes and cooled to 37 °C at a rate of 1 °C / second to disrupt the internal structure of the RNA fragment and anneal to form a DNA / RNA hybrid structure. ("With annealing"). Alternatively, this step was skipped ("Without annealing step").

[0174] Next, the RNA / DNA mixture with or without annealing was diluted with T4 RNA ligase II reaction buffer (New England Biolabs (NEB)) containing 50 U of T4 RNA ligase II at a final concentration of 10 μM per each RNA and DNA splint oligonucleic acid. The ligation reaction was prepared by adding MgCl 2 to a final concentration of 12.5 mM, or without adding MgCl 2 . The solution was incubated at 37 °C for 16 hours. The reaction was stopped by adding proteinase K or quenching with EDTA. Next, the reaction mixture was analyzed by ion exchange HPLC for the presence of full-length gRNA product and partial ligation products (RNA2-RNA3 and RNA1-RNA2).

[0175] As shown in Figure 10, in the ligation reaction of each condition evaluated, the full-length RNA product (RNA1-2-3 gRNA, retention time 30.58 minutes) was detected. The results indicate that the splint-mediated ligation reaction of the modified RNA fragment can be achieved without the first annealing step and can be carried out normally regardless of the addition of magnesium salts.

[0176] Example 4: Design Variations for Achieving Splint-Mediated Ligation Reaction Additional designs were developed to prepare the final sgRNA product (the modified sequence shown in SEQ ID NO: 20) described in Example 3 using a splinted ligation reaction. This design is based on the ligation of three RNA fragments using two splint oligonucleotides. One difference in the design is the degree of complementarity between the first splint oligonucleotide and the first RNA fragment. As further described below, the first RNA fragment contains a variable spacer sequence and a portion of the invariant sequence of the final sgRNA product. The first design has a first splint oligonucleotide that is complementary only to the invariant segment of the first RNA fragment, and the second design has a first splint oligonucleotide that is complementary to both the invariant segment and a portion of the variable segment. As a result, the first design provides a set of components in which only the first RNA fragment is changed to prepare sgRNAs with different target specificities. Based on the second design, both the first RNA fragment and the first splint oligonucleotide are changed to prepare sgRNAs with different target specificities. However, the advantage of this design is that the overlap between the first splint oligonucleotide and the first RNA fragment increases, increasing the stability (i.e., melting temperature) of the RNA / DNA heteroduplex formed between these components and promoting heteroduplex formation at the temperature used for the ligation reaction (e.g., 37°C).

[0177] The first design includes the RNA fragments shown in Table 5, shown as either the modified or unmodified versions.. Specifically, the RNA fragments are (i) 34mer RNA1 incorporating the spacer sequence (SEQ ID NO: 20), the crRNA repeat sequence, and a portion of the tetraloop of the final repeat-inverted repeat stem-loop from 5' to 3', and (ii) 34mer RNA2, which includes the remaining part of the tetraloop of the repetitive inverted repeat stem-loop, the tracrRNA inverted repeat sequence, and a part of tracrRNA extending to the bases of the second stem-loop, and (iii) 32mer RNA3, which includes the remaining 3’ part of tracrRNA.

[0178] DNA splint 1 oligonucleotide (SEQ ID NO: 44) is designed using segments that are complementary to the 3’ segment of RNA1 and the 5’ segment of RNA2. Specifically, the sequence 5’-TCTAGCTCTAAAAC-3’ (SEQ ID NO: 30) of DNA splint 1 is complementary to the sequence 5’-GUUUUAGAGCUAGA-3’ (SEQ ID NO: 31) of RNA1, and the sequence 5’-TTATTTTAACTTGCTATT-3’ (SEQ ID NO: 32) of DNA splint 1 is complementary to the sequence 5’-AAUAGCAAGUUAAAAUAA-3’ (SEQ ID NO: 33) of RNA2.

[0179] Furthermore, DNA splint 2 oligonucleotide (SEQ ID NO: 45) is designed using segments that are complementary to the 3’ segment of RNA2 and the 5’ segment of RNA3. Specifically, the sequence 5’-TGATAACGGACTAGCC-3’ (SEQ ID NO: 34) of DNA splint 2 is complementary to the sequence 5’-GGCUAGUCCGUUAUCA-3’ (SEQ ID NO: 35) of RNA2, and the sequence 5’-TCGGTGCCACTTTTTCAAGT-3’ (SEQ ID NO: 36) of DNA splint 2 is complementary to the sequence 5’-ACUUGAAAAAGUGGCACCGA-3’ (SEQ ID NO: 37) of RNA3.

[0180] The advantage of this design is that the first sprint oligonucleotide (DNA sprint 1) is complementary to a segment of the first RNA fragment (RNA1) that does not contain a spacer sequence. Thus, the remaining first and second sprint oligonucleotides (DNA sprint 1 and DNA sprint 2), as well as the second and third RNA fragments (RNA2 and RNA3), are "universal" in that they are used to prepare the sgRNA for use with SpyCas9 having the invariant backbone sequence shown in SEQ ID NO: 17. Only the first RNA fragment containing the spacer sequence of the final sgRNA is customized according to the target specificity of the sgRNA.

Table 5

[0181] The second design includes the RNA fragments shown in Table 6, shown as either a modified version or an unmodified version, and is identical to those used in the first design above. A schematic of the second design is shown in FIG. 11, providing sequence alignments of the first, second, and third RNA fragments to the first and second DNA sprints. The DNA version of the nucleotide sequence of the final sgRNA product is shown in the 5' to 3' direction (the RNA version of the nucleotide sequence is shown in SEQ ID NO: 19), and segments of the corresponding nucleotide sequence are shown for the first, second, and third RNA fragments (the RNA versions of the first, second, and third RNA fragments are shown in SEQ ID NOs: 38, 40, and 42, respectively). Also shown is the alignment of the first and second DNA sprints with the first, second, and third RNA fragments, forming an RNA / DNA duplex, with the nucleotide sequences of the first and second DNA sprints shown in the 3' to 5' direction (nucleotide sequences shown in SEQ ID NOs: 52 and 53, respectively).

[0182] The second-designed DNA splint 1 oligonucleotide (SEQ ID NO: 52) has segments complementary to the 3’ segment of RNA1 and the 5’ segment of RNA2. Specifically, the sequence 5’-TCTAGCTCTAAAACACCAGTATG-3’ (SEQ ID NO: 46) of DNA splint 1 is complementary to the sequence 5’-CAUACUGGUGUUUUAGAGCUAGA-3’ (SEQ ID NO: 47) of RNA1, and 5’-TATTTTAACTTGCTATT-3’ (SEQ ID NO: 48) of DNA splint 1 is complementary to the sequence 5’-AAUAGCAAGUUAAAAUA-3’ (SEQ ID NO: 49) of RNA2. According to this design, DNA splint 1 overlaps with 9 nucleotides of RNA1 present at the 3’ end of the spacer sequence. The extended overlap with the first RNA fragment enhances the stability of the heteroduplex formed between the first splint oligonucleotide and the first RNA fragment, particularly raising the melting temperature of the heteroduplex and promoting the formation of the heteroduplex under the conditions used in the ligation reaction.

[0183] Furthermore, the DNA splint 2 oligonucleotide (SEQ ID NO: 53) is designed using segments complementary to the 3’ portion of RNA2 and the 5’ portion of RNA3. Specifically, the sequence 5’-TGATAACGGACTAGCCT-3’ (SEQ ID NO: 50) of DNA splint 2 is complementary to the sequence 5’-AGGCUAGUCCGUUAUCA-3’ (SEQ ID NO: 51) of RNA2, and the sequence 5’-GACTCGGTGCCACTTTTTCAAGT-3’ (SEQ ID NO: 54) of DNA splint 2 is complementary to the sequence 5’-ACUUGAAAAAGUGGCACCGAGUC-3’ (SEQ ID NO: 55) of RNA3.

Table 6

Table 7-1

Table 7-2

Table 7-3

Claims

**Claim 1** A method for synthesizing a guide RNA (gRNA), the method comprising: (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) (i) a first splint oligonucleotide comprising 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; (e) (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 of a second splint oligonucleotide; (f) an RNA ligase, hybridizing the first, second, and third RNA fragments and the first and second splint oligonucleotides results in the formation of a complex having a first ligation site between the 3'-hydroxyl group of the first RNA fragment and the 5'-phosphate group of the second RNA fragment, and a second ligation site between the 3'-hydroxyl group of the second RNA fragment and the 5'-phosphate group of the third RNA fragment; the ligase effecting ligation of the first and second RNA fragments at the first ligation site and the second and third RNA fragments at the second ligation site, thereby synthesizing the gRNA. **Claim 2** (a) the gRNA comprises, from 5' to 3', the first RNA fragment linked to the second RNA fragment by a first phosphodiester bond and the second RNA fragment linked to the third RNA fragment by a second phosphodiester bond; (b) the 5'-phosphate moiety is 5'-phosphate or 5'-phosphorothioate; (c) the length of the gRNA is 30 to 160 nucleotides; (d) the gRNA is a single molecule gRNA (sgRNA); (e) the first RNA fragment, the second RNA fragment, and / or the third RNA fragment comprises at least one secondary structure; (f) the complex formed by hybridizing the first, second, and third RNA fragments and the first and second splint oligonucleotides has a lower free energy than that of the secondary structure having the lowest free energy; and / or (g) the gRNA comprises a sequence complementary to a sequence in the target DNA, the method according to claim 1.

3. (1) 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 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; and / or, (2) the target DNA is mammalian DNA or human DNA, the method according to claim 2.

4. A method for synthesizing a single molecule guide RNA (sgRNA) for use with an RNA-induced endonuclease, the method comprising providing a complex formed between a first RNA fragment, a second RNA fragment, a third RNA fragment, a first splint oligonucleotide, and a second splint oligonucleotide, and an RNA ligase, (a) the first RNA fragment comprises (i) a terminal region comprising a 3'-hydroxyl group, (b) the second RNA fragment comprises (i) a first terminal region comprising a 5'-phosphate moiety, and (ii) a second terminal region comprising a 3'-hydroxyl group, (c) the third RNA fragment comprises (i) a terminal region comprising a 5'-phosphate moiety, (d) the first splint oligonucleotide comprises (i) a first portion complementary to the terminal region comprising the 3'-hydroxyl group of the first RNA fragment, and (ii) a second portion complementary to the first terminal region comprising the 5'-phosphate moiety of the second RNA fragment, (e) the second splint oligonucleotide includes (i) a first portion complementary to the second terminal region including the 3'-hydroxyl group of the second RNA fragment, and (ii) a second portion complementary to the terminal region including the 5'-phosphate portion 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 ligation 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 ligation site present between the 3'-hydroxyl group of the second RNA fragment and the 5'-phosphate group of the third RNA fragment; the ligase results in ligation at the first ligation site and ligation at the second ligation site to form, from 5' to 3', a spacer sequence and a constant sequence that binds to an RNA-guided endonuclease, the constant sequence including a stem-loop formed between a crRNA repeat sequence and a tracrRNA reverse repeat sequence, and a 3'-tracrRNA sequence including at least one stem-loop, thereby synthesizing the sgRNA for use with the RNA-guided endonuclease. **Claim 5** (a) the first ligation site corresponds to a site within a stem-loop formed between a crRNA repeat sequence and a tracrRNA reverse repeat sequence; (b) the 3'-tracrRNA sequence includes a first stem-loop, a second stem-loop, and a third stem-loop; (c) the first ligation site corresponds to a site in the 5'-stem of the stem-loop, the tetraloop of the stem-loop, the helical portion, or the 3'-stem of the stem-loop; (d) the second ligation site corresponds to a site in the first stem-loop, the second stem-loop, or the third stem-loop; (e) the second ligation site corresponds to a site adjacent to the 5' base of the second stem loop or a site adjacent to the 3' base of the second stem loop; and / or (f) the first RNA fragment includes a nucleotide sequence that is 5' to the first ligation site, the second RNA fragment includes a nucleotide sequence that is between the first ligation site and the second ligation site, and the third RNA fragment includes a nucleotide sequence that is 3' to the second ligation site, the method according to claim 4.

6. The method according to claim 5, wherein the second ligation site corresponds to a site of the second stem loop, or the second ligation site corresponds to a site within the 5' stem of the second stem loop, a site within the tetraloop of the second stem loop, a helical portion, or a site within the 3' stem of the second stem loop.

7. (a) the terminal region containing the 3' hydroxyl group of the first RNA located at the 3' end of the first RNA fragment includes a nucleotide sequence of 10 to 30 nucleotides; (b) the terminal region containing the 5' phosphate moiety of the second RNA located at the 5' end of the second RNA fragment and the terminal region containing the 3' hydroxyl group of the second RNA located at the 3' end of the second RNA fragment include a nucleotide sequence of 10 to 30 nucleotides; (c) the terminal region containing the 5' phosphate moiety of the third RNA located at the 5' end of the third RNA fragment includes a nucleotide sequence of 10 to 40 nucleotides; (d) 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, or 30 to 40 nucleotides in length; and / or (e) the second RNA fragment and / or the third RNA fragment is 40 nucleotides or less, the method according to any one of claims 1 to 6.

8. (a) The first portion of the first splint oligonucleotide is completely complementary to the terminal region containing the 3'-hydroxyl group of the first RNA, or has 1, 2, or 3 mismatches with respect to the terminal region containing the 3'-hydroxyl group of the first RNA; (b) The second portion of the first splint oligonucleotide is completely complementary to the first terminal region containing the 5'-phosphate portion of the second RNA, or has 1, 2, or 3 mismatches with respect to the terminal region containing the 5'-phosphate portion of the second RNA; (c) The first portion of the second splint oligonucleotide is completely complementary to the second terminal region containing the 3'-hydroxyl group of the second RNA, or has 1, 2, or 3 mismatches with respect to the terminal region containing the 3'-hydroxyl group of the second RNA; (d) The second portion of the second splint oligonucleotide is completely complementary to the terminal region containing the 5'-phosphate portion of the third RNA, or has 1, 2, or 3 mismatches with respect to the terminal region containing the 5'-phosphate portion of the third RNA; (e) The first splint oligonucleotide is a DNA or RNA oligonucleotide; (f) The second splint oligonucleotide is a DNA or RNA oligonucleotide; (g) The first splint oligonucleotide and the second splint oligonucleotide are each 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 in length; and / or (h) The first and / or the second splint oligonucleotide is bound to a solid support. The method according to any one of claims 1 to 7.

9. (a) The RNA-induced endonuclease is a Cas nuclease or a small RNA-induced endonuclease; and / or (b) The ligase is T4 RNA ligase I or T4 RNA ligase II. The method according to any one of claims 1 to 8. **Claim 10**: The method according to claim 9, wherein the Cas nuclease is selected from the group consisting of Cas9, Cas12, Cas13, and variants thereof, and the Cas endonuclease is (i) Streptococcus pyogenes Cas9 (SpyCas9) or Staphylococcus aureus (SaCas9), and / or (ii) selected from the group consisting of small Cas9, inactive Cas9 (dCas9), and Cas9 nickase. **Claim 11** (A) The invariant sequence comprises the nucleotide sequence of SEQ ID NO: 17 or a nucleotide sequence having a deletion, insertion, or substitution of at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides relative to SEQ ID NO: 17; (B) Each of the first RNA fragment, the second RNA fragment, and the third RNA fragment is (a)(i) N １５－３０ GUUUUAGAGCUAG (SEQ ID NO: 56) (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) (N １５－３０ corresponding to the spacer sequence), (ii) SEQ ID NO: 40, and (iii) SEQ ID NO: 42, (c)(i) N １５－３０ GUUUUAGAGCUAG (SEQ ID NO: 56) (N １５－３０ corresponding to the spacer sequence), (ii) SEQ ID NO: 58, and (iii) SEQ ID NO: 42, or (d)(i) N １５－３０ GUUUUAGAGCUAGA (SEQ ID NO: 57) (N １５－３０ corresponding to the spacer sequence), (ii) SEQ ID NO: 59, and (iii) SEQ ID NO: 4, and is selected from the nucleotide sequences comprising the same; (C) The first split oligonucleotide comprises the nucleotide sequence shown in SEQ ID NO: 60, SEQ ID NO: 44, or SEQ ID NO: 61, and the second split oligonucleotide comprises the nucleotide sequence shown in SEQ ID NO: 6, SEQ ID NO: 45, or SEQ ID NO: 53; and / or (D) The method according to any one of claims 4 to 6, wherein the first split oligonucleotide further comprises a 3' end having a nucleotide sequence that is not complementary to the spacer sequence or is complementary to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides present at the 3' end of the spacer sequence or the spacer sequence. **Claim 12** (a) Providing the first RNA fragment, the second RNA fragment, and the third RNA fragment comprises the synthesis of the RNA fragments using enzymatic synthesis or phosphoramidite chemistry, and / or The method according to any one of claims 1 to 11, wherein providing the first and second splint oligonucleotides comprises synthesizing the oligonucleotides using enzymatic synthesis or phosphoramidite chemistry. **Claim 13**: (1) Providing the first RNA fragment, the second RNA fragment, and the third RNA fragment comprises purifying the RNA fragments. (2) The synthesis of the RNA fragments using phosphoramidite chemistry (i) Synthesizing the first RNA fragment, the second RNA fragment, and the third RNA fragment in the 5' to 3' or 3' to 5' direction, or (ii) Synthesizing the first RNA fragment in the 5' to 3' or 3' to 5' direction, and synthesizing the second RNA fragment and the third RNA fragment in the 3' to 5' direction, and / or (3) The method according to claim 12, wherein providing the first and second splint oligonucleotides comprises purifying the oligonucleotides after synthesis. **Claim 14** The method according to any one of claims 1 to 13, wherein the first RNA fragment, the second RNA fragment, and / or the third RNA fragment comprises at least one modification and / or at least one phosphorothioate bond. **Claim 15**: (a) The modification is in the RNA backbone and / or is a base modification; (b) 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), bridged nucleic acid, 2'-deoxy nucleic acid (DNA), and peptide nucleic acid (PNA); or (c) The base modification is selected from the group consisting of 2-aminopurine, inosine, thymine, 2,6-diaminopurine, 2-pyrimidinone, and 5-methylcytosine. The method according to claim 14. **Claim 16** (a) Hybridizing is performed in solution. (b) Hybridizing is performed with or without an annealing step, and / or The method according to any one of claims 1 to 15, wherein the concentration of the first split oligonucleotide, the concentration of the second split oligonucleotide, the concentration of the first RNA fragment, the concentration of the second RNA fragment, and the concentration of the third RNA fragment in the solution are equal. **Claim 17**: The annealing step according to claim 16, comprising: (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 used for the ligation at a rate of 0.1°C to 2°C / second. **Claim 18** (a) The ligation is carried out at 15°C to 45°C; (b) The ligation is carried out for 0.1 to 48 hours; (c) The ligation includes using a protease or a chelating agent; and / or (d) The ligation includes using one or more crowding agents. The method according to any one of claims 1 to 17. **Claim 19**: (i) The ligation is carried out at 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C; (ii) The ligation is carried out for 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 hours; (iii) The chelating agent is EDTA, EGTA, or a combination of both; and / or (iv) The one or more crowding agents include polyethylene glycol (PEG), Ficoll (registered trademark), ethylene glycol, dextran, or any combination thereof. The method according to claim 18. **Claim 20** The method according to any one of claims 1 to 19, further comprising purifying the gRNA after synthesis. **Claim 21**: Purifying the gRNA includes purifying using a chromatography method, and the chromatography method is reverse phase HPLC, ion exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, affinity chromatography, polyacrylamide gel purification, or any combination thereof. The method according to claim 20. **Claim 22** The method according to any one of claims 1 to 21, wherein the gRNA is tracrRNA. Providing a crRNA comprising a sequence complementary to a sequence in a target DNA; enabling hybridization of the tracrRNA and the crRNA, thereby generating a bimolecular gRNA; and the method according to claim 22, wherein providing the crRNA comprises synthesizing the crRNA via enzymatic synthesis or phosphoramidite chemistry.

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