Synthetic guide molecules, compositions and methods relating thereto

Abstract

To provide chemical syntheses of guide molecules, as well as compositions and methods relating thereto.SOLUTION: This disclosure addresses the need for a cost-effective and straightforward chemical synthesis of high-purity unimolecular guide molecules with minimal n-1 and / or n+1 species, truncation species, and other contaminants by providing, among other things, methods for synthesizing unimolecular guide molecules that involve cross-linking two or more pre-annealed guide fragments. In some embodiments, a unimolecular guide molecule provided herein has improved sequence fidelity at the 5' end, reducing undesired off-target editing. Also provided herein are compositions comprising, or consisting essentially of, the full length unimolecular guide molecules, which are substantially free of n-1 and / or n+1 contamination.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 441,046, filed December 30, 2016, and U.S. Provisional Patent Application No. 62 / 492,001, filed April 28, 2017, the disclosures of each of which are incorporated by reference in their entirety herein.

[0002] The present disclosure relates to CRISPR / Cas-related methods and components for editing a target nucleic acid sequence or regulating the expression of a target nucleic acid sequence. More particularly, the present disclosure relates to synthetic guide molecules, and related systems, methods, and compositions. [Background technology]

[0003] CRISPR (clustered regularly interspaced short palindromic repeats) evolved in bacteria and archaea as an adaptive immune system to defend against viral attack. Upon exposure to a virus, a short segment of viral DNA is integrated into the CRISPR locus. RNA is transcribed from the part of the CRISPR locus that contains the viral sequence. That RNA, which contains a sequence complementary to the viral genome, mediates the targeting of an RNA-guided nuclease protein, such as Cas9 or Cpf1, to a target sequence in the viral genome. The RNA-guided nuclease then cleaves the viral target, thereby silencing it.

[0004] Recently, CRISPR systems have been adapted for genome editing in eukaryotic cells. These systems generally include a protein component (RNA-guided nuclease) and a nucleic acid component (commonly referred to as guide molecule, guide RNA, or "gRNA"). These two components interact with a specific target DNA sequence that is recognized by or complementary to the two components of the system, and optionally form a complex that edits or modifies the target sequence, for example, by site-specific DNA cleavage. Editing or modifying the target sequence may also involve the recruitment of cellular DNA repair mechanisms, such as non-homologous end joining (NHEJ) or homology-directed repair (HDR).

[0005] Although the value of CRISPR systems as a means to treat genetic diseases is widely recognized, certain technical challenges must be addressed for therapeutics based on these systems to achieve widespread clinical application, particularly the need for cost-effective, straightforward, commercial-scale synthesis of high-quality CRISPR system components.

[0006] For example, most guide molecules are currently synthesized by one of two methods: in vitro transcription (IVT) and chemical synthesis. IVT typically involves transcription of RNA from a DNA template by a bacterial RNA polymerase, such as T7 polymerase. IVT production of guide molecules according to Good Manufacturing Practice (GMP), currently required by U.S. and foreign regulatory agencies, can be costly and limited in scale. Furthermore, IVT synthesis is not suitable for all guide RNA sequences: T7 polymerase tends to transcribe sequences beginning with a 5' guanine more efficiently than sequences beginning with another 5' base, and it can recognize a stem-loop structure followed by a polyuracil tract, present in certain guide molecules, as a signal to terminate transcription, resulting in transcription of a truncated guide molecule. On the other hand, chemical synthesis is inexpensive and GMP production for shorter oligonucleotides (e.g., less than 100 nucleotides in length) is readily available. Chemical synthesis methods are described throughout the literature, for example by Beaucage and Carruthers, Curr Protoc Nucleic Acid Chem. 2001 May; Chapter 3: Unit 3.3 (Beaucage & Carruthers), which is incorporated herein by reference in its entirety for all purposes herein. These methods typically involve the stepwise addition of activated nucleotide monomers until an oligonucleotide sequence of the desired length is reached. In the most commonly used synthesis methods (such as the phosphoramidite method), monomers are added to the 5' end of the oligonucleotide. These monomers are often 3'-functionalized (e.g., with a phosphoramidite) and can be represented, for example, by the following formula I: [ka] and 5' protecting groups (such as 4,4' dimethoxytrityl) according to the above. In formula I, DMTr is 4,4'-dimethoxytrityl, R is a group compatible with oligonucleotide synthesis conditions, non-limiting examples of which include H, F, O-alkyl, or a protected hydroxyl group, and B is any suitable nucleobase (Beaucage & Carruthers). The use of 5' protected monomers requires a deprotection step after each round of addition to remove the 5' protecting group leaving a hydroxyl group. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Beaucage and Carruthers,Curr Protoc Nucleic Acid Chem.2001 May;Chapter 3:Unit 3.3(Beaucage&Carruthers) Summary of the Invention [Problem to be solved by the invention]

[0008] Whatever chemistry is utilized, the stepwise addition of 5' residues does not occur quantitatively; some oligonucleotides will "fail" to add some residues. This results in a synthesis product that contains the desired oligonucleotide but is contaminated with shorter oligonucleotides lacking various residues (termed "n-1 species," but which may include n-2, n-3, etc., as well as other truncated or deleted species). To minimize contamination by n-1 species, many chemical synthesis schemes include a "capping" reaction between the stepwise addition and deprotection steps. In the capping reaction, a non-reactive moiety is added to the 5' end of those oligonucleotides that are not terminated by a 5' protecting group; this non-reactive moiety prevents further addition to the monomeric oligonucleotide and is effective in reducing n-1 contamination to an acceptably low level during the synthesis of oligonucleotides about 60 or 70 bases long. However, the capping reaction is also not quantitative and may be ineffective in preventing n-1 contamination in longer oligonucleotides, such as unimolecular guide RNAs. On the other hand, DMT protection may be lost during the coupling reaction to yield longer oligonucleotides (termed "n+1 species," but which may include n+2, n+3, etc.). Single-molecule guide RNAs contaminated with n-1 and / or n+1 species may not perform as well as full-length guide RNAs prepared by other means, potentially complicating the use of synthetic guide RNAs in therapy. [Means for solving the problem]

[0009] The present disclosure addresses the need for cost-effective and straightforward chemical synthesis of high-purity unimolecular guide molecules with minimal n-1 and / or n+1 species, truncated species, and other contaminants, by providing, inter alia, a method for synthesizing unimolecular guide molecules comprising crosslinking two or more pre-annealed guide fragments. In some embodiments, the unimolecular guide molecules provided herein have improved sequence fidelity at the 5' end, reducing undesired off-target editing. Also provided herein are compositions comprising or consisting essentially of full-length unimolecular guide molecules substantially free of n-1 and / or n+1 contamination.

[0010] Certain aspects of the present disclosure encompass the recognition that pre-annealing of guide fragments may be particularly useful when the guide fragments are homomultifunctional (e.g., homobifunctional), such as the amine-functionalized fragments used in the urea-based cross-linking methods described herein. Indeed, pre-annealing homomultifunctional guide fragments to heterodimers can reduce the formation of undesired homodimers. Thus, the present disclosure also provides compositions that comprise or consist essentially of full-length unimolecular guide molecules that are substantially free of by-products (e.g., homodimers).

[0011] In one aspect, the present disclosure relates to a method of synthesizing a single-molecule guide molecule for a CRISPR system, the method comprising: annealing a first oligonucleotide and a second oligonucleotide to form a duplex between a 3' region of the first oligonucleotide and a 5' region of the second oligonucleotide, wherein the first oligonucleotide comprises a first reactive group that is at least one of a 2' reactive group and a 3' reactive group, and the second oligonucleotide comprises a second reactive group that is a 5' reactive group; and conjugating the annealed first and second oligonucleotides via the first and second reactive groups to form a unimolecular guide RNA molecule that includes a covalent bond linking the first and second oligonucleotides. Includes.

[0012] In one aspect, the present disclosure relates to a single molecule guide molecule for CRISPR system. In some embodiments, the single molecule guide molecule provided herein is for type II CRISPR system.

[0013] In some embodiments, the 5' region of the first oligonucleotide comprises a target domain that is fully or partially complementary to a target domain within a target sequence (eg, a target sequence within a eukaryotic gene).

[0014] In some embodiments, the 3' region of the second oligonucleotide comprises one or more stem-loop structures.

[0015] In some embodiments, the unimolecular guide molecules provided herein are capable of interacting with a Cas9 molecule and mediating the formation of a Cas9 / guide molecule complex.

[0016] In some embodiments, the unimolecular guide molecules provided herein are complexed with Cas9 or an RNA-guided nuclease.

[0017] In some embodiments, the unimolecular guide molecules provided herein comprise, 5' to 3': A first guide molecule fragment, target domain sequence; 1st lower stem sequence; The first bulge sequence; First upper stem array a first guide molecule fragment comprising: Non-nucleotide chemical bonds; and A second guide molecule fragment, second upper stem array; A second bulge arrangement; and Second Lower Stem Array a second guide molecule fragment comprising Including, (a) at least one nucleotide in the first lower stem sequence base pairs with a nucleotide in the second lower stem sequence, and (b) at least one nucleotide in the first upper stem sequence base pairs with a nucleotide in the second upper stem sequence.

[0018] In some embodiments, the unimolecular guide molecule does not include a tetraloop sequence between the first and second upper stem sequences. In some embodiments, the first and / or second upper stem sequences include between 4 and 22 nucleotides (inclusive).

[0019] In some embodiments, the unimolecular guide molecule has the formula: [ka] is a single-molecule guide molecule, In the formula, (N) c and (N) t wherein each N is independently a nucleotide residue, optionally modified, each independently linked to its adjacent nucleotide via a phosphodiester, phosphorothioate, phosphonoacetate, thiophosphonoacetate, or phosphoramidate linkage; (N) c is (N) t (N) t a 3' region that forms a duplex with the 5' region of c is an integer equal to or greater than 20; t is an integer equal to or greater than 20; The linker is a non-nucleotidic chemical bond; B1 and B2 are each independently a nucleobase; R2' and R3' are each independently H, OH, fluoro, chloro, bromo, NH2, SH, S-R', or O-R'; wherein each R' is independently a protecting group or an alkyl group, which alkyl group may be optionally substituted; [ka] each independently represents a phosphodiester bond, a phosphorothioate bond, a phosphonoacetate bond, a thiophosphonoacetate bond, or a phosphoramidate bond.

[0020] In some embodiments, (N) c In some embodiments, (N) comprises a 3' region that includes at least a portion of a repeat from a type II CRISPR system. c In some embodiments, (N) comprises a 3' region that includes a target domain that is fully or partially complementary to a target domain in the target sequence. t comprises a 3' region which contains one or more stem-loop structures.

[0021] In some embodiments, the unimolecular guide molecule has the formula: [ka] is a single-molecule guide molecule, During the ceremony: N is each independently a nucleotide residue, optionally modified, linked to its adjacent nucleotide via a phosphodiester, phosphorothioate, phosphonoacetate, thiophosphonoacetate, or phosphoramidate linkage; N----N each independently represent two complementary nucleotides, optionally two complementary nucleotides base-paired by hydrogen bonds; p and q are each 0; u is an integer from 2 to 22, inclusive; s is an integer from 1 to 10, inclusive; x is an integer from 1 to 3, inclusive; y is >x and is an integer from 3 to 5, inclusive; m is an integer greater than or equal to 15; and n is an integer of 30 or greater.

[0022] In some embodiments, u is an integer from 2 to 22, inclusive; s is an integer from 1 to 8, inclusive; x is an integer from 1 to 3, inclusive; y is >x and is an integer from 3 to 5, inclusive; m is an integer between 15 and 50, inclusive; and n is an integer between 30 and 70 (inclusive).

[0023] In some embodiments, the guide molecule does not contain a tetraloop (p and q are each 0). In some embodiments, the lower stem sequence and the upper stem sequence do not contain an identical sequence of four or more nucleotides. In some embodiments, u is an integer between 3 and 22, inclusive.

[0024] In some embodiments, the first reactive group and the second reactive group are each an amino group, and the conjugating step comprises crosslinking the amine moieties of the first and second reactive groups with a carbonate-containing bifunctional crosslinker to form a urea bond. In some embodiments, the first reactive group and the second reactive group are a bromoacetyl group and a sulfhydryl group. In some embodiments, the first reactive group and the second reactive group are a phosphate group and a hydroxyl group.

[0025] In some embodiments, the unimolecular guide molecule has the formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein L and R are each independently a non-nucleotidic chemical linker.

[0026] In some embodiments, the unimolecular guide molecule has the formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein L and R are each independently a non-nucleotidic chemical linker.

[0027] In some embodiments, the unimolecular guide molecule has the formula: [ka] or a pharma- ceutically acceptable salt thereof; in the presence of an activator to form a phosphodiester bond, [ka] or prepared by a process including a reaction between salts thereof.

[0028] In one aspect, the present disclosure provides a compound of formula: [ka] or a pharmaceutically acceptable salt thereof. In some embodiments, less than about 10% of the guide molecules comprise a truncation at the 5' end relative to the reference guide molecule sequence. In some embodiments, at least about 99% of the guide molecules comprise a 5' sequence comprising nucleotides 1-20 of the guide molecule that is 100% identical to the corresponding 5' sequence of the reference guide molecule sequence.

[0029] In some embodiments, the composition of the guide molecule has the formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein the composition has the formula: [ka] or a pharma- ceutically acceptable salt thereof.

[0030] In some embodiments, the composition has the formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein the composition has the formula: [ka] or a pharma- ceutically acceptable salt thereof.

[0031] In some embodiments, the composition has the formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein the composition has the formula: [ka] or a pharma- ceutically acceptable salt thereof; During the ceremony: a is not equal to c; and / or b is not equal to t.

[0032] In some embodiments, the composition has the formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein the composition has the formula: [ka] or a pharma- ceutically acceptable salt thereof; During the ceremony: a is not equal to c; and / or b is not equal to t.

[0033] In some embodiments, the composition has the formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein the composition has the formula: [ka] is substantially free of molecules of

[0034] In some embodiments, the composition has the formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein the composition has the formula: [ka] is substantially free of molecules of

[0035] In some embodiments, the composition has the formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein the composition has the formula: [ka] or a pharma- ceutically acceptable salt thereof; During the ceremony: a is not equal to c; and / or b is not equal to t.

[0036] In some embodiments, the composition has the formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein the composition has the formula: [ka] or a pharma- ceutically acceptable salt thereof; During the ceremony: a is not equal to c; and / or b is not equal to t.

[0037] In some embodiments, the composition has the formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein the composition has the formula: [ka] or a pharma- ceutically acceptable salt thereof; During the ceremony: a is not equal to c; and / or b is not equal to t.

[0038] In some embodiments, the composition has the formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein the composition has the formula: [ka] or a pharma- ceutically acceptable salt thereof; During the ceremony: a is not equal to c; and / or b is not equal to t.

[0039] In some embodiments, the composition comprises: (a) a synthetic unimolecular guide molecule for a CRISPR system, the unimolecular guide molecule having the formula: [ka] or a pharma- ceutically acceptable salt thereof; and (b) one or more of: (i) a carbodiimide or a salt thereof; (ii) imidazole, cyanoimidazole, pyridine, and dimethylaminopyridine, or salts thereof; Formula (iii): [ka] or a salt thereof wherein R4 and R5 are each independently substituted or unsubstituted alkyl, or substituted or unsubstituted carbocyclic.

[0040] In some embodiments, the composition comprises: Included is a synthetic unimolecular guide molecule for a CRISPR system, the guide molecule having the formula: [ka] or a pharma- ceutically acceptable salt thereof, having the formula: [ka] or a pharma- ceutically acceptable salt thereof; During the ceremony: a+b is c+tk, where k is 1 to 10.

[0041] In some embodiments, the composition comprises, or consists essentially of, a synthetic unimolecular guide molecule for a CRISPR system, the guide molecule having the formula: [ka] or a pharma- ceutically acceptable salt thereof; Here, the 2' to 5' phosphodiester bond shown in this formula is (N) c and the 3' region of (N) t The 5′ region of the nucleotide sequence is between two nucleotides in the duplex formed between the nucleotide sequence of the 5′ region of the nucleotide sequence of the

[0042] In one aspect, the disclosure relates to an oligonucleotide for synthesizing a unimolecular guide molecule provided herein and / or for synthesizing a unimolecular guide molecule by the methods provided herein. In some embodiments, the oligonucleotide has the formula: [ka] or a salt thereof.

[0043] In some embodiments, the oligonucleotide has the formula: [ka] or a salt thereof.

[0044] In some embodiments, the oligonucleotide has the formula: [ka] or a salt thereof.

[0045] In some embodiments, the composition has the formula: [ka] or a salt thereof.

[0046] In some embodiments, the oligonucleotide has the formula: [ka] or a salt thereof.

[0047] In some embodiments, the oligonucleotide has the formula: [ka] or a salt thereof.

[0048] In some embodiments, the composition has the formula: [ka] or a salt thereof.

[0049] In one aspect, the present disclosure provides a compound of formula: [ka] The present invention relates to a compound of the formula:

[0050] In one aspect, the disclosure relates to a method of modifying nucleic acid in a cell or a subject comprising administering to the subject a guide molecule or composition provided herein.

[0051] In some embodiments, the compositions provided herein have not been subjected to any purification steps.

[0052] In some embodiments, the compositions provided herein comprise a single guide RNA molecule suspended in a solution or pharma- ceutically acceptable carrier.

[0053] In one aspect, the present disclosure relates to a genome editing system comprising the guide molecule provided herein. In some embodiments, the genome editing system and / or the guide molecule are for use in therapy. In some embodiments, the genome editing system and / or the guide molecule are for use in the manufacture of medicines.

[0054] The accompanying drawings are intended to provide illustrative and schematic examples of certain aspects and embodiments of the present disclosure, not exhaustive.The drawings are not intended to be limited or bound to any particular theory or model, and are not necessarily drawn to scale.Without limiting the above, nucleic acid and polypeptide can be depicted as linear sequences or as schematic two-dimensional or three-dimensional structures; these depictions are for illustrative purposes, but are not limited or bound to any particular model or theory regarding their structures. In an embodiment of the present invention, for example, the following items are provided: (Item 1) 1. A synthetic unimolecular guide molecule for a CRISPR system, the unimolecular guide molecule having the formula: [ka] containing chemical bonds of During the ceremony: R2' and R3' are each independently H, OH, fluoro, chloro, bromo, NH2, SH, S-R', or O-R', where each R' is independently a protecting group or an alkyl group, where said alkyl group may be optionally substituted; L and R are each independently a non-nucleotidic linker; and B1 and B2 are each independently a synthetic unimolecular guide molecule that is a nucleobase. (Item 2) R 2’ is selected from the group consisting of H, fluoro, and O-R', where R' is a protecting group or an optionally substituted alkyl group. formula: [ka] The During the ceremony: (N) c and (N) t wherein each N is independently a nucleotide residue, optionally modified, each independently linked to its adjacent nucleotide via a phosphodiester, phosphorothioate, phosphonoacetate, thiophosphonoacetate, or phosphoramidate linkage; (N) c is (N) t and (N) is complementary or partially complementary to the 5' region of t a 3' region that forms a duplex with the 5' region of c is an integer equal to or greater than 20; t is an integer equal to or greater than 20; and [ka] represents a phosphodiester bond, a phosphorothioate bond, a phosphonoacetate bond, a thiophosphonoacetate bond, or a phosphoramidate bond. (Item 4) (N) c and (N) t wherein each N is independently a ribonucleotide residue or a sugar-modified ribonucleotide residue. (Item 5) (N) c OR (N) t The guide molecule of item 3 or 4, comprising one or more deoxyribonucleotide residues. (Item 6) (N) c OR (N) t 6. The guide molecule according to any one of items 3 to 5, comprising one or more 2'-O-methyl modified ribonucleotide residues. (Item 7) (N) c and / or each of the three nucleotides at the 5' end of (N) t 7. The guide molecule according to any one of items 3 to 6, wherein each of the three nucleotides at the 3' end of the guide molecule comprises a 2'-O-methyl modified ribonucleotide residue linked to its adjacent nucleotide via a phosphorothioate bond. (Item 8) 8. The guide molecule of any one of items 1 to 7, wherein L and R are each independently a non-nucleotidic linker comprising a moiety selected from the group consisting of polyethylene, polypropylene, polyethylene glycol, and polypropylene glycol. (Item 9) (N) for a type II CRISPR system; c 9. The guide molecule of any one of items 3 to 8, comprising a 5' region that includes a target domain that is fully or partially complementary to a target domain in the target sequence. (Item 10) (N) t10. The guide molecule according to any one of items 3 to 9, comprising a 3' region comprising one or more stem-loop structures. (Item 11) 11. The guide molecule according to any one of items 1 to 10, which is capable of interacting with a Cas9 molecule and mediating the formation of a Cas9 / guide ribonucleoprotein complex. (Item 12) (N) c 12. The guide molecule of any one of items 3 to 11, comprising a 3' region that includes at least a portion of the repeats from a type II CRISPR system. (Item 13) formula: [ka] The During the ceremony: L1 and R1 are each independently a non-nucleotidic linker; Each R2 is independently O or S; R3 each independently represents O - or COO - and; p and q are each independently an integer from 0 to 6 (inclusive), and p+q is an integer from 0 to 6 (inclusive); u is an integer from 2 to 22, inclusive; s is an integer from 1 to 10, inclusive; x is an integer from 1 to 3, inclusive; y is >x and is an integer from 3 to 5, inclusive; m is an integer equal to or greater than 15; n is an integer equal to or greater than 30; N is each independently a nucleotide residue, optionally a modified nucleotide residue, each independently linked to its adjacent nucleotide via a phosphodiester, phosphorothioate, phosphonoacetate, thiophosphonoacetate, or phosphoramidate bond; and 13. The guide molecule of any one of items 1 to 12, wherein N----N each independently represents two complementary nucleotides, optionally two complementary nucleotides base-paired by hydrogen bonds. (Item 14) 14. The guide molecule of item 13, wherein p and q are each 0. (Item 15) 14. The guide molecule according to item 13, wherein u is an integer from 3 to 22 (inclusive). (Item 16) formula: [ka] wherein: u' is an integer from 2 to 22, inclusive; and 16. The guide molecule according to any one of items 1 to 15, wherein p' and q' are each independently an integer from 0 to 4 (inclusive), and p'+q' is an integer from 0 to 4 (inclusive). (Item 17) formula: [ka] The During the ceremony: u' is an integer from 2 to 22, inclusive; and 16. The guide molecule according to any one of items 1 to 15, wherein p' and q' are each independently an integer from 0 to 4 (inclusive), and p'+q' is an integer from 0 to 4 (inclusive). (Item 18) 16. The guide molecule of item 14 or 15, wherein p' and q' are each 0. (Item 19) 16. The guide molecule according to item 14 or 15, wherein u' is an integer from 3 to 22 (inclusive). (Item 20) L1 is -(CH2) W- and w is an integer from 1 to 20 (inclusive). (Item 21) R1 is -(CH2CH2O) v - and v is an integer from 1 to 10 (inclusive). (Item 22) 22. The guide molecule of item 21, wherein w is 6 and v is 4. (Item 23) 23. The guide molecule according to any one of items 1 to 22, comprising a sequence selected from SEQ ID NOs: 36 to 56. (Item 24) 1. A synthetic unimolecular guide molecule for a CRISPR system, the unimolecular guide molecule having the formula: [ka] containing chemical bonds of During the ceremony: R2' and R3' are each independently H, OH, fluoro, chloro, bromo, NH2, SH, S-R', or O-R', where each R' is independently a protecting group or an alkyl group, where said alkyl group may be optionally substituted; L and R are each independently a non-nucleotidic linker; and B1 and B2 are each independently a synthetic unimolecular guide molecule that is a nucleobase. (Item 25) R 2’ is selected from the group consisting of H, fluoro, and O-R', where R' is a protecting group or an optionally substituted alkyl group. (Item 26) formula: [ka] The During the ceremony: (N) c and (N) t wherein each N is independently a nucleotide residue, optionally modified, each independently linked to its adjacent nucleotide via a phosphodiester, phosphorothioate, phosphonoacetate, thiophosphonoacetate, or phosphoramidate linkage; (N) c is (N) t and (N) is complementary or partially complementary to the 5' region of t a 3' region that forms a duplex with the 5' region of c is an integer equal to or greater than 20; t is an integer equal to or greater than 20; and [ka] 26. The guide molecule according to item 24 or 25, wherein each independently represents a phosphodiester bond, a phosphorothioate bond, a phosphonoacetate bond, a thiophosphonoacetate bond, or a phosphoramidate bond. (Item 27) (N) c and (N) t 27. The guide molecule of item 26, wherein each N is independently a ribonucleotide residue or a sugar-modified ribonucleotide residue. (Item 28) (N) c OR (N) t 28. The guide molecule of item 26 or 27, comprising one or more deoxyribonucleotide residues. (Item 29) (N) c OR (N) t 29. The guide molecule of any one of items 26 to 28, comprising one or more 2'-O-methyl modified ribonucleotide residues. (Item 30) (N) c and / or each of the three nucleotides at the 5' end of (N) t30. The guide molecule according to any one of items 26 to 29, wherein each of the three nucleotides at the 3' end of the guide molecule comprises a 2'-O-methyl modified ribonucleotide residue linked to its adjacent nucleotide via a phosphorothioate bond. (Item 31) 31. The guide molecule of any one of items 24 to 30, wherein L and R are each independently a non-nucleotide linker comprising a moiety selected from the group consisting of polyethylene, polypropylene, polyethylene glycol, and polypropylene glycol. (Item 32) (N) for a type II CRISPR system; c 32. The guide molecule of any one of items 26 to 31, comprising a 5' region that includes a target domain that is fully or partially complementary to a target domain in the target sequence. (Item 33) (N) t 33. The guide molecule of any one of items 26 to 32, comprising a 3' region comprising one or more stem-loop structures. (Item 34) 34. The guide molecule of any one of items 24 to 33, which is capable of interacting with a Cas9 molecule and mediating the formation of a Cas9 / guide ribonucleoprotein complex. (Item 35) (N) c 35. The guide molecule of any one of items 26 to 34, comprising a 3' region that includes at least a portion of the repeats from a type II CRISPR system. (Item 36) formula: [ka] The During the ceremony: L1 and R1 are each independently a non-nucleotidic linker; Each R2 is independently O or S; R3 each independently represents O - or COO -and; p and q are each independently an integer from 0 to 6 (inclusive), and p+q is an integer from 0 to 6 (inclusive); u is an integer from 2 to 22, inclusive; s is an integer from 1 to 10, inclusive; x is an integer from 1 to 3, inclusive; y is >x and is an integer from 3 to 5, inclusive; m is an integer equal to or greater than 15; n is an integer equal to or greater than 30; N is each independently a nucleotide residue, optionally a modified nucleotide residue, each independently linked to its adjacent nucleotide via a phosphodiester, phosphorothioate, phosphonoacetate, thiophosphonoacetate, or phosphoramidate bond; and 36. The guide molecule of any one of items 24 to 35, wherein N----N each independently represents two complementary nucleotides, optionally two complementary nucleotides base-paired by hydrogen bonds. (Item 37) formula: [ka] The During the ceremony: u' is an integer from 2 to 22, inclusive; and 37. The guide molecule according to any one of items 24 to 36, wherein p' and q' are each independently an integer from 0 to 4 (inclusive), and p'+q' is an integer from 0 to 4 (inclusive). (Item 38) formula: [ka] [ka] The During the ceremony: u' is an integer from 2 to 22, inclusive; and 37. The guide molecule according to any one of items 24 to 36, wherein p' and q' are each independently an integer from 0 to 4 (inclusive), and p'+q' is an integer from 0 to 4 (inclusive). (Item 39) L1 is -(CH2) w -NH-C(O)-(CH2) w -NH- or -(OCH2CH2) v -NH-C(O)-(CH2) w -, wherein each w is independently 1 to 20; and each v is independently 1 to 10. (Item 40) R1 is -(CH2) w -NH-C(O)-(CH2) w -or-(OCH2CH2) v -NH-C(O)-(CH2) w -, wherein each w is independently 1 to 20; and each v is independently 1 to 10. (Item 41) 41. The guide molecule according to item 40, wherein L1 is -(CH2)6-NH-C(O)-(CH2)1-NH- and R1 is -(OCH2CH2)4-NH-C(O)-(CH2)2-. (Item 42) 42. The guide molecule according to any one of items 24 to 41, comprising a sequence selected from SEQ ID NO: 35. (Item 43) 43. A composition comprising a plurality of synthetic guide molecules according to any one of items 1 to 42, wherein less than about 10% of the guide molecules comprise a truncation at the 5' end relative to a reference guide molecule sequence. (Item 44) 40. The composition of claim 39, wherein at least about 99% of the guide molecules comprise a 5' sequence comprising nucleotides 1 to 20 of the guide molecule that is 100% identical to the corresponding 5' sequence of the reference guide molecule sequence. (Item 45) formula: [ka] 24. A composition comprising or consisting essentially of a guide molecule according to any one of items 1 to 23, or a pharma- ceutically acceptable salt thereof, comprising a compound represented by the formula: [ka] or a pharma- ceutically acceptable salt thereof. (Item 46) formula: [ka] 24. A composition comprising or consisting essentially of a guide molecule according to any one of items 1 to 23, or a pharma- ceutically acceptable salt thereof, comprising a compound represented by the formula: [ka] or a pharma- ceutically acceptable salt thereof. (Item 47) 47. The composition according to item 45 or 46, which has not been subjected to any purification step. (Item 48) formula: [ka] 24. A composition comprising or consisting essentially of a guide molecule according to any one of items 1 to 23, or a pharma- ceutically acceptable salt thereof, comprising a compound represented by the formula: [ka] or a pharma- ceutically acceptable salt thereof; During the ceremony: a is not equal to c; and / or b is not equal to t, composition. (Item 49) formula: [ka] 24. A composition comprising or consisting essentially of a guide molecule according to any one of items 1 to 23, or a pharma- ceutically acceptable salt thereof, comprising a compound represented by the formula: [ka] or a pharma- ceutically acceptable salt thereof; During the ceremony: a is not equal to c; and / or b is not equal to t, composition. (Item 50) 50. The composition according to item 48 or 49, wherein a is less than c and / or b is less than t. (Item 51) 51. The composition according to any one of items 48 to 50, which has not been subjected to any purification step. (Item 52) 52. The composition of any one of items 45 to 51, comprising a complex of the guide molecule and Cas9 or an RNA-guided nuclease. (Item 53) 53. The composition according to any one of items 45 to 52, wherein the guide molecule is suspended in a solution or in a pharma- ceutically acceptable carrier. (Item 54) (N) c54. The composition of any one of items 45 to 53, comprising a 3' region that includes at least a portion of the repeat from a type II CRISPR system. (Item 55) formula: [ka] A composition comprising or consisting essentially of a guide molecule according to any one of items 24 to 42, or a pharma- ceutically acceptable salt thereof, comprising a compound represented by the formula: [ka] or a pharma- ceutically acceptable salt thereof; During the ceremony: a is not equal to c; and / or b is not equal to t, composition. (Item 56) formula: [ka] A composition comprising or consisting essentially of a guide molecule according to any one of items 24 to 42, or a pharma- ceutically acceptable salt thereof, comprising a compound represented by the formula: [ka] or a pharma- ceutically acceptable salt thereof; During the ceremony: a is not equal to c; and / or b is not equal to t, composition. (Item 57) formula: [ka] A composition comprising or consisting essentially of a guide molecule according to any one of items 24 to 42, or a pharma- ceutically acceptable salt thereof, comprising a compound of the formula: [ka] or a pharma- ceutically acceptable salt thereof; During the ceremony: a is not equal to c; and / or b is not equal to t, composition. (Item 58) formula: [ka] A composition comprising or consisting essentially of a guide molecule according to any one of items 24 to 42, or a pharma- ceutically acceptable salt thereof, comprising a compound of the formula: [ka] or a pharma- ceutically acceptable salt thereof; During the ceremony: a is not equal to c; and / or b is not equal to t, composition. (Item 59) 59. The composition according to any one of items 55 to 58, wherein a is less than c and / or b is less than t. (Item 60) 59. The composition according to any one of items 55 to 58, which has not been subjected to any purification step. (Item 61) 61. The composition of any one of items 55 to 60, comprising a complex of the guide molecule and Cas9 or an RNA-guided nuclease. (Item 62) 62. The composition according to any one of items 55 to 61, wherein the guide molecule is suspended in a solution or in a pharma- ceutically acceptable carrier. (Item 63) (N) c 63. The composition of any one of items 55 to 62, comprising a 3' region that includes at least a portion of the repeat from a type II CRISPR system. (Item 64) (a) a synthetic unimolecular guide molecule for a CRISPR system, the unimolecular guide molecule having the formula: [ka] or a pharma- ceutically acceptable salt thereof; During the ceremony: (N) c and (N) t wherein each N is independently a nucleotide residue, optionally modified, each independently linked to its adjacent nucleotide via a phosphodiester, phosphorothioate, phosphonoacetate, thiophosphonoacetate, or phosphoramidate linkage; (N) c is (N) t and (N) is complementary or partially complementary to the 5' region of t a 3' region that forms a duplex with the 5' region of c is an integer equal to or greater than 20; t is an integer equal to or greater than 20; B1 and B2 are each independently a nucleobase; and [ka] each independently represent a phosphodiester linkage, a phosphorothioate linkage, a phosphonoacetate linkage, a thiophosphonoacetate linkage, or a phosphoramidate linkage; and (b) One or more of the following: (i) a carbodiimide or a salt thereof; (ii) imidazole, cyanoimidazole, pyridine, and dimethylaminopyridine, or salts thereof; and Formula (iii): [ka] or a salt thereof wherein R4 and R5 are each independently substituted or unsubstituted alkyl, or substituted or unsubstituted carbocyclyl. (Item 65) 65. The composition of claim 64, wherein the carbodiimide is EDC, DCC, or DIC. (Item 66) 1. A composition comprising a synthetic unimolecular guide molecule for a CRISPR system, the unimolecular guide molecule having the formula: [ka] or a pharma- ceutically acceptable salt thereof; During the ceremony: (N) c and (N) t wherein each N is independently a nucleotide residue, optionally modified, each independently linked to its adjacent nucleotide via a phosphodiester, phosphorothioate, phosphonoacetate, thiophosphonoacetate, or phosphoramidate linkage; (N) c is (N) t and (N) is complementary or partially complementary to the 5' region of t a 3' region that forms a duplex with the 5' region of c is an integer equal to or greater than 20; t is an integer equal to or greater than 20; B1 and B2 are each independently a nucleobase; and [ka] each independently represents a phosphodiester bond, a phosphorothioate bond, a phosphonoacetate bond, a thiophosphonoacetate bond, or a phosphoramidate bond; The composition has the formula: [ka] or a pharma- ceutically acceptable salt thereof; During the ceremony: The composition, a+b=c+tk, where k is an integer from 1 to 10, inclusive. (Item 67) 1. A composition comprising, or consisting essentially of, a synthetic unimolecular guide molecule for a CRISPR system, comprising: [ka] or a pharma- ceutically acceptable salt thereof; During the ceremony: (N) c and (N) t wherein each N is independently a nucleotide residue, optionally modified, each independently linked to its adjacent nucleotide via a phosphodiester, phosphorothioate, phosphonoacetate, thiophosphonoacetate, or phosphoramidate linkage; (N) c is (N) t and (N) is complementary or partially complementary to the 5' region of t a 3' region that forms a duplex with the 5' region of The 2'-5' phosphodiester bond shown in the formula is between two nucleotides in the duplex; c is an integer equal to or greater than 20; t is an integer equal to or greater than 20; B1 and B2 are each independently a nucleobase; R2' and R3' are each independently H, OH, fluoro, chloro, bromo, NH2, SH, S-R', or O-R', where each R' is independently a protecting group or an alkyl group, where the alkyl group may be optionally substituted; and [ka] each independently represents a phosphodiester linkage, a phosphorothioate linkage, a phosphonoacetate linkage, a thiophosphonoacetate linkage, or a phosphoramidate linkage. (Item 68) The guide molecule is for a type II CRISPR system, and (N) c 68. The composition of claim 67, comprising a 5' region that includes a targeting domain that is fully or partially complementary to a targeting domain within the target sequence. (Item 69) (N) t 69. The composition according to item 67 or 68, comprising a 3' region comprising one or more stem-loop structures. (Item 70) 70. The composition of any one of items 67 to 69, wherein the guide molecule is capable of interacting with a Cas9 molecule and mediating the formation of a Cas9 / guide molecule complex. (Item 71) The guide molecule has the formula: [ka] or a pharma- ceutically acceptable salt thereof; During the ceremony: Z represents a nucleotide loop that is 4 to 6 nucleotides in length, optionally 4 or 6 nucleotides in length; u is an integer from 0 to 22, inclusive, optionally 2; s is an integer from 1 to 10, inclusive, optionally 4; x is an integer from 1 to 3 (inclusive), optionally 2; y is >x and is an integer from 3 to 5 (inclusive), optionally 4; m is an integer equal to or greater than 15; n is an integer equal to or greater than 30; N----N each independently represent two complementary nucleotides, optionally base-paired by hydrogen bonds; 71. The composition of any one of items 67 to 70, wherein at least one phosphodiester bond between two nucleotides in the duplex region shown in said formula is a 2'-5' phosphodiester bond; and N is each independently a nucleotide residue, optionally modified, each independently linked to its adjacent nucleotide via a phosphodiester bond, a phosphorothioate bond, a phosphonoacetate bond, a thiophosphonoacetate bond, or a phosphoramidate bond. (Item 72) 72. The composition according to any one of items 67 to 71, wherein the 2'-5' phosphodiester bond is between two nucleotides located 5' of the bulge as shown in the formula according to item 71. (Item 73) 72. The composition according to any one of items 67 to 71, wherein the 2'-5' phosphodiester bond is between two nucleotides located 5' of the nucleotide loop Z and 3' of the bulge according to the formula according to item 71. (Item 74) 72. The composition according to any one of items 67 to 71, wherein the 2'-5' phosphodiester bond is between two nucleotides located 3' of the nucleotide loop Z and 5' of the bulge according to the formula according to item 71. (Item 75) 72. The composition according to any one of items 67 to 71, wherein the 2'-5' phosphodiester bond is between two nucleotides located 3' of the bulge according to the formula according to item 71. (Item 76) 76. The composition of any one of items 45 to 75, wherein less than about 10% of the guide molecules comprise a truncation at the 5' end relative to the reference guide molecule sequence. (Item 77) 77. The composition of claim 76, wherein at least about 99% of the guide molecules comprise a 5' sequence comprising nucleotides 1 to 20 of the guide molecule that is 100% identical to the corresponding 5' sequence of the reference guide molecule sequence. (Item 78) 1. A composition of a guide molecule for a CRISPR system, comprising the formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein (N) c and (N) t wherein each N is independently a nucleotide residue, optionally modified, each independently linked to its adjacent nucleotide via a phosphodiester, phosphorothioate, phosphonoacetate, thiophosphonoacetate, or phosphoramidate linkage; (N) c is (N) t and (N) is complementary or partially complementary to the 5' region of t a 3' region that forms a duplex with the 5' region of c is an integer equal to or greater than 20; t is an integer equal to or greater than 20; and [ka] each independently represent a phosphodiester bond, a phosphorothioate bond, a phosphonoacetate bond, a thiophosphonoacetate bond, or a phosphoramidate bond; The linker is a non-nucleotidic chemical bond; B1 and B2 are each independently a nucleobase; and A composition, wherein R2' and R3' are each independently H, OH, fluoro, chloro, bromo, NH2, SH, S-R', or O-R', where each R' is independently a protecting group or an alkyl group, wherein the alkyl group may be optionally substituted. (Item 79) formula: [ka] or a salt thereof, wherein p and q are each independently an integer from 0 to 6 (inclusive), and p+q is an integer from 0 to 6 (inclusive); u' is an integer from 2 to 22, inclusive; m is an integer equal to or greater than 15; n is an integer equal to or greater than 30; N----N each independently represent two complementary nucleotides, optionally two complementary nucleotides base-paired by hydrogen bonds; and 79. The composition of the guide molecule of claim 78, wherein each N is independently a nucleotide residue, optionally modified, linked to its adjacent nucleotide via a phosphodiester bond, a phosphorothioate bond, a phosphonoacetate bond, a thiophosphonoacetate bond, or a phosphoramidate bond. (Item 80) formula: [ka] or a salt thereof, wherein p and q are each independently an integer from 0 to 6 (inclusive), and p+q is an integer from 0 to 6 (inclusive); u' is an integer from 2 to 22, inclusive; m is an integer equal to or greater than 15; n is an integer equal to or greater than 30; N----N each independently represent two complementary nucleotides, optionally two complementary nucleotides base-paired by hydrogen bonds; and 79. The composition of the guide molecule of claim 78, wherein each N is independently a nucleotide residue, optionally modified, linked to its adjacent nucleotide via a phosphodiester bond, a phosphorothioate bond, a phosphonoacetate bond, a thiophosphonoacetate bond, or a phosphoramidate bond. (Item 81) formula: [ka] [ka] or a salt thereof, During the ceremony: Z represents a nucleotide loop 4 to 6 nucleotides in length, optionally 4 or 6 nucleotides in length; p and q are each independently an integer from 0 to 2, inclusive, optionally 0; p' is an integer from 0 to 4, inclusive, optionally 0; q' is an integer from 0 to 4, inclusive, optionally 2; x is an integer from 1 to 3 (inclusive), optionally 2; y is >x and is an integer from 3 to 5 (inclusive), optionally 4; u is an integer from 2 to 22, inclusive, optionally 2; s is an integer from 1 to 10, inclusive, optionally 4; m is an integer equal to or greater than 15; n is an integer equal to or greater than 30; B1 and B2 are each independently a nucleobase; N is each independently a nucleotide residue, optionally modified, linked to its adjacent nucleotide via a phosphodiester, phosphorothioate, phosphonoacetate, thiophosphonoacetate, or phosphoramidate linkage; N1 and N2 are each independently a nucleotide residue; N----N each independently represent two complementary nucleotides, optionally two complementary nucleotides base-paired by hydrogen bonds; and [ka] 79. The composition of the guide molecule of item 78, wherein each independently represents a phosphodiester bond, a phosphorothioate bond, a phosphonoacetate bond, a thiophosphonoacetate bond, or a phosphoramidate bond. (Item 82) 1. A composition of guide molecules for a CRISPR system, the guide molecule having the formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein: (N) c and (N) t wherein each N is independently a nucleotide residue, optionally modified, each independently linked to its adjacent nucleotide via a phosphodiester, phosphorothioate, phosphonoacetate, thiophosphonoacetate, or phosphoramidate linkage; (N) c is (N) t and (N) is complementary or partially complementary to the 5' region of t a 3' region that forms a duplex with the 5' region of c is an integer equal to or greater than 20; t is an integer equal to or greater than 20; and [ka] each independently represent a phosphodiester bond, a phosphorothioate bond, a phosphonoacetate bond, a thiophosphonoacetate bond, or a phosphoramidate bond; The linker is a non-nucleotidic chemical bond; B1 and B2 are each independently a nucleobase; and R2' and R3' are each independently H, OH, fluoro, chloro, bromo, NH2, SH, S-R', or O-R', where each R' is independently a protecting group or an alkyl group, where said alkyl group may be optionally substituted; wherein less than about 10% of the guide molecules comprise a truncation at the 5' end relative to the reference guide molecule sequence; wherein at least about 99% of the guide molecules comprise a 5' sequence comprising nucleotides 1-20 of the guide molecule that is 100% identical to the corresponding 5' sequence of the reference guide molecule sequence. (Item 83) The guide molecule has the formula: [ka] or a salt thereof, wherein: p and q are each independently an integer from 0 to 6 (inclusive), and p+q is an integer from 0 to 6 (inclusive); u' is an integer from 2 to 22, inclusive; m is an integer equal to or greater than 15; n is an integer equal to or greater than 30; N----N each independently represent two complementary nucleotides, optionally two complementary nucleotides base-paired by hydrogen bonds; and 83. The composition of the guide molecule of item 82, wherein each N is independently a nucleotide residue, optionally a modified nucleotide residue, linked to its adjacent nucleotide via a phosphodiester bond, a phosphorothioate bond, a phosphonoacetate bond, a thiophosphonoacetate bond, or a phosphoramidate bond. (Item 84) The guide molecule has the formula: [ka] or a salt thereof, wherein: p and q are each independently an integer from 0 to 6 (inclusive), and p+q is an integer from 0 to 6 (inclusive); u' is an integer from 2 to 22, inclusive; m is an integer equal to or greater than 15; n is an integer equal to or greater than 30; N----N each independently represent two complementary nucleotides, optionally two complementary nucleotides base-paired by hydrogen bonds; and 83. The composition of the guide molecule of claim 82, wherein each N is independently a nucleotide residue, optionally modified, linked to its adjacent nucleotide via a phosphodiester bond, a phosphorothioate bond, a phosphonoacetate bond, a thiophosphonoacetate bond, or a phosphoramidate bond. (Item 85) The guide molecule has the formula: [ka] [ka] or a salt thereof, During the ceremony: Z represents a nucleotide loop 4 to 6 nucleotides in length, optionally 4 or 6 nucleotides in length; p and q are each independently an integer from 0 to 2, inclusive, optionally 0; p' is an integer from 0 to 4, inclusive, optionally 0; q' is an integer from 0 to 4, inclusive, optionally 2; x is an integer from 1 to 3 (inclusive), optionally 2; y is >x and is an integer from 3 to 5 (inclusive), optionally 4; u is an integer from 2 to 22, inclusive, optionally 2; s is an integer from 1 to 10, inclusive, optionally 4; m is an integer equal to or greater than 15; n is an integer equal to or greater than 30; B1 and B2 are each independently a nucleobase; N is each independently a nucleotide residue, optionally modified, linked to its adjacent nucleotide via a phosphodiester, phosphorothioate, phosphonoacetate, thiophosphonoacetate, or phosphoramidate linkage; N1 and N2 are each independently a nucleotide residue; N----N each independently represent two complementary nucleotides, optionally two complementary nucleotides base-paired by hydrogen bonds; and [ka] 83. The composition of the guide molecule of item 82, wherein each independently represents a phosphodiester bond, a phosphorothioate bond, a phosphonoacetate bond, a thiophosphonoacetate bond, or a phosphoramidate bond. (Item 86) 1. A method for synthesizing a single-molecule guide molecule for a CRISPR system, comprising: annealing a first oligonucleotide and a second oligonucleotide to form a duplex between a 3' region of the first oligonucleotide and a 5' region of the second oligonucleotide, wherein the first oligonucleotide comprises a first reactive group that is at least one of a 2' reactive group and a 3' reactive group, and the second oligonucleotide comprises a second reactive group that is a 5' reactive group; and conjugating the annealed first and second oligonucleotides via the first and second reactive groups to form a unimolecular guide molecule that includes a covalent bond linking the first and second oligonucleotides. The method includes: (Item 87) 87. The method of claim 86, wherein the guide molecule is for a type II CRISPR system and the 5' region of the first oligonucleotide comprises a target domain that is fully or partially complementary to a target domain within a target sequence. (Item 88) 88. The method of claim 86 or 87, wherein the 3' region of the second oligonucleotide comprises one or more stem-loop structures. (Item 89) 89. The method of any one of items 86 to 88, wherein the guide molecule is capable of interacting with a Cas9 molecule and mediating the formation of a Cas9 / guide molecule complex. (Item 90) 89. The method of any one of items 86 to 88, wherein the first and second reactive groups both comprise amine moieties, and the conjugating step comprises crosslinking the amine moieties of the first and second reactive groups with a carbonate-containing bifunctional crosslinker to form a urea bond. (Item 91) Item 91. The method of item 90, wherein the carbonate-containing bifunctional crosslinker is disuccinimidyl carbonate, diimidazole carbonate, or bis-(p-nitrophenyl) carbonate. (Item 92) 92. The method according to any one of items 86 to 91, wherein the concentration of each of the first and second oligonucleotides is in the range of 10 μM to 1 mM. (Item 93) 93. The method according to any one of items 90 to 92, wherein the concentration of the carbonate-containing bifunctional crosslinker is in the range of 1 mM to 100 mM. (Item 94) 94. The method according to any one of items 90 to 93, wherein the concentration of the carbonate-containing bifunctional crosslinker is 100 to 1,000 times higher than the concentration of each of the first and second oligonucleotides. (Item 95) 95. The method according to any one of items 86 to 94, wherein the conjugating step is carried out at a pH in the range of 7 to 9. (Item 96) 96. The method according to any one of items 86 to 95, wherein the conjugating step is carried out in water using DMSO, DMF, NMP, DMA, morpholine, pyridine, or MeCN as a co-solvent. (Item 97) 97. The method according to any one of items 86 to 96, wherein the conjugating step is carried out in the presence of a divalent metal cation. (Item 98) 98. The method according to any one of items 86 to 97, wherein the conjugating step is carried out at a temperature ranging from 0°C to 40°C. (Item 99) The first oligonucleotide has the formula: [ka] or a salt thereof; and said second oligonucleotide has the formula: [ka] or a salt thereof; During the ceremony: (N) c and (N) twherein each N is independently a nucleotide residue, optionally modified, each independently linked to its adjacent nucleotide via a phosphodiester, phosphorothioate, phosphonoacetate, thiophosphonoacetate, or phosphoramidate linkage; (N) c is (N) t and (N) is complementary or partially complementary to the 5' region of t a 3' region that forms a duplex with the 5' region of c is an integer equal to or greater than 20; t is an integer equal to or greater than 20; R2' and R3' are each independently H, OH, fluoro, chloro, bromo, NH2, SH, S-R', or O-R', where each R' is independently a protecting group or an alkyl group, where said alkyl group may be optionally substituted; L and R are each independently a non-nucleotidic linker; and B1 and B2 are each independently a nucleobase; and [ka] each independently represents a phosphodiester bond, a phosphorothioate bond, a phosphonoacetate bond, a thiophosphonoacetate bond, or a phosphoramidate bond. (Item 100) The unimolecular guide molecule has the formula: [ka] or a salt thereof. (Item 101) The unimolecular guide molecule has the formula: [ka] or a salt thereof, During the ceremony: L1 and R1 are each independently a non-nucleotidic linker; Each R2 is independently O or S; R3 each independently represents O - or COO - and; p and q are each independently an integer from 0 to 6 (inclusive), and p+q is an integer from 0 to 6 (inclusive); u is an integer from 2 to 22, inclusive; s is an integer from 1 to 10, inclusive; x is an integer from 1 to 3, inclusive; y is >x and is an integer from 3 to 5, inclusive; m is an integer equal to or greater than 15; n is an integer equal to or greater than 30; N is each independently a nucleotide residue, optionally modified, linked to its adjacent nucleotide via a phosphodiester, phosphorothioate, phosphonoacetate, thiophosphonoacetate, or phosphoramidate linkage; 97. The method of any one of items 82 to 96, wherein N----N each independently represents two complementary nucleotides, optionally two complementary nucleotides base-paired by hydrogen bonds. (Item 102) The unimolecular guide molecule has the formula: [ka] or a salt thereof, wherein p' and q' are each independently an integer from 0 to 4 (inclusive), and p'+q' is an integer from 0 to 4 (inclusive); and u' is an integer from 2 to 14 (inclusive). (Item 103) The unimolecular guide molecule has the formula: [ka] or a salt thereof, wherein p' and q' are each independently an integer from 0 to 4 (inclusive), and p'+q' is an integer from 0 to 4 (inclusive); and u' is an integer from 2 to 22 (inclusive). (Item 104) The method according to item 102 or 103, wherein p'=q', optionally p'=q'=0, p'=q'=1, or p'=q'=2. (Item 105) L1 is -(CH2) W - and w is 1 to 20. (Item 106) R1 is -(CH2CH2O) v - and v is 1 to 10. (Item 107) The unimolecular guide molecule has the formula: [ka] or a salt thereof, wherein p' and q' are each independently an integer from 0 to 4 (inclusive), and p'+q' is an integer from 0 to 4 (inclusive); and u' is an integer from 2 to 14 (inclusive). (Item 108) The unimolecular guide molecule has the formula: [ka] or a salt thereof, wherein p' and q' are each independently an integer from 0 to 4 (inclusive), and p'+q' is an integer from 0 to 4 (inclusive); and u' is an integer from 2 to 14 (inclusive). (Item 109) 90. The method of any one of items 86-89, wherein (a) the first reactive group comprises a bromoacetyl moiety and the second reactive group comprises a sulfhydryl moiety, or (b) the first reactive group comprises a sulfhydryl moiety and the second reactive group comprises a bromoacetyl moiety, and the conjugating step comprises reacting the bromoacetyl moiety with the sulfhydryl moiety to form a bromoacetyl-thiol bond. (Item 110) Item 109. The method according to item 109, wherein the concentration of each of the first and second oligonucleotides is in the range of 10 μM to 1 mM. (Item 111) 111. The method according to item 109 or 110, wherein the conjugating step is carried out at a pH in the range of 7 to 9. (Item 112) 112. The method according to any one of items 109 to 111, wherein the conjugating step is carried out under argon. (Item 113) 113. The method according to any one of items 109 to 112, wherein the conjugating step is carried out in the presence of a chelating agent, optionally ethylenediaminetetraacetic acid (EDTA) or a salt thereof. (Item 114) 114. The method according to any one of items 109 to 113, wherein the conjugating step is carried out at a temperature ranging from 0°C to 40°C. (Item 115) (a) the first oligonucleotide has the formula: [ka] or a salt thereof, and said second oligonucleotide has the formula: [ka] or a salt thereof; or (b) the first oligonucleotide has the formula: [ka] or a salt thereof, and said second oligonucleotide has the formula: [ka] or a salt thereof; During the ceremony: (N) c and (N) t wherein each N is independently a nucleotide residue, optionally modified, each independently linked to its adjacent nucleotide via a phosphodiester, phosphorothioate, phosphonoacetate, thiophosphonoacetate, or phosphoramidate linkage; (N) c is (N) t and (N) is complementary or partially complementary to the 5' region of t a 3' region that forms a duplex with the 5' region of c is an integer equal to or greater than 20; t is an integer equal to or greater than 20; R2' and R3' are each independently H, OH, fluoro, chloro, bromo, NH2, SH, S-R', or O-R', where each R' is independently a protecting group or an alkyl group, where said alkyl group may be optionally substituted; L and R are each independently a non-nucleotidic linker; and B1 and B2 are each independently a nucleobase; and [ka] each independently represents a phosphodiester bond, a phosphorothioate bond, a phosphonoacetate bond, a thiophosphonoacetate bond, or a phosphoramidate bond. (Item 116) The unimolecular guide molecule has the formula: [ka] or a salt thereof. (Item 117) The unimolecular guide molecule has the formula: [ka] or a salt thereof, During the ceremony: L1 and R1 are each independently a non-nucleotidic linker; Each R2 is independently O or S; R3 each independently represents O - or COO - and; p and q are each independently an integer from 0 to 6 (inclusive), and p+q is an integer from 0 to 6 (inclusive); u is an integer from 2 to 22, inclusive; s is an integer from 1 to 10, inclusive; x is an integer from 1 to 3, inclusive; y is >x and is an integer from 3 to 5, inclusive; m is an integer equal to or greater than 15; n is an integer equal to or greater than 30; N is each independently a nucleotide residue, optionally a modified nucleotide residue, each independently linked to its adjacent nucleotide via a phosphodiester, phosphorothioate, phosphonoacetate, thiophosphonoacetate, or phosphoramidate bond; and 117. The method according to any one of items 109 to 116, wherein N----N each independently represent two complementary nucleotides, optionally two complementary nucleotides base-paired by hydrogen bonds. (Item 118) The unimolecular guide molecule has the formula: [ka] or a salt thereof, wherein p' and q' are each independently an integer from 0 to 4 (inclusive), and p'+q' is an integer from 0 to 4 (inclusive); and u' is an integer from 2 to 14 (inclusive). (Item 119) The unimolecular guide molecule has the formula: [ka] or a salt thereof, wherein p' and q' are each independently an integer from 0 to 4 (inclusive), and p'+q' is an integer from 0 to 4 (inclusive); and u' is an integer from 2 to 22 (inclusive). (Item 120) 120. The method of claim 118 or 119, wherein p'=q', optionally p'=q'=0, p'=q'=1, or p'=q'=2. (Item 121) L1 and R1 each independently represent -(CH2) W -NH-C(O)-(CH2) w -NH- or -(OCH2CH2)v -NH-C(O)-(CH2) w - each w is independently 1 to 20; and each v is independently 1 to 10. (Item 122) The unimolecular guide molecule has the formula: [ka] or a salt thereof, wherein p' and q' are each independently an integer from 0 to 4 (inclusive), and p'+q' is an integer from 0 to 4 (inclusive); and u' is an integer from 2 to 14 (inclusive). (Item 123) 90. The method of any one of items 86-89, wherein (a) the first reactive group comprises a 2' or 3' hydroxyl moiety and the second reactive group comprises a 5' phosphate moiety, or (b) the first reactive group comprises a 5' phosphate moiety and the second reactive group comprises a 2' or 3' hydroxyl moiety, and the conjugating step comprises reacting the hydroxyl moiety and the phosphate moiety in the presence of an activating agent to form a phosphodiester bond. (Item 124) Item 124. The method of item 123, wherein the activating agent is a carbodiimide or a salt thereof. (Item 125) Item 125. The method of item 124, wherein the carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N,N'-dicyclohexylcarbodiimide (DCC), or N',N'-diisopropylcarbodiimide (DIC), or a salt thereof. (Item 126) 126. The method of any one of items 123 to 125, wherein the conjugating step comprises reacting the hydroxyl moiety and the phosphate moiety in the presence of an activating agent and a stabilizing agent. (Item 127) Item 127. The method of item 126, wherein the stabilizer is imidazole, cyanoimidazole, pyridine, or dimethylaminopyridine, or a salt thereof. (Item 128) 128. The method according to any one of items 123 to 127, wherein the concentration of each of the first and second oligonucleotides is in the range of 10 μM to 1 mM. (Item 129) 129. The method according to any one of items 123 to 128, wherein the concentration of the activator is in the range of 1 mM to 100 mM. (Item 130) 130. The method according to any one of items 123 to 129, wherein the concentration of the activator is 100 to 1,000 times higher than the concentration of each of the first and second oligonucleotides. (Item 131) 131. The method according to any one of items 123 to 130, wherein the conjugating step is carried out at a pH in the range of 5 to 9. (Item 132) 132. The method according to any one of items 123 to 131, wherein the conjugating step is carried out in the presence of a divalent metal cation. (Item 133) 133. The method according to any one of items 123 to 132, wherein the conjugating step is carried out at a temperature ranging from 0°C to 40°C. (Item 134) The first oligonucleotide has the formula: [ka] or a salt thereof; and said second oligonucleotide has the formula: [ka] or a salt thereof; During the ceremony: (N) c and (N) twherein each N is independently a nucleotide residue, optionally modified, each independently linked to its adjacent nucleotide via a phosphodiester, phosphorothioate, phosphonoacetate, thiophosphonoacetate, or phosphoramidate linkage; (N) c is (N) t and (N) is complementary or partially complementary to the 5' region of t a 3' region that forms a duplex with the 5' region of B1 and B2 are each independently a nucleobase; c is an integer equal to or greater than 20; t is an integer equal to or greater than 20; R' is independently H, OH, fluoro, chloro, bromo, NH, SH, S-R', or O-R', where each R' is independently a protecting group or an alkyl group, where said alkyl group may be optionally substituted; and [ka] each independently represents a phosphodiester bond, a phosphorothioate bond, a phosphonoacetate bond, a thiophosphonoacetate bond, or a phosphoramidate bond. (Item 135) The unimolecular guide molecule has the formula: [ka] or a pharma- ceutically acceptable salt thereof. (Item 136) The unimolecular guide molecule has the formula: [ka] or a pharma- ceutically acceptable salt thereof; During the ceremony: Z represents a nucleotide loop 4 to 6 nucleotides in length, optionally 4 or 6 nucleotides in length; u is an integer from 2 to 22, inclusive; s is an integer from 1 to 10, inclusive; x is an integer from 1 to 3, inclusive; y is >x and is an integer from 3 to 5, inclusive; m is an integer equal to or greater than 15; n is an integer equal to or greater than 30; N is each independently a nucleotide residue, optionally modified, linked to its adjacent nucleotide via a phosphodiester, phosphorothioate, phosphonoacetate, thiophosphonoacetate, or phosphoramidate linkage; 136. The method of any one of items 123 to 135, wherein N----N each independently represent two complementary nucleotides, optionally comprising at least one 2'-5' phosphodiester bond in the duplex region, optionally two complementary nucleotides base-paired by hydrogen bonds. (Item 137) 137. A composition comprising a plurality of guide molecules produced by the method of any one of items 86 to 136, wherein less than about 10% of the guide molecules comprise a truncation at the 5' end relative to a reference guide molecule sequence. (Item 138) 138. The composition of claim 137, wherein at least about 99% of the guide molecules comprise a 5' sequence comprising nucleotides 1 to 20 of the guide molecule that is 100% identical to the corresponding 5' sequence of the reference guide molecule sequence. (Item 139) formula: [ka] A composition comprising a synthetic unimolecular guide molecule for a CRISPR system or a pharma- ceutical acceptable salt thereof, wherein the synthetic unimolecular guide molecule is, in the presence of an activator, capable of forming a phosphodiester bond: [ka] or a salt thereof, During the ceremony: (N) c and (N) t wherein each N is independently a nucleotide residue, optionally modified, each independently linked to its adjacent nucleotide via a phosphodiester, phosphorothioate, phosphonoacetate, thiophosphonoacetate, or phosphoramidate linkage; (N) c is (N) t and (N) is complementary or partially complementary to the 5' region of t a 3' region that forms a duplex with the 5' region of c is an integer equal to or greater than 20; t is an integer equal to or greater than 20; B1 and B2 are each independently a nucleobase; and [ka] each independently represents a phosphodiester linkage, a phosphorothioate linkage, a phosphonoacetate linkage, a thiophosphonoacetate linkage, or a phosphoramidate linkage. (Item 140) 1. An oligonucleotide for synthesizing a unimolecular guide molecule for a type II CRISPR system, comprising the formula: [ka] or a salt thereof, wherein: R2' and R3' are each independently H, OH, fluoro, chloro, bromo, NH2, SH, S-R', or O-R', where each R' is independently a protecting group or an alkyl group, where said alkyl group may be optionally substituted; (N) c and (N) t wherein each N is independently a nucleotide residue, optionally modified, each independently linked to its adjacent nucleotide via a phosphodiester, phosphorothioate, phosphonoacetate, thiophosphonoacetate, or phosphoramidate linkage; (N) c comprises a 5' region that includes a target domain that is fully or partially complementary to a target domain within the target sequence, and a 3' region that includes at least a portion of a repeat from a type II CRISPR system; (N) t comprises a 5' region that includes at least a portion of an anti-repeat from a type II CRISPR system; c is an integer equal to or greater than 20; t is an integer equal to or greater than 20; L and R are each independently a non-nucleotidic linker; B1 and B2 are each independently a nucleobase; and [ka] each independently represents a phosphodiester linkage, a phosphorothioate linkage, a phosphonoacetate linkage, a thiophosphonoacetate linkage, or a phosphoramidate linkage. (Item 141) (N) c 141. The oligonucleotide of claim 140, comprising a 3' region that includes at least a portion of a repeat from a type II CRISPR system. (Item 142) (N) t142. The oligonucleotide according to claim 140 or 141, comprising a 3' region comprising one or more stem-loop structures. (Item 143) 1. An oligonucleotide intermediate for synthesizing a unimolecular guide molecule for a type II CRISPR system, comprising the formula: [ka] or a salt thereof, wherein: R2' and R3' are each independently H, OH, fluoro, chloro, bromo, NH2, SH, S-R', or O-R', where each R' is independently a protecting group or an alkyl group, where said alkyl group may be optionally substituted; (N) c and (N) t wherein each N is independently a nucleotide residue, optionally modified, each independently linked to its adjacent nucleotide via a phosphodiester, phosphorothioate, phosphonoacetate, thiophosphonoacetate, or phosphoramidate linkage; (N) c comprises a 5' region that includes a target domain that is fully or partially complementary to a target domain within the target sequence, and a 3' region that includes at least a portion of a repeat from a type II CRISPR system; (N) t comprises a 5' region that includes at least a portion of an anti-repeat from a type II CRISPR system; c is an integer equal to or greater than 20; t is an integer equal to or greater than 20; L and R are each independently a non-nucleotidic linker; B1 and B2 are each independently a nucleobase; and [ka] each independently represents a phosphodiester linkage, a phosphorothioate linkage, a phosphonoacetate linkage, a thiophosphonoacetate linkage, or a phosphoramidate linkage, an oligonucleotide intermediate. (Item 144) (N) t 144. The oligonucleotide according to claim 143, comprising a 3' region comprising one or more stem-loop structures. (Item 145) 1. An oligonucleotide intermediate for synthesizing a unimolecular guide molecule for a type II CRISPR system, comprising the formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein: R2' and R3' are each independently H, OH, fluoro, chloro, bromo, NH2, SH, S-R', or O-R', where each R' is independently a protecting group or an alkyl group, where said alkyl group may be optionally substituted; (N) c and (N) t wherein each N is independently a nucleotide residue, optionally modified, each independently linked to its adjacent nucleotide via a phosphodiester, phosphorothioate, phosphonoacetate, thiophosphonoacetate, or phosphoramidate linkage; (N) c comprises a 5' region that includes a target domain that is fully or partially complementary to a target domain within the target sequence, and a 3' region that includes at least a portion of a repeat from a type II CRISPR system; (N) t comprises a 5' region that includes at least a portion of an anti-repeat from a type II CRISPR system; c is an integer equal to or greater than 20; t is an integer equal to or greater than 20; L and R are each independently a non-nucleotidic linker; B1 and B2 are each independently a nucleobase; and [ka] each independently represents a phosphodiester linkage, a phosphorothioate linkage, a phosphonoacetate linkage, a thiophosphonoacetate linkage, or a phosphoramidate linkage, an oligonucleotide intermediate. (Item 146) (N) t 146. The oligonucleotide according to claim 145, comprising a 3' region comprising one or more stem-loop structures. (Item 147) 1. A composition for synthesizing a unimolecular guide molecule for a type II CRISPR system, comprising an intermediate having an annealed duplex, the intermediate having the formula: [ka] or a salt thereof, wherein: L1 and R1 are each independently a non-nucleotidic linker; Each R2 is independently O or S; R3 each independently represents O - or COO - and; p and q are each independently an integer from 0 to 6 (inclusive), and p+q is an integer from 0 to 6 (inclusive); u is an integer from 2 to 22, inclusive; s is an integer from 1 to 10, inclusive; x is an integer from 1 to 3, inclusive; y is >x and is an integer from 3 to 5, inclusive; m is an integer equal to or greater than 15; n is an integer equal to or greater than 30; N is each independently a nucleotide residue, optionally a modified nucleotide residue, each independently linked to its adjacent nucleotide via a phosphodiester, phosphorothioate, phosphonoacetate, thiophosphonoacetate, or phosphoramidate bond; and A composition in which N----N each independently represents two complementary nucleotides, optionally two complementary nucleotides base paired by hydrogen bonds. (Item 148) Item 148. The composition according to item 147, wherein p and q are each 0. (Item 149) Item 148. The composition according to item 147, wherein u is an integer from 3 to 22. (Item 150) The intermediate has the formula: [ka] or a salt thereof. (Item 151) The intermediate has the formula: [ka] or a salt thereof. (Item 152) 152. The composition according to item 150 or 151, wherein p' and q' are each 0. (Item 153) 152. The composition according to item 150 or 151, wherein u′ is an integer from 3 to 22. (Item 154) (N) m 154. The composition of any one of items 147 to 153, comprising a 5' region that includes a targeting domain that is fully or partially complementary to a targeting domain in the target sequence. (Item 155) (N) n155. The composition according to any one of items 147 to 154, comprising a 3' region comprising one or more stem-loop structures. (Item 156) 1. An oligonucleotide for synthesizing a unimolecular guide molecule for a type II CRISPR system, comprising the formula: [ka] or a salt thereof, wherein: R2' and R3' are each independently H, OH, fluoro, chloro, bromo, NH2, SH, S-R', or O-R', where each R' is independently a protecting group or an alkyl group, where said alkyl group may be optionally substituted; L and R are each independently a non-nucleotidic linker; B1 and B2 are each independently a nucleobase; (N) c and (N) t wherein each N is independently a nucleotide residue, optionally modified, each independently linked to its adjacent nucleotide via a phosphodiester, phosphorothioate, phosphonoacetate, thiophosphonoacetate, or phosphoramidate linkage; (N) c is (N) t (N) t a 3' region that forms a duplex with the 5' region of c is an integer equal to or greater than 20; t is an integer equal to or greater than 20; and [ka] each independently represents a phosphodiester linkage, a phosphorothioate linkage, a phosphonoacetate linkage, a thiophosphonoacetate linkage, or a phosphoramidate linkage. (Item 157) (N) c 157. The oligonucleotide of claim 156, comprising a 3' region that includes at least a portion of a repeat from a type II CRISPR system. (Item 158) (N) t 158. The oligonucleotide according to item 156 or 157, comprising a 3' region comprising one or more stem-loop structures. (Item 159) 1. An oligonucleotide intermediate for synthesizing a unimolecular guide molecule for a type II CRISPR system, comprising the formula: [ka] or a salt thereof, During the ceremony: R6 and R7 are each independently substituted or unsubstituted alkyl, or substituted or unsubstituted carbocyclyl; (N) t comprises a 5' region containing at least a portion of an anti-repeat from a type II CRISPR system, wherein (N) t wherein each N is independently a nucleotide residue, optionally modified, each independently linked to its adjacent nucleotide via a phosphodiester, phosphorothioate, phosphonoacetate, thiophosphonoacetate, or phosphoramidate linkage; t is an integer equal to or greater than 20; B2 is a nucleobase; and [ka] each independently represents a phosphodiester linkage, a phosphorothioate linkage, a phosphonoacetate linkage, a thiophosphonoacetate linkage, or a phosphoramidate linkage, an oligonucleotide intermediate. (Item 160) 1. A composition for synthesizing a unimolecular guide molecule for a type II CRISPR system, comprising an intermediate having an annealed duplex, the intermediate having the formula: [ka] or a salt thereof, During the ceremony: Z represents a nucleotide loop 4 to 6 nucleotides in length, optionally 4 or 6 nucleotides in length; p and q are each independently an integer from 0 to 6 (inclusive), and p+q is an integer from 0 to 6 (inclusive); u is an integer from 2 to 22, inclusive; s is an integer from 1 to 10, inclusive; x is an integer from 1 to 3, inclusive; y is >x and is an integer from 3 to 5, inclusive; m is an integer equal to or greater than 15; n is an integer equal to or greater than 30; N is each independently a nucleotide residue, optionally a modified nucleotide residue, each independently linked to its adjacent nucleotide via a phosphodiester, phosphorothioate, phosphonoacetate, thiophosphonoacetate, or phosphoramidate bond; and A composition in which N----N each independently represents two complementary nucleotides, optionally two complementary nucleotides base paired by hydrogen bonds. (Item 161) (N) m 161. The composition of claim 160, comprising a 5' region that includes a targeting domain that is fully or partially complementary to a targeting domain in the target sequence. (Item 162) (N) n 162. The composition according to item 160 or 161, comprising a 3' region comprising one or more stem-loop structures. (Item 163) 163. The composition according to any one of items 160 to 162, further comprising a carbodiimide or a salt thereof, and / or imidazole, cyanoimidazole, pyridine, and dimethylaminopyridine, or a salt thereof. (Item 164) Item 164. The composition of item 163, wherein the carbodiimide is EDC, DCC, or DIC. (Item 165) 165. The composition according to any one of items 160 to 164, comprising a carbodiimide or a salt thereof, and an imidazole or a salt thereof. (Item 166) formula: [ka] A compound of the formula: During the ceremony: (N) c and (N) t wherein each N is independently a nucleotide residue, optionally modified, each independently linked to its adjacent nucleotide via a phosphodiester, phosphorothioate, phosphonoacetate, thiophosphonoacetate, or phosphoramidate linkage; (N) c is (N) t (N) t a 3' region that forms a duplex with the 5' region of c is an integer equal to or greater than 20; t is an integer equal to or greater than 20; and [ka] each independently represent a phosphodiester bond, a phosphorothioate bond, a phosphonoacetate bond, a thiophosphonoacetate bond, or a phosphoramidate bond; L and R are each independently a non-nucleotidic linker; and The compound, wherein B1 and B2 are each independently a nucleobase. (Item 167) formula: [ka] 24. A composition comprising or consisting essentially of a guide molecule according to any one of items 1 to 23, or a pharma- ceutically acceptable salt thereof, comprising a compound represented by the formula: [ka] and / or or a pharma- ceutically acceptable salt thereof; During the ceremony: (N) c and (N) t wherein each N is independently a nucleotide residue, optionally modified, each independently linked to its adjacent nucleotide via a phosphodiester, phosphorothioate, phosphonoacetate, thiophosphonoacetate, or phosphoramidate linkage; (N) c is (N) t (N) t a 3' region that forms a duplex with the 5' region of c is an integer equal to or greater than 20; t is an integer equal to or greater than 20; and [ka] each independently represent a phosphodiester bond, a phosphorothioate bond, a phosphonoacetate bond, a thiophosphonoacetate bond, or a phosphoramidate bond; L and R are each independently a non-nucleotidic linker; and The composition, wherein B1 and B2 are each independently a nucleobase. (Item 168) formula: [ka] 24. A composition comprising or consisting essentially of a guide molecule according to any one of items 1 to 23, or a pharma- ceutically acceptable salt thereof, comprising a compound represented by the formula: [ka] and / or or a pharma- ceutically acceptable salt thereof; During the ceremony: (N) c and (N) t wherein each N is independently a nucleotide residue, optionally modified, each independently linked to its adjacent nucleotide via a phosphodiester, phosphorothioate, phosphonoacetate, thiophosphonoacetate, or phosphoramidate linkage; (N) c is (N) t (N) t a 3' region that forms a duplex with the 5' region of c is an integer equal to or greater than 20; t is an integer equal to or greater than 20; and [ka] each independently represent a phosphodiester bond, a phosphorothioate bond, a phosphonoacetate bond, a thiophosphonoacetate bond, or a phosphoramidate bond; L and R are each independently a non-nucleotidic linker; and The composition, wherein B1 and B2 are each independently a nucleobase. (Item 169) formula: [ka] A synthetic single-molecule guide molecule for a CRISPR system, comprising: During the ceremony: N is each independently a nucleotide residue, optionally a modified nucleotide residue, each independently linked to its adjacent nucleotide via a phosphodiester, phosphorothioate, phosphonoacetate, thiophosphonoacetate, or phosphoramidate bond; and N----N each independently represent two complementary nucleotides, optionally base-paired by hydrogen bonds; [ka] each independently represent a phosphodiester bond, a phosphorothioate bond, a phosphonoacetate bond, a thiophosphonoacetate bond, or a phosphoramidate bond; p and q are each 0; u is an integer from 2 to 22, inclusive; s is an integer from 1 to 10, inclusive; x is an integer from 1 to 3, inclusive; y is >x and is an integer from 3 to 5, inclusive; m is an integer equal to or greater than 15; n is an integer equal to or greater than 30; The linker is a non-nucleotidic chemical bond; B1 and B2 are each independently a nucleobase; and R2' and R3' are each independently H, OH, fluoro, chloro, bromo, NH2, SH, S-R', or O-R', where R' is each independently a protecting group or an alkyl group, where the alkyl group may be optionally substituted; (Item 170) Item 169. The guide molecule of item 169, wherein R2' and R3' are each independently selected from the group consisting of H, fluoro, and O-R', where R' is a protecting group or an optionally substituted alkyl group. (Item 171) 171. The guide molecule of item 169 or 170, wherein each N is independently a ribonucleotide residue or a sugar-modified ribonucleotide residue. (Item 172) 172. The guide molecule of any one of items 169 to 171, wherein one or more N is a deoxyribonucleotide residue. (Item 173) 173. The guide molecule of any one of items 169 to 172, wherein one or more N is a 2'-O-methyl modified ribonucleotide residue. (Item 174) (N) m and / or each of the three nucleotides at the 5' end of (N) n 174. The guide molecule according to any one of items 169 to 173, wherein each of the three nucleotides at the 3' end of said guide molecule comprises a 2'-O-methyl modified ribonucleotide residue linked to its adjacent nucleotide via a phosphorothioate bond. (Item 175) (N) for a type II CRISPR system; m 175. The guide molecule of any one of items 169 to 174, comprising a 5' region that includes a target domain that is fully or partially complementary to a target domain in the target sequence. (Item 176) (N) n 176. The guide molecule of any one of items 169 to 175, comprising a 3' region comprising one or more stem-loop structures. (Item 177) 177. The guide molecule of any one of items 169 to 176, which is capable of interacting with a Cas9 molecule and mediating the formation of a Cas9 / guide ribonucleoprotein complex. (Item 178) (N) m178. The guide molecule of any one of items 169 to 177, comprising a 3' region that includes at least a portion of the repeats from a type II CRISPR system. (Item 179) (N‐‐‐N) u and (N---N) s 179. The guide molecule of any one of items 169 to 178, which does not contain an identical sequence of 3 or more nucleotides. (Item 180) (N‐‐‐N) u and (N---N) s 180. The guide molecule of item 179, wherein the guide molecule does not contain an identical sequence of 4 or more nucleotides. (Item 181) (N‐‐‐N) s contains the sequence N'UUU, UN'UU, UUN'U, or UUUN', and (N---N) u 181. The guide molecule of item 180, comprising the sequence UUUU, where N' is A, G, or C. (Item 182) 182. The guide molecule according to item 181, wherein N' is A. (Item 183) (N‐‐‐N) s The lower stem sequence of contains the sequence UUUU and (N---N) u 181. The guide molecule of item 180, comprising an N'UUU, UN'UU, UUN'U, or UUUN' sequence, where N' is A, G, or C. (Item 184) 184. The guide molecule according to item 183, wherein N' is A. (Item 185) formula: [ka] The During the ceremony: u' is an integer from 2 to 22, inclusive; and 185. The guide molecule according to any one of items 169 to 184, wherein p' and q' are each 0. (Item 186) formula: [ka] or a covariant thereof, wherein: p' and q' are each 0; and 186. The guide molecule according to item 185, wherein u' is an integer from 0 to 15 (inclusive). (Item 187) (N----N) u’ is the expression: [ka] or its covariants; 187. The guide molecule of item 186, wherein B1 is a cytosine residue and B2 is a guanine residue. (Item 188) (N----N) u’ is the expression: [ka] or its covariants; 187. The guide molecule of item 186, wherein B1 is a guanine residue and B2 is a cytosine residue. (Item 189) (N----N) u’ is the expression: [ka] or its covariants; 187. The guide molecule of item 186, wherein B1 is a guanine residue and B2 is a cytosine residue. (Item 190) formula: [ka] or a covariant thereof, wherein: p' and q' are each 0; and 186. The guide molecule according to item 185, wherein u' is an integer from 0 to 15 (inclusive). (Item 191) (N----N) u’ is the expression: [ka] or its covariants; 191. The guide molecule of item 190, wherein B1 is a cytosine residue and B2 is a guanine residue. (Item 192) (N----N) u’ is the expression: [ka] or its covariants; 191. The guide molecule of item 190, wherein B1 is a guanine residue and B2 is a cytosine residue. (Item 193) (N----N) u’ is the expression: [ka] or its covariants; 191. The guide molecule of item 190, wherein B1 is a guanine residue and B2 is a cytosine residue. (Item 194) formula: [ka] The During the ceremony: u' is an integer from 2 to 22, inclusive; and 185. The guide molecule according to any one of items 169 to 184, wherein p' and q' are each 0. (Item 195) formula: [ka] or its covariants, During the ceremony: u' is an integer between 0 and 19, inclusive; and 195. The guide molecule of item 194, wherein p' and q' are each 0. (Item 196) (N----N) u’ is the expression: [ka] or its covariants; 196. The guide molecule of item 195, wherein B1 is an adenine residue and B2 is a uracil residue. (Item 197) (N----N) u’ is the expression: [ka] or its covariants; 196. The guide molecule of item 195, wherein B1 is a uracil residue and B2 is an adenine residue. (Item 198) (N----N) u’ is the expression: [ka] or its covariants; 196. The guide molecule of item 195, wherein B1 is a guanine residue and B2 is a cytosine residue. (Item 199) formula: [ka] or its covariants, During the ceremony: u' is an integer between 0 and 19, inclusive; and 185. The guide molecule according to any one of items 169 to 184, wherein p' and q' are each 0. (Item 200) (N----N) u’ is the expression: [ka] or its covariants; 199. The guide molecule of claim 199, wherein B1 is an adenine residue and B2 is a uracil residue. (Item 201) (N----N) u’ is the expression: [ka] or its covariants; 199. The guide molecule of claim 199, wherein B1 is a uracil residue and B2 is an adenine residue. (Item 202) (N----N) u’ is the expression: [ka] or its covariants; 199. The guide molecule of claim 199, wherein B1 is a guanine residue and B2 is a cytosine residue. (Item 203) formula: [ka] The During the ceremony: L1 and R1 are each independently a non-nucleotidic linker; Each R2 is independently O or S; R3 each independently represents O - or COO - 185. The guide molecule according to any one of items 169 to 184, (Item 204) formula: [ka] wherein: u' is an integer from 2 to 22, inclusive; and 204. The guide molecule of item 203, wherein p' and q' are each 0. (Item 205) formula: [ka] or a covariant thereof, wherein: p' and q' are each 0; and 205. The guide molecule according to item 204, wherein u' is an integer from 0 to 19, inclusive. (Item 206) (N----N) u’ is the expression: [ka] or its covariants; 206. The guide molecule of item 205, wherein B1 is a cytosine residue and B2 is a guanine residue. (Item 207) (N----N) u’ is the expression: [ka] or its covariants; 206. The guide molecule of item 205, wherein B1 is a guanine residue and B2 is a cytosine residue. (Item 208) (N----N) u’ is the expression: [ka] or its covariants; 206. The guide molecule of item 205, wherein B1 is a guanine residue and B2 is a cytosine residue. (Item 209) formula: [ka] or a covariant thereof, wherein: p' and q' are each 0; and 204. The guide molecule according to item 203, wherein u' is an integer from 0 to 19, inclusive. (Item 210) (N----N) u’ is the expression: [ka] or its covariants; 209. The guide molecule of claim 209, wherein B1 is a cytosine residue and B2 is a guanine residue. (Item 211) (N----N) u’ is the expression: [ka] or its covariants; 209. The guide molecule of claim 209, wherein B1 is a guanine residue and B2 is a cytosine residue. (Item 212) (N----N) u’ is the expression: [ka] or its covariants; 209. The guide molecule of claim 209, wherein B1 is a guanine residue and B2 is a cytosine residue. (Item 213) formula: [ka] wherein: u' is an integer between 0 and 22, inclusive; and 204. The guide molecule of item 203, wherein p' and q' are each 0. (Item 214) formula: [ka] or its covariants, During the ceremony: u' is an integer between 0 and 19, inclusive; and 214. The guide molecule of item 213, wherein p' and q' are each 0. (Item 215) (N----N) u’ is the expression: [ka] or its covariants; 215. The guide molecule of item 214, wherein B1 is an adenine residue and B2 is a uracil residue. (Item 216) (N----N) u’ is the expression: [ka] or its covariants; 215. The guide molecule of item 214, wherein B1 is a uracil residue and B2 is an adenine residue. (Item 217) (N----N) u’ is the expression: [ka] or its covariants; 215. The guide molecule according to item 214, wherein B1 is a guanine residue and B2 is a cytosine residue. (Item 218) formula: [ka] or its covariants, During the ceremony: u' is an integer between 0 and 19, inclusive; and 204. The guide molecule of item 203, wherein p' and q' are each 0. (Item 219) (N----N) u’ is the expression: [ka] or its covariants; 219. The guide molecule of item 218, wherein B1 is an adenine residue and B2 is a uracil residue. (Item 220) (N----N) u’ is the expression: [ka] or its covariants; 219. The guide molecule of item 218, wherein B1 is a uracil residue and B2 is an adenine residue. (Item 221) (N----N) u’ is the expression: [ka] or its covariants; 219. The guide molecule of item 218, wherein B1 is a guanine residue and B2 is a cytosine residue. (Item 222) 222. A composition comprising a plurality of synthetic guide molecules according to any one of items 169 to 221, wherein less than about 10% of the guide molecules comprise a truncation at the 5' end relative to a reference guide molecule sequence. (Item 223) 223. The composition of claim 222, wherein at least about 99% of the guide molecules comprise a 5' sequence comprising nucleotides 1 to 20 of the guide molecule that is 100% identical to the corresponding 5' sequence of the reference guide molecule sequence. (Item 224) From 5' to 3': A first guide molecule fragment, target domain sequence; 1st lower stem sequence; First bulge arrangement; First upper stem array a first guide molecule fragment comprising: Non-nucleotide chemical bonds; and A second guide molecule fragment, second upper stem array; A second bulge arrangement; and Second Lower Stem Array a second guide molecule fragment comprising Including, wherein (a) at least one nucleotide in the first lower stem sequence base pairs with a nucleotide in the second lower stem sequence, and (b) at least one nucleotide in the first upper stem sequence base pairs with a nucleotide in the second upper stem sequence. (Item 225) (c) the guide molecule does not include a tetraloop sequence between the first and second upper stem sequences. (Item 226) 226. The guide molecule of item 224 or 225, wherein the first and / or second upper stem sequence comprises 4 to 22 nucleotides (inclusive). (Item 227) 227. The guide molecule according to any one of items 224 to 226, characterized in that the Gibbs free energy (ΔG) for the formation of a duplex between the first and second guide molecule fragments is smaller than the ΔG for the formation of a duplex between two first guide molecule fragments. (Item 228) 228. The guide molecule of item 227, wherein the ΔG for formation of a duplex between the first and second guide molecule fragments characterized by greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, or greater than 95% base pairing between (i) the first and second upper stem sequences and (ii) the first and second lower stem sequences is less than the ΔG for formation of a duplex characterized by less than 50%, less than 60%, less than 70%, less than 80%, less than 90%, or less than 95% base pairing between (i) and (ii). (Item 229) 226. The guide molecule of claim 224 or 225, wherein the non-nucleotide chemical bond covalently links a first nucleotide at or near the 3' end of the first upper stem sequence to a second nucleotide at or near the 5' end of the second upper stem sequence. (Item 230) 230. The guide molecule of claim 229, wherein the first and second nucleotides are base paired. (Item 231) 230. The guide molecule of claim 229, wherein the Gibbs free energy (ΔG) for formation of a duplex between the first and second guide molecule fragments including base pairing of the first and second nucleotides is less than ΔG for formation of a duplex between the first and second guide molecule fragments where the first and second nucleotides are not base paired. (Item 232) 232. The guide molecule of any one of items 224 to 231, wherein the non-nucleotide chemical bond comprises urea. (Item 233) A composition comprising a guide molecule according to any one of items 224 to 232. (Item 234) 234. The composition of claim 233, wherein more than 90% of the guide molecules in the composition are full-length guide molecules. (Item 235) 235. The composition according to item 233 or 234, characterized in that more than 85% of the guide molecules in the composition contain identical target domain sequences. (Item 236) 236. The composition according to any one of items 233 to 235, wherein the target domain sequence is a predetermined target sequence. (Item 237) 237. The composition of any one of claims 233 to 236, further comprising a Cas9 protein, wherein the guide molecule and the Cas9 protein form a complex capable of interacting with a target nucleic acid comprising (x) a sequence complementary to the target domain sequence, and (y) a protospacer adjacent motif (PAM) sequence recognized by the Cas9 protein. (Item 238) 238. The composition of claim 237, wherein the complex forms a single-stranded or double-stranded break in the target nucleic acid. (Item 239) 238. The composition of claim 237, wherein the complex chemically modifies the target nucleic acid or a protein associated with the target nucleic acid. (Item 240) A genome editing system comprising the guide molecule described in any one of items 224 to 232. (Item 241) A guide molecule according to any one of items 224 to 232, a composition according to any one of items 233 to 239, or a genome editing system according to item 240 for use in therapy. (Item 242) 239. A guide molecule according to any one of items 224 to 232, a composition according to any one of items 233 to 239, or a genome editing system according to item 240 for use in the manufacture of a medicament. (Item 243) A guide molecule according to any one of items 224 to 232, a composition according to any one of items 233 to 239, or a genome editing system according to item 240 for use in modifying a cell of a subject ex vivo. (Item 244) A guide molecule according to any one of items 224 to 232, a composition according to any one of items 233 to 239, or a genome editing system according to item 240 for use in modifying a cell of a subject in vivo. (Item 245) A method for altering a nucleic acid in a cell or a subject, the method comprising administering to the subject a guide molecule according to any one of items 1-42, 169-221, or 224-232, or a composition according to any one of items 43-85, 137-139, 147-155, 161-165, 222, 223, or 233-239. (Item 246) 3. A composition consisting essentially of a plurality of synthetic molecular guide molecules according to any one of items 1-42, 169-221, or 224-232. (Item 247) A composition consisting essentially of a plurality of guide molecules produced by the method of any one of items 86 to 137 and a pharma- ceutically acceptable carrier. (Item 248) 138. The method according to any one of items 86 to 137, wherein the guide molecule is capable of acting as a substrate for an enzyme acting on RNA. (Item 249) 249. The method of claim 248, wherein the enzyme is a reverse transcriptase. [Brief description of the drawings]

[0055] [Figure 1A] FIG. 1A illustrates an exemplary cross-linking reaction process according to certain embodiments of the present disclosure. [Figure 1B] FIG. 1B illustrates a two-dimensional schematic of an exemplary S. pyogenes guide molecule highlighting the positions (marked with stars) where the first and second guide molecule fragments are crosslinked to each other, according to various embodiments of the present disclosure. [Figure 1C] FIG. 1C illustrates a two-dimensional schematic of an exemplary S. aureus guide molecule highlighting the positions (marked with stars) where the first and second guide molecule fragments are crosslinked to each other, according to various embodiments of the present disclosure. [Figure 2A] FIG. 2A illustrates steps in an exemplary cross-linking reaction process according to certain embodiments of the present disclosure. [Figure 2B] FIG. 2B illustrates steps in an exemplary cross-linking reaction process according to certain embodiments of the present disclosure. [Figure 2C] FIG. 2C illustrates an additional step in an exemplary cross-linking reaction process using the reaction products from FIGS. 2A and 2B. [Figure 3A] FIG. 3A illustrates an exemplary cross-linking reaction process according to certain embodiments of the present disclosure. [Figure 3B] FIG. 3B illustrates steps in an exemplary cross-linking reaction process according to certain embodiments of the present disclosure. [Figure 3C]FIG. 3C shows a two-dimensional schematic of an exemplary S. pyogenes guide molecule highlighting the positions where the first and second guide molecule fragments are crosslinked to each other, according to various embodiments of the present disclosure. [Figure 3D] FIG. 3D shows a two-dimensional schematic of an exemplary S. aureus guide molecule highlighting the positions where the first and second guide molecule fragments are crosslinked to each other, according to various embodiments of the present disclosure. [Figure 4] Figure 4 shows the DNA cleavage dose-response curves of synthetic unimolecular guide molecules according to certain embodiments of the present disclosure compared to unligated annealed guide molecule fragments and guide molecules prepared by IVT obtained from a commercial supplier. DNA cleavage was assayed by the T7E1 assay described herein. As the graph shows, the conjugated guide molecules supported cleavage in HEK293 cells in a dose-dependent manner consistent with that observed with unimolecular guide molecules prepared by IVT or synthetic unimolecular guide molecules. It should be noted that the annealed unconjugated guide molecule fragments supported a similar dose-dependent, but lower level, cleavage. [Figure 5AB] Figure 5A shows a representative ion chromatograph, and Figure 5B shows a deconvoluted mass spectrum of an ion-exchange purified guide molecule conjugated with a urea linker according to the process of Example 1. Figure 5C shows a representative ion chromatograph, and Figure 5D shows a deconvoluted mass spectrum of a commercially available synthetic unimolecular guide molecule. The mass spectrum was evaluated for the peaks highlighted in the ion chromatograph. Figure 5E shows a magnified version of the mass spectrum. The mass spectrum of the commercially available synthetic unimolecular guide molecule is on the left (34% purity relative to total mass), and the mass spectrum of the guide molecule conjugated with a urea linker according to the process of Example 1 is on the right (72% purity relative to total mass). [Figure 5C-D] Same as above. [Figure 5E] Same as above. [Figure 6AB]FIG. 6A shows a plot representing the frequency of individual base and length changes at each position from the 5' end of a complementary DNA (cDNA) made from a synthetic unimolecular guide molecule containing a urea bond, and FIG. 6B shows a plot representing the frequency of individual base and length changes at each position from the 5' end of a cDNA made from a commercially available synthetic unimolecular guide molecule (i.e., prepared without conjugation). The box encloses the 20 bp target domain of the guide molecule. FIG. 6C shows a plot representing the frequency of individual base and length changes at each position from the 5' end of a cDNA made from a synthetic unimolecular guide molecule containing a thioether bond. [Figure 6C] Same as above. [Figure 7A] Figures 7A and 7B are graphs showing the change in internal sequence length (+5 to -5) at the first 41 positions counting from the 5' end of cDNA made from various synthetic unimolecular guide molecules containing urea linkages (Figure 7A) and from a commercially available synthetic unimolecular guide molecule (i.e., prepared without conjugation) (Figure 7B). [Figure 7B] Same as above. [Figure 8A] 8A-8H show in two-dimensional schematic diagrams the structures of certain exemplary guide molecules according to various embodiments of the present disclosure. Complementary bases capable of base pairing are indicated by one (AU or AT pairing) or two (GC) horizontal lines between the bases. Bases capable of non-Watson-Crick pairing are indicated by a circle and a single horizontal line. [Figure 8B] Same as above. [Figure 8C] Same as above. [Figure 8D] Same as above. [Figure 8E] Same as above. [Figure 8F] Same as above. [Figure 8G] Same as above. [Figure 8H] Same as above. [Figure 9A]9A-9D show in two-dimensional schematic diagrams the structure of certain exemplary guide molecules according to various embodiments of the present disclosure. Complementary bases capable of base pairing are indicated by one (AU or AT pairing) or two (GC) horizontal lines between the bases. Bases capable of non-Watson-Crick pairing are indicated by a circle and a single horizontal line. [Figure 9B] Same as above. [Figure 9C] Same as above. [Figure 9D] Same as above. [Figure 10A] 10A-10D show in two-dimensional schematic diagrams the structure of certain exemplary guide molecules according to various embodiments of the present disclosure. Complementary bases capable of base pairing are indicated by one (AU or AT pair) or two (GC) horizontal lines between the bases. Bases capable of non-Watson-Crick pairing are indicated by a circle and a single horizontal line. [Figure 10B] Same as above. [Figure 10C] Same as above. [Figure 10D] Same as above. [Figure 11] FIG. 11 shows a graph of DNA cleavage in CD34+ cells using a series of ribonucleoprotein complexes containing conjugated guide molecules from Table 10. Cleavage was assessed using next-generation sequencing technology to quantify insertion and deletion (indel) rates relative to the wild-type human reference sequence. Ligated guide molecules made according to Example 1 support DNA cleavage in CD34+ cells. Indel rates were found to increase with increasing stem-loop length, but incorporation of UA exchanges adjacent to the stem-loop sequence (see gRNAs 1E, 1F, and 2D) mitigates the effect. [Figure 12A] Figure 12A shows the liquid chromatography-mass spectrometry (LC-MS) trace after T1 endonuclease digestion of gRNA 1A, and Figure 12B shows the mass spectrum of the peak at a retention time of 4.50 min (A34:G39). In particular, a fragment containing a urea bond, A-[UR]-AAUAG (A34:G39), was detected at m / z=1190.7 with a retention time of 4.50 min. [Figure 12B] Same as above. [Figure 13A] FIG. 13A shows LC-MS data for an unpurified composition of urea-linked guide molecule, with both a major product (A-2, retention time 3.25 min) and a minor product (A-1, retention time 3.14 min) present. Note that the minor product (A-1) in FIG. 13A has been enriched for illustrative purposes and is typically detected at up to 10% yield in the synthesis of guide molecules by the method of Example 1. FIG. 13B shows the deconvoluted mass spectrum of peak A-2 (retention time 3.25 min) and FIG. 13C shows the deconvoluted mass spectrum of peak A-1 (retention time 3.14 min). Analysis of each peak by mass spectrometry showed that both products have the same molecular weight. [Figure 13B] Same as above. [Figure 13C] Same as above. [Figure 14A] Figure 14A shows LC-MS data for the guide molecule composition after chemical modification as described in Example 10. The major product (B-1, urea) has the same retention time as in the original analysis (3.26 min), while the retention time of the minor product (B-2, carbamate) has shifted to 3.86 min, consistent with chemical functionalization of the free amine moiety. Figure 14B shows the mass spectrum of peak B-2 (retention time 3.86 min). Analysis of the peak at 3.86 min (M+134) indicates that the predicted functionalization has occurred. [Figure 14B] Same as above. [Figure 15A]Figure 15A shows the LC-MS trace of the fragment mixture after digestion with T1 endonuclease of the reaction mixture containing both the major product (urea) and the chemically modified minor product (carbamate). Both the urea bond (G35-[UR]-C36) and the chemically modified carbamate bond (G35-[CA+PAA]-C36) were detected at retention times of 4.31 minutes and 5.77 minutes, respectively. Figure 15B shows the mass spectrum of the peak at 4.31 minutes, where m / z=532.13 is assigned to [M-2H]2-, and Figure 15C shows the mass spectrum of the peak at 5.77 minutes, where m / z=599.15 is assigned to [M-2H]2-. Figure 15D and Figure 15E show LC / MS-MS collision-induced dissociation (CID) experiments of m / z=532.1 from Figure 15B and m / z=599.1 from Figure 15C. In Figure 15D, typical ad and xz ions were observed, as well as MS / MS fragment ions on both sides of the UR bond from the 5' end (m / z=487.1 and 461.1) and 3' end (m / z=603.1 and 577.1). In Figure 15E, only two product ions were observed, including MS / MS fragment ions from the carbamate bond 5' end (m / z=595.2) and the CA bond 3' end (m / z=603.1). [Figure 15B] Same as above. [Figure 15C] Same as above. [Figure 15D] Same as above. [Figure 15E] Same as above. [Figure 16A]FIG. 16A shows LC-MS data of the crude reaction mixture for reaction with the 2'-H modified 5' guide molecule fragment (upper spectrum) compared to the crude reaction mixture for reaction with the unmodified version of the same 5' guide molecule (lower spectrum). With the 2'-H modified 5' guide molecule fragment, no formation of the carbamate by-product is observed (upper spectrum). In contrast, the crude reaction mixture for reaction with the unmodified version of the same 5' guide molecule fragment (lower spectrum) contained a mixture of the major urea coupling product (A-2) and a trace of the carbamate by-product (A-1). Note that unlike Example 10, the carbamate by-product was not concentrated and was therefore detected at a much lower level than in FIG. 13A of Example 10. Figure 16B shows the deconvoluted mass spectrum of peak B (retention time 3.14 min, upper spectrum in Figure 16A) and Figure 16C shows the deconvoluted mass spectrum of peak A-2 (retention time 3.45 min, lower spectrum in Figure 16A). Analysis of the products of the reaction with the 2'-H modified 5' guide molecule fragment (B) gave M-16 (compared to A-2, the unmodified urea-linked main product) as expected for a molecule in which the 2'-OH was replaced with a 2'-H (see Figures 16B and 16C). [Figure 16B] Same as above. [Figure 16C] Same as above. [Figure 17A] Figure 17A shows the LC-MS trace after T1 endonuclease digestion of gRNA 1L, and Figure 17B shows the mass spectrum of the peak at a retention time of 4.65 min (A34:G39). In particular, a fragment containing a urea bond, A-[UR]-AAUAG (A34:G39), was detected at a retention time of 4.65 min at m / z=1182.7. [Figure 17B] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0056] Definitions and Abbreviations Unless otherwise stated, each of the following terms has the meaning associated with it in this section.

[0057] The indefinite articles "a" and "an" refer to at least one of the associated noun and are used interchangeably with the terms "at least one" and "one or more." For example, "a module" means at least one module, or one or more modules.

[0058] The conjunctions "or" and "and / or" are used interchangeably as non-exclusive disjunctions.

[0059] The phrase "consisting essentially of" means that the recited species is the predominant species, but other species may be present in minor amounts or in amounts that do not affect the structure, function, or behavior of the subject composition. For example, a composition consisting essentially of a particular species will generally comprise 90%, 95%, 96%, or more (by mass or molar concentration) of that species.

[0060] The phrase "substantially free of a molecule" means that the molecule is not the major component in the recited composition. For example, a composition that is substantially free of a molecule means that the molecule is present in the composition at less than 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% (by mass or molar concentration). The amount of a molecule can be determined by various analytical techniques, for example, as described in the Examples. In some embodiments, the compositions provided herein are substantially free of a particular molecule, and the molecule is present at less than 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% (by mass or molar concentration) as determined by gel electrophoresis. In some embodiments, the compositions provided herein are substantially free of a particular molecule, and the molecule is present at less than 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% (by mass or molar concentration) as determined by mass spectrometry.

[0061] "Domain" is used to describe a segment of a protein or nucleic acid. Unless otherwise specified, a domain does not necessarily have any particular functional properties.

[0062] The term "complementary" refers to a pair of nucleotides that can form a stable base pair through hydrogen bonds. For example, U is complementary to A, and G is complementary to C. As will be understood by those skilled in the art, whether a particular complementary nucleotide pair is bound through hydrogen bond base pairing (e.g., within a guide molecule duplex) may depend on the context (e.g., surrounding nucleotides and chemical bonds) and external conditions (e.g., temperature and pH). Therefore, it should be understood that complementary nucleotides are not necessarily associated through hydrogen bond base pairing.

[0063] A "covariant" sequence differs from a reference sequence by the substitution of one or more nucleotides in the reference sequence with complementary nucleotides (e.g., one or more U's are substituted with A's, one or more G's are substituted with C's, etc.). When used in reference to a region that contains two complementary sequences that form a duplex (e.g., the upper stem of a guide molecule), the term "covariant" encompasses duplexes that have one or more nucleotide exchanges (i.e., one or more AU exchanges and / or one or more GC exchanges) between the two complementary sequences of the reference duplex, as illustrated in Table 1 below.

[0064] [Table 1]

[0065] In some embodiments, a covariant sequence may exhibit substantially the same energetic favorability of a particular annealing reaction (e.g., duplex formation in the context of a guide molecule of the present disclosure) as a reference sequence. As described elsewhere in this disclosure, the energetic favorability of a particular annealing reaction may be measured empirically or predicted using computational models.

[0066] "Indel" is an insertion and / or deletion in a nucleic acid sequence.Indel can be the product of repair of DNA double-strand break, such as the double-strand break formed by the genome editing system of the present disclosure.Indel is most commonly formed when the break is repaired by "error-prone" repair pathway, such as the NHEJ pathway described below.

[0067] "Gene conversion" refers to the modification of DNA sequence by the incorporation of endogenous homologous sequence (e.g., homologous sequence in gene array). "Gene correction" refers to the modification of DNA sequence by the incorporation of exogenous homologous sequence, such as exogenous single-stranded or double-stranded donor template DNA. Gene conversion and gene correction are the products of DNA double-strand break repair by HDR pathway as described below.

[0068] The results of indels, gene conversions, gene corrections, and other genome editing are typically assessed by sequencing (most commonly "next-generation" or "sequencing by synthesis" methods, although Sanger sequencing can still be used) and quantified by the relative frequency of numerical changes (e.g., ±1, ±2, or more bases) at the site of interest among all sequencing reads. DNA samples for sequencing can be prepared by a variety of methods known in the art, including amplification of the site of interest by polymerase chain reaction (PCR), capture of DNA ends created by double-strand breaks as in the GUIDEseq process described in Tsai et al. (Nat. Biotechnol. 34(5):483 (2016), incorporated herein by reference), or by other means known in the art. The results of genome editing can also be assessed by in situ hybridization methods, such as the FiberComb™ system commercially available from Genomic Vision (Bagneux, France), and other suitable methods known in the art.

[0069] "Alt-HDR", "alternative homology directed repair" or "alternative HDR" are used interchangeably to mean the repair process of DNA damage using homologous nucleic acid (e.g., endogenous homologous sequence, e.g., sister chromatid, or foreign nucleic acid, e.g., template nucleic acid). Alt-HDR differs from standard HDR in that the process utilizes a different pathway from standard HDR and can be inhibited by standard HDR mediators, RAD51 and BRCA2. Alt-HDR is also distinguished by the involvement of single-stranded or nicked homologous nucleic acid template, whereas standard HDR generally involves double-stranded homologous template.

[0070] "Standard HDR", "standard homologous recombination repair" or "cHDR" refers to the repair process of DNA damage using a homologous nucleic acid (e.g., an endogenous homologous sequence, e.g., a sister chromatid, or a foreign nucleic acid, e.g., a template nucleic acid). Standard HDR typically functions upon significant excision at a double-stranded break, forming at least one single-stranded portion of DNA. In normal cells, cHDR typically includes a series of steps, such as recognition of the break, stabilization of the break, excision, stabilization of single-stranded DNA, formation of a DNA crossover intermediate, resolution of this crossover intermediate, and ligation. This process requires RAD51 and BRCA2, and the homologous nucleic acid is typically double-stranded.

[0071] Unless otherwise specified, the term "HDR", as used herein, encompasses both standard HDR and alt-HDR.

[0072] "Non-homologous end joining" or "NHEJ" refers to ligation-mediated repair and / or non-template-mediated repair, including canonical NHEJ (cNHEJ) and alternative NHEJ (altNHEJ), which in turn includes microhomology-mediated end joining (MMEJ), single-strand annealing (SSA), and synthesis-dependent microhomology-mediated end joining (SD-MMEJ).

[0073] "Substitution" or "substituted," when used in reference to modification of a molecule (eg, a nucleic acid or protein), does not require a restriction of the process but merely indicates that a replacement entity is present.

[0074] "Subject" means a human or non-human animal. A human subject may be of any age (e.g., infant, child, young adult, or adult) and may be suffering from a disease or in need of genetic modification. Alternatively, the subject may be an animal, which term includes, but is not limited to, mammals, birds, fish, reptiles, amphibians, more specifically non-human primates, rodents (such as mice, rats, hamsters), rabbits, guinea pigs, dogs, cats, and the like. In certain embodiments of the present disclosure, the subject is a livestock animal, such as a cow, horse, sheep, or goat. In certain embodiments, the subject is a poultry animal.

[0075] "Treat", "treating", and "treatment" refer to the treatment of a disease in a subject (e.g., a human subject), including one or more of: inhibiting the disease, i.e., halting or preventing its onset or progression; relieving the disease, i.e., relieving the pathology; alleviating one or more symptoms of the disease; curing the disease.

[0076] "Prevent," "preventing," and "prevention" refer to the prevention of disease in a mammal, e.g., a human, including (a) avoiding or eliminating disease; (b) affecting predisposition to illness; or (c) preventing or delaying the onset of at least one symptom of the disease.

[0077] A "kit" refers to any collection of two or more components that together constitute a functional unit that can be utilized for a particular purpose. By way of example (and not limitation), a kit according to the present disclosure may include a guide RNA that is complexed or can be complexed with an RNA-guided nuclease, and is associated with (e.g., suspended or capable of being suspended in) a pharma- ceutically acceptable carrier. The kit can be used, for example, to introduce the complex into a cell or subject to cause a desired genomic modification in such a cell or subject. The components of the kit may be packaged together or separately. A kit according to the present disclosure also optionally includes a instructions for use (DFU) that describes the use of the kit, for example, according to the methods of the present disclosure. The DFU may be physically packaged with the kit or may be made available to a user of the kit, for example, by electronic means.

[0078] The terms "polynucleotide", "nucleotide sequence", "nucleic acid", "nucleic acid molecule", "nucleic acid sequence", and "oligonucleotide" refer to a series of nucleotide bases (also called "nucleotides") in DNA and RNA, meaning any chain of two or more nucleotides. Polynucleotides, nucleotide sequences, nucleic acids, etc. can be single-stranded or double-stranded chimeric mixtures or derivatives or modified versions thereof. They can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve the stability of the molecule, its hybridization parameters, etc. Nucleotide sequences typically carry genetic information, including but not limited to information used by cellular machinery to produce proteins and enzymes. These terms include single-stranded or double-stranded genomic DNA, RNA, any synthetic and genetically engineered polynucleotides, and both sense and antisense polynucleotides. These terms also include nucleic acids containing modified bases.

[0079] Conventional IUPAC nomenclature is used for the nucleotide sequences presented herein, as shown in Table 2 below (see also Cornish-Bowden A, Nucleic Acids Res. 1985 May 10; 13(9): 3021-30, which is incorporated herein by reference). However, it should be noted that, for example, in the guide molecule targeting domain, "T" means "thymine or uracil" when the sequence can be encoded by either DNA or RNA.

[0080] [Table 2]

[0081] The terms "protein," "peptide," and "polypeptide" are used interchangeably to refer to a continuous chain of amino acids linked together through peptide bonds. The term includes individual proteins, groups or complexes of proteins associated with each other, as well as fragments or portions, variants, derivatives, and analogs of such proteins. Peptide sequences are presented herein using conventional notation starting from the amino or N-terminus on the left and proceeding to the carboxyl or C-terminus on the right. Standard one-letter or three-letter abbreviations can be used.

[0082] The term "variant" refers to an entity such as a polypeptide, polynucleotide, or small molecule that exhibits significant structural identity with a reference entity, but is structurally different from the reference entity in the presence or level of one or more chemical moieties compared to the reference entity.In many embodiments, the variant also differs functionally from the reference entity.In general, whether a particular entity is strictly considered to be a "variant" of a reference entity is based on the degree of structural identity with the reference entity.

[0083] summary Certain embodiments of the present disclosure generally relate to a method for synthesizing guide molecules, in which two or more guide fragments are (a) annealed to each other and then (b) crosslinked using an appropriate crosslinking chemistry. The inventors have found that a method that includes a step of pre-annealing the guide fragments before crosslinking them tends to improve the efficiency of crosslinking and favor the formation of the desired heterodimer product, even when homo-multifunctional crosslinkers are used. Without wishing to be bound by any theory, it is believed that the improvement in crosslinking efficiency and the resulting yield of the desired reaction product is due to the increased stability of the annealed heterodimer as a crosslinking substrate compared to the unannealed homodimer, achieved by pre-annealing, and / or the reduced fraction of free RNA fragments available to form homodimers, etc.

[0084] Methods of the present disclosure that involve pre-annealing the guide fragments have numerous advantages, including, but not limited to: allowing for the achievement of high yields even when the fragments are homomultifunctional (e.g., homobifunctional), such as the amine-functionalized fragments used in the urea-based cross-linking methods described herein; the reduction or absence of unwanted homodimers and other reaction products may in turn simplify downstream purification; and because the fragments used for cross-linking tend to be shorter than full-length guide molecules, they may exhibit lower levels of contamination with n-1 species, truncated species, n+1 species, and other contaminants than observed with full-length synthetic guide molecules.

[0085] With respect to pre-annealing, one of skill in the art will understand that longer tracts of annealed bases may be more stable than shorter tracts, and that a greater degree of annealing between two tracts of similar length is generally associated with greater stability. Thus, in certain embodiments of the present disclosure, fragments are designed to maximize the degree of annealing between the fragments and / or to place functionalized 3' or 5' ends in close proximity to annealed bases and / or to each other.

[0086] As discussed in more detail below, certain unimolecular guide molecules, particularly unimolecular Cas9 guide molecules, are characterized by a relatively large stem-loop structure. For example, Figures 1B and 1C show the two-dimensional structure of unimolecular S. pyogenes and S. aureus gRNAs, from which it is apparent that both gRNAs generally contain a relatively long stem-loop structure with a "bulge". In certain embodiments, the synthetic guide molecule includes a bridge between fragments within this stem-loop structure. This is optionally achieved by bridging a first fragment and a second fragment that have complementary regions at or near their 3' and 5' ends, respectively; the 3' and 5' ends of these fragments may be, for example, represented by the following formulas II and III: [ka] As shown in Figure 1, the fluorophenyl ethers have been functionalized to facilitate cross-linking reactions. In these formulas, p and q are each independently 0 to 6, and p+q is 0 to 6; m is 20 to 40; n is 30 to 70; ---- each independently represents a hydrogen bond between corresponding nucleotides; [ka] represents a phosphodiester bond, a phosphorothioate bond, a phosphonoacetate bond, a thiophosphonoacetate bond, or a phosphoramidate bond, respectively; N----N independently represents two complementary nucleotides, two complementary nucleotides base-paired with optional hydrogen bonds; and F1 and F2 each contain a functional group such that they can undergo a cross-linking reaction to cross-link two guide fragments. Exemplary cross-linking chemistries are described in Table 3 below.

[0087] [Table 3]

[0088] [Table 4]

[0089] Although formulas II and III show a crosslinker located within (or replacing) a "tetraloop" structure in the guide molecule repeat-antirepeat duplex, it should be understood that the crosslinker can be located anywhere within the molecule, for example, at any stem-loop structure present in the guide molecule, including naturally occurring stem-loops and engineered stem-loops. In particular, certain embodiments of the present disclosure relate to guide molecules that lack a tetraloop structure and include crosslinkers located at the ends of the first and second complementary regions (e.g., at the 3' end of the first upper stem region and the 5' end of the second upper stem region).

[0090] Formulas II and III represent guide molecules that may or may not (p>0 and q>0) include a "tetraloop" structure in the repeat-anti-repeat duplex. One aspect of the invention is the recognition that guide molecules lacking a "tetraloop" may exhibit enhanced ligation efficiency as a result of having functionalized 3' and 5' ends in close proximity and in the appropriate orientation.

[0091] Alternatively, or in addition, cross-linking reactions according to the present disclosure may include a "splint" or single-stranded oligonucleotide that hybridizes to sequences at or near the functionalized 3' and 5' ends such that the functionalized ends are stably close to one another.

[0092] Another aspect of the present invention is the recognition that guide molecules with longer duplexes (e.g., with extended upper stems) may exhibit enhanced ligation efficiency compared to guide molecules with shorter duplexes. These longer duplex structures are interchangeably referred to in the present disclosure as "extended duplexes" and are generally (but not necessarily) located in the vicinity of the functionalized nucleotide in the guide fragment. Thus, in some embodiments, the present disclosure provides a method for the preparation of a guide molecule having a longer duplex, such as the following formulas VIII and IX: [ka] The present invention provides a guide molecule for

[0093] In formulas VIII and IX, p' and q' are each independently an integer between 0 and 4, inclusive, p'+q' is an integer between 0 and 4, inclusive, u' is an integer between 2 and 22, inclusive, and the other variables are defined as in formulas II and III. Formulas VIII and IX show a duplex with an optionally extended upper stem, as well as an optional tetraloop (i.e., when p and q are 0). Guide molecules of formulas VIII and IX may be advantageous due to the increased ligation efficiency resulting from a longer upper stem. Additionally, the combination of a longer upper stem and the absence of a tetraloop may be beneficial to achieve the proper orientation of reactive groups F1 and F2 for the ligation reaction.

[0094] Another aspect of the present invention relates to the recognition that guide fragments may contain multiple regions of complementarity within a single guide fragment and / or between different guide fragments. For example, in certain embodiments of the present disclosure, the first and second guide fragments are designed with complementary upper and lower stem regions, and when fully annealed, a heterodimer is formed in which (a) the first and second functional groups are located at the ends of the duplex upper stem regions in a suitable vicinity for cross-linking reaction, and / or (b) a duplex structure is formed between the first and second guide fragments that can support the formation of a complex between the guide molecule and the RNA-guided nuclease. However, it may be possible that the first and second guide fragments anneal incompletely with each other or form an internal duplex or homodimer, thereby not resulting in (a) and / or (b). As an example, in S. pyogenes guide molecules based on wild-type crRNA and tracrRNA sequences, there may be multiple highly complementary sequences, such as poly-U or poly-A tracts in the lower and upper stems, that may result in improper "staggered" heterodimers involving annealing between the upper and lower stem regions, rather than the desired annealing of the upper stem regions to each other. Similarly, undesired duplexes may form between the target domain sequences of the guide fragments and another region of the same or a different fragment, and mispairing may occur between otherwise complementary regions of the first and second guide fragments, resulting in incomplete duplexing, bulges, and / or unpaired segments.

[0095] While it is not practical to predict all possible undesired intra- or intermolecular duplex structures that may form between guide fragments, the inventors have found that in some cases, modifications made to reduce or prevent the formation of certain mismatches or undesired duplexes can have a significant impact on the yield of the desired guide molecule product in a crosslinking reaction and / or can result in a reduction of one or more contaminating species from the same reaction. Thus, in some embodiments, the present disclosure provides guide molecules and methods in which the primary sequence of the guide fragments is designed to avoid two or more of the mismatches or undesired duplexes (e.g., by exchanging two complementary nucleotides between the first and second guide fragments). For example, an AU exchange in the upper stem of the wild-type S. pyogenes guide fragment described above would result in a first guide fragment containing non-identical UUUU and UAUU sequences, and a second guide fragment containing sequences complementary to the modified sequences of the first fragment, i.e., AAAA and AUAA sequences. More broadly, the guides may encompass sequence alterations, such as nucleotide exchanges between the two duplex portions of the upper or lower stem, insertions, deletions, or substitutions of sequences in the upper or lower stem, or structural alterations such as the incorporation of locked nucleic acids (LNAs) at positions selected to reduce or eliminate the formation of secondary structures.

[0096] Without wishing to be bound by any theory, it is believed that the duplex extensions, sequence modifications, and structural modifications described herein promote the formation of desired duplexes and reduce the formation of mismatches and undesired duplexes by increasing the energy favorability of the formation of desired duplexes over the formation of mismatches or undesired duplexes. The energy favorability of a particular annealing reaction can be expressed by Gibbs free energy (ΔG); negative ΔG values ​​are associated with spontaneous reactions, and a first annealing reaction is energetically more favorable than a second reaction when the ΔG of the first reaction is smaller (i.e., more negative) than the ΔG of the second reaction. ΔG can be empirically evaluated based on the thermal stability (melting behavior) of a particular duplex using, for example, NMR, fluorescence quenching, UV absorption, calorimetry, etc., as described in You, Tatourov and Owczarzy, “Measuring Thermodynamic Details of DNA Hybridization Using Fluorescence” Biopolymers Vol. 95, No. 7, pp. 472-486 (2011), which is incorporated by reference for all purposes (see, for example, “Introduction” at pp. 472-73 and “Materials and Methods” at pp. 473-475). However, it may be more practical to design guide fragments and annealing reactions to utilize computational models to estimate the free energies of correct duplexing and selected mismatches or undesired duplexing reactions, and numerous tools are available to perform such modeling, including the biophysics.idtdna.com tool provided by Integrated DNA Technologies (Coralville, Iowa). Alternatively, or in addition, a number of algorithms have been described in the literature that utilize thermodynamic nearest neighbor models (TNNs).See, e.g., Tulpan, Andronescu and Leger, “Free energy estimation of short DNA duplex hybridizations,” BMC Bioinformatics, Vol. 11, No. 105 (2010) (see “Background Art” on pages 1-2, which describes TNN models, as well as the MultiRNAFold package, the Vienna package and the UNAFold package). Other algorithms are also described, e.g., in Kim et al. “An evolutionary Monte Carlo algorithm for predicting DNA. hybridization,” J. Biosystems Vol. 7, No. 5 (2007) (see section 2 on page 71 which describes this model). The aforementioned documents are each incorporated herein by reference in their entirety for all purposes.

[0097] The arrangement shown in formulas II and III may be particularly advantageous when the functional group is located in a linking group containing multiple carbons. For less bulky crosslinkers, it may be desirable to achieve close juxtaposition between the functionalized 3' and 5' ends. Figures 3C and 3D identify the duplex portions of S. pyogenes and S. aureus gRNAs that are amenable to the use of shorter linkers, including but not limited to phosphodiester bonds. These positions are generally selected to allow annealing between the fragments and to position the functionalized 3' and 5' ends so that they are directly adjacent to one another prior to crosslinking. Exemplary 3' and 5' positions located within (rather than adjacent to) a tract of annealed residues are shown in formulas IV, V, VI, and VII below: [ka] [ka] As shown in Z represents a nucleotide loop 4 to 6 nucleotides in length, optionally 4 or 6 nucleotides in length; p and q are each independently an integer from 0 to 2 (inclusive), optionally 0; p' is an integer from 0 to 4 (inclusive), optionally 0; q' is an integer from 2 to 4 (inclusive), optionally 2; x is an integer from 0 to 6 (inclusive), optionally 2; y is an integer from 0 to 6 (inclusive), optionally 4; u is an integer from 0 to 4 (inclusive), optionally 2; s is an integer from 2 to 6 (inclusive), optionally 4; m is an integer from 20 to 40 (inclusive); n is an integer from 30 to 70 (inclusive); B1 and B2 are each independently a nucleobase; (N) m and (N) n wherein N is each independently a nucleotide residue: N1 and N2 are each independently a nucleotide residue; N----N independently represent two complementary nucleotides, optionally two complementary nucleotides base-paired by hydrogen bonds; [ka] represent a phosphodiester bond, a phosphorothioate bond, a phosphonoacetate bond, a thiophosphonoacetate bond, or a phosphoramidate bond, respectively.

[0098] Another aspect of the present invention is the recognition that the configurations shown in any of formulas II, III, IV, V, VI, or VII may be advantageous to avoid by-products in crosslinking reactions and to allow homobifunctionalization reactions without homodimerization. Pre-annealing of the two heterodimer strands directs the reactive groups toward the desired coupling and avoids reaction with other potential reactive groups in the guide molecule.

[0099] Another aspect of the present invention is the recognition that a hydroxyl group proximal to a reactive group (e.g., the 2'-OH at the 3' end of the first fragment) is preferably modified to avoid the formation of certain by-products. In particular, as exemplified below, the inventors have found that carbamate by-products can be formed when amine-functionalized fragments are used in the urea-based cross-linking methods described herein: [ka]

[0100] Thus, in certain embodiments, the 2'-OH at the 3' end of the first fragment is modified (e.g., to H, halogen, O-Me, etc.) to prevent the formation of a carbamate by-product. For example, the 2'-OH is modified to 2'-H: [ka]

[0101] Turning now to crosslinking, the selection of the crosslinker linking moiety, functional group, and reactive group involves several considerations, including the size, aqueous solubility, and biocompatibility of the linker, as well as the reactivity of the functional groups, optimal reaction conditions for crosslinking, and any reagents or catalysts required for crosslinking.

[0102] In general, the size and solubility of the linker are selected to preserve or achieve the desired RNA secondary structure and to avoid disrupting or destabilizing the complex between the guide molecule and the RNA-guided nuclease. These two factors are somewhat related in that organic linkers beyond a certain length may be poorly soluble in aqueous solution and may sterically interfere with the surrounding nucleotides in the guide molecule and / or the amino acids of the RNA-guided nuclease complexed with the guide molecule.

[0103] A variety of linkers are suitable for use in various embodiments of the present disclosure. Certain embodiments utilize common linking moieties, including but not limited to polyvinyl ether, polyethylene, polypropylene, polyethylene glycol (PEG), polypropylene glycol (PEG), polyvinyl alcohol (PVA), polyglycolide (PGA), polylactide (PLA), polycaprolactone (PCL), and copolymers thereof. In some embodiments, no linker is used.

[0104] With respect to functional groups, in embodiments in which a bifunctional crosslinker is used to link the 5' and 3' ends of the guide fragments, the 3' or 5' ends of the guide fragments to be linked are modified with functional groups that react with the reactive groups of the crosslinker. Generally, these modifications include one or more of amines, sulfhydryls, carboxyls, hydroxyls, alkenes (e.g., terminal alkenes), azides, and / or other suitable functional groups. Multifunctional (e.g., bifunctional) crosslinkers are also generally known in the art and may be either heterofunctional or homofunctional, and include isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonyl chlorides, tosyl esters, tresyl esters, aldehydes, amines, epoxides, carbonates (e.g., bis(p-nitrophenyl)carbonate), aryl halides, alkyl halides, imido esters, carboxylates, alkyl phosphates, anhydrides, fluorophenyl esters, HOBt esters, hydroxymethylphosphine, O-methylisourea, DSC, NHS carbamates, glutaraldehyde, etc. The functional groups may include any suitable functional group, including, but not limited to, aryl, aryl ethers, aryl groups, aryl ester ...

[0105] These and other crosslinking chemistries are known in the art and are described in Bioconjugate Techniques, 3 by Greg T. Hermanson, published by Academic Press, which is incorporated herein by reference in its entirety for all purposes. rd This has been summarized in the literature, including Ed.2013.

[0106] Compositions comprising guide molecules synthesized by the methods provided by the present disclosure are characterized in certain embodiments by high purity of the desired guide molecule reaction product with low levels of contamination from undesired species including n-1 species, truncated species, n+1 species, guide fragment homodimers, unreacted functionalized guide fragments, etc. In certain embodiments of the present disclosure, purified compositions comprising synthetic guide molecules may include multiple species in the composition (i.e., the guide molecule is the most prevalent species in the composition by mass or molar concentration). Alternatively, or in addition, compositions according to embodiments of the present disclosure may include 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and / or 99% or more guide molecules having the desired length (e.g., no truncation at the 5' end relative to the reference guide molecule sequence) and the desired sequence (e.g., including the 5' sequence of the reference guide molecule sequence).

[0107] For example, in some embodiments, compositions comprising guide molecules according to the present disclosure (e.g., guide molecules comprising fragments cross-linked using suitable cross-linking chemistries described herein) comprise less than about 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less than about 20% of guide molecules comprise truncations at the 5' end relative to the reference guide molecule sequence. Alternatively, or in addition, compositions comprising guide molecules according to the present disclosure (e.g., guide molecules comprising fragments cross-linked using suitable cross-linking chemistries described herein) comprise at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% of guide molecules having a 5' sequence that is 100% identical to the corresponding 5' sequence of the reference guide molecule sequence (e.g., a 5' sequence that includes or consists of nucleotides 1-30, 1-25, or 1-20 of the guide molecule). In some embodiments, a composition comprises a guide molecule having a 5' sequence that is less than 100% identical to the corresponding 5' sequence of a reference guide molecule sequence, and when such guide molecules are present at levels of 0.1% or greater, such guide molecules do not contain a targeting domain of a potential off-target site. In some embodiments, a composition comprising guide molecules according to the present disclosure comprises at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more guide molecules that do not comprise a truncation at the 5' end (relative to a reference guide molecule sequence), and such guide molecules (i.e., such guide molecules that do not comprise a truncation at the 5' end) have at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% 5' sequences that are 100% identical to the corresponding 5' sequence of the reference guide molecule sequence (e.g., a 5' sequence that comprises, or consists of, nucleotides 1-30, 1-25, or 1-20 of the guide molecule), and the composition comprises guide molecules having a 5' sequence that is less than 100% identical to the corresponding 5' sequence of the reference guide molecule sequence, and when such guide molecules are present at a level of 0.1% or greater, such guide molecules do not comprise a targeting domain of a potential off-target site.In some embodiments, a composition comprising a guide molecule according to the present disclosure comprises less than about 10% of guide molecules that comprise a truncation at the 5' end relative to a reference guide molecule sequence and exhibit an acceptable level of activity / efficacy. In some embodiments, a composition comprising a guide molecule according to the present disclosure comprises (i) at least about 99% of guide molecules having a 5' sequence that is 100% identical to the corresponding 5' sequence of the reference guide molecule sequence (e.g., a 5' sequence that comprises or consists of nucleotides 1-30, 1-25, 1-20 of the guide molecule), and (ii) when the composition comprises a guide molecule having a 5' sequence that is less than 100% identical to the corresponding 5' sequence of the reference guide molecule sequence, and such guide molecules are present at a level of 0.1% or greater, such guide molecules do not comprise a targeting domain of a potential off-target site, and the composition exhibits an acceptable level of specificity / safety. The purity of a composition can be expressed as a percentage of total guide molecules in the composition (by mass or molar concentration), as a percentage of total RNA or total nucleic acids in the composition (by mass or molar concentration), as a percentage of all solutes in the composition (by mass), and / or as a percentage of the total mass of the composition.

[0108] The purity of the composition comprising the guide molecule according to the present disclosure is assessed by any suitable means known in the art. For example, the relative abundance of the desired guide molecule species can be qualitatively or semi-quantitatively assessed by gel electrophoresis. Alternatively, or in addition, the purity of the desired guide molecule species is assessed by chromatography (e.g., liquid chromatography, HPLC, FPLC, gas chromatography), spectrometry (e.g., time of flight, sector field, quadrupole mass, ion trap, orbitrap, Fourier transform ion cyclotron resonance, or mass spectrometry that may or may not be based on other techniques), nuclear magnetic resonance (NMR) spectroscopy (e.g., visible, infrared, or ultraviolet), thermal stability methods (e.g., differential scanning calorimetry, etc.), sequencing methods (e.g., using template-switching oligonucleotides), and combinations thereof (e.g., chromatography-mass spectrometry, etc.).

[0109] The synthetic guide molecules provided herein function in substantially the same manner as any other guide molecule (e.g., gRNA), generally by (a) forming a complex with an RNA-guided nuclease such as Cas9, (b) interacting with a target sequence that contains a region complementary to the targeting sequence of the guide molecule and a protospacer adjacent motif (PAM) that is recognized by the RNA-guided nuclease, and optionally (c) modifying DNA within or adjacent to the target sequence, e.g., creating a DNA double-strand break, single-strand break, etc. that can be repaired by a DNA repair pathway operative in the cell that contains the guide molecule and the RNA-guided nuclease.

[0110] In some embodiments, guide molecules described herein, such as those generated using the methods described herein, can act as substrates for enzymes that act on RNA (e.g., reverse transcriptase). Without wishing to be bound by theory, crosslinkers present within the guide molecules described herein may be compatible with such processive enzymes due to the close juxtaposition of reactive ends facilitated by pre-annealing according to the methods of the present disclosure.

[0111] The above exemplary embodiment has focused on the application of the synthesis and crosslinking methods described herein to the construction of a guide molecule from two guide fragments. However, the methods described herein have a variety of applications, many of which will be apparent to one of skill in the art. These applications are within the scope of this disclosure. As an example, the methods of this disclosure can be utilized to link heterologous sequences to a guide molecule. Heterologous sequences can include, but are not limited to, DNA donor templates as described in WO 2017 / 180711 by Cotta-Ramusino et al., incorporated herein by reference for all purposes (see, for example, Section I, p. 23, "gRNA Fusion Molecules," which describes the use of covalently linked template nucleic acids and splint oligos to facilitate ligation of the template to the 3' end of the guide molecule). Heterologous sequences can also include nucleic acid sequences recognized by peptide DNA or RNA binding domains, such as the MS2 loop, as also described in Section I of WO 2017 / 180711, supra.

[0112] This overview has focused on a handful of exemplary embodiments that illustrate certain principles related to the synthesis of guide molecules and compositions that include such guide molecules. However, for clarity, this disclosure encompasses modifications and variations that are not described but would be apparent to those skilled in the art. With that in mind, the following disclosure is intended to more generally describe the working principles of genome editing systems. The following should not be understood as limiting, but rather as an illustration of certain principles of genome editing systems that, in combination with this disclosure, inform those skilled in the art of further implementations and modifications that are within the scope of this disclosure.

[0113] Genome editing system The term "genome editing system" refers to any system that has RNA-guided DNA editing activity. The genome editing system of the present disclosure includes at least two components that are compatible with the CRISPR system of natural origin: guide molecule (e.g., guide RNA or gRNA) and RNA-guided nuclease. These two components form a complex that can associate with a specific nucleic acid sequence and edit the DNA in or near the nucleic acid sequence, for example, by generating one or more of single-strand breaks (SSB or nick), double-strand breaks (DSB), and / or point mutations.

[0114] Naturally occurring CRISPR systems have been evolutionarily classified into two classes and five types (Makarova et al. Nat Rev Microbiol. 2011 Jun;9(6):467-477 (Makarova), incorporated herein by reference), and the genome editing system of the present disclosure can accommodate components of any type or class of naturally occurring CRISPR system, and the embodiments presented herein can be implemented by class 2, and type II or V CRISPR systems in general. Class 2 systems, including types II and V, are characterized by a relatively large multi-domain RNA-guided nuclease protein (e.g., Cas9 or Cpf1) and one or more guide RNAs (e.g., crRNA and optionally tracrRNA) that form a ribonucleoprotein (RNP) complex that associates with (i.e., targets) and cleaves a specific locus complementary to the target (or spacer) sequence of the crRNA. The genome editing system according to the present disclosure similarly targets and edits cellular DNA sequences, but is significantly different from naturally occurring CRISPR systems. For example, the unimolecular guide molecules described herein do not occur in nature, and both the guide molecules and RNA-guided nucleases according to the present disclosure can incorporate any number of non-naturally occurring modifications.

[0115] The genome editing system can be implemented (e.g., administered or delivered to a cell or subject) in a variety of ways, and different implementations may be suitable for different applications. For example, the genome editing system, in certain embodiments, is implemented as a protein / RNA complex (ribonucleoprotein, or RNP), which complex can be included in a pharmaceutical composition, optionally including a pharma- ceutically acceptable carrier and / or encapsulating agent, such as lipid or polymer microparticles or nanoparticles, micelles, liposomes, and the like. In certain embodiments, the genome editing system is implemented as one or more nucleic acids encoding the above-mentioned RNA-guided nuclease and guide molecule components (optionally including one or more additional components); in certain embodiments, the genome editing system is implemented as one or more vectors, such as viral vectors, such as adeno-associated viruses, that include such nucleic acids; in certain embodiments, the genome editing system is implemented as any combination of the foregoing. Additional or modified implementations that operate according to the principles described herein will be apparent to those of skill in the art and are within the scope of the present disclosure.

[0116] It should be noted that the genome editing system of the present disclosure may target a single specific nucleotide sequence, or may target two or more specific nucleotide sequences by the use of two or more guide molecules, which can be edited in parallel. The use of multiple guide molecules, referred to throughout this disclosure as "multiplexing," can be utilized to target multiple unrelated target sequences of interest, or to form multiple SSBs or DSBs within a single target domain, and in some cases, to perform specific edits within such a target domain. For example, WO 2015 / 138510 by Maeder et al. (Maeder), incorporated herein by reference, describes a genome editing system for correcting a point mutation (C.2991+1655A-G) in the human CEP290 gene that creates a cryptic splice site, thereby reducing or eliminating the function of the gene. Maeder's genome editing system utilizes two guide RNAs that target sequences on either side (i.e., adjacent) of the point mutation to form DSBs adjacent to the mutation. This facilitates the deletion of the intervening sequence containing the mutation, thereby eliminating the cryptic splice site and restoring normal gene function.

[0117] As another example, WO 2016 / 073990 by Cotta-Ramusino et al. ("Cotta-Ramusino"), incorporated herein by reference, describes a genome editing system that utilizes two gRNAs in combination with a Cas9 nickase (a single-stranded nick-generating Cas9, such as S. pyogenes D10A), an arrangement referred to as the "dual-nickase system." The Cotta-Ramusino dual-nickase system is configured to generate two nicks on opposite strands of the sequence of interest, offset by one or more nucleotides, which join to generate a double-stranded break with an overhang (5' in the case of Cotta-Ramusino, but a 3' overhang is also possible). The overhang can then facilitate homologous recombination repair events in some circumstances. And, as another example, WO 2015 / 070083 by Palestrant et al. ("Palestrant") describes gRNAs (called "governing RNAs") that target nucleotide sequences encoding Cas9, which can be included in a genome editing system that includes one or more additional gRNAs that would allow for transient expression of Cas9, for example in some virally transduced cells, and constitutive expression if not transient expression. These multiplexing applications are intended to be exemplary, not limiting, and one of skill in the art will understand that other multiplexing applications are generally compatible with the genome editing systems described herein.

[0118] Genome editing systems can, in some cases, form double-strand breaks that are repaired by cellular DNA double-strand break mechanisms, such as NHEJ or HDR. These mechanisms are described throughout the literature, for example, by Davis & Maizels, PNAS, 111(10):E924-932, March 11, 2014 (Davis) (describing Alt-HDR); Frit et al. DNA Repair 17(2014)81-97 (Frit) (describing Alt-NHEJ); and Iyama and Wilson III, DNA Repair (Amst.) 2013-Aug; 12(8):620-636 (Iyama) (describing generally canonical HDR and NHEJ pathways).

[0119] When genome editing system functions by forming DSB, such system optionally includes one or more components that promote or facilitate a specific mode of double-strand break repair or a specific repair outcome.For example, Cotta-Ramusino also describes a genome editing system that is added with a single-stranded oligonucleotide "donor template"; the donor template can be integrated into the target region of the cellular DNA that is cut by genome editing system and cause the change of the target sequence.

[0120] In certain embodiments, the genome editing system modifies the target sequence or alters the expression of a gene within or near the target sequence without causing single- or double-strand breaks. For example, the genome editing system may include an RNA-guided nuclease fused to a functional domain that acts on DNA, thereby altering the target sequence or its expression. As an example, the RNA-guided nuclease may be bound (e.g., fused) to a cytidine deaminase functional domain and may function by generating a C to A substitution in the target. Exemplary nuclease / deaminase fusions are described in Komor et al. Nature 533, 420-424 (19 May 2009), incorporated herein by reference. (2016) ("Komor"). Alternatively, genome editing systems can utilize cleavage-inactivating (i.e., "dead") nucleases, such as deadCas9 (dCas9), which can function by forming a stable complex at one or more target regions of cellular DNA, thereby inhibiting functions involving the target regions, including, but not limited to, mRNA transcription, chromatin remodeling, and the like.

[0121] Guide molecule The term "guide molecule" is used herein to refer to any nucleic acid that facilitates the specific association (or "targeting") of an RNA-guided nuclease, such as Cas9 or Cpf1, with a target sequence, such as a genomic or episomal sequence in a cell. A guide molecule can be an RNA molecule or a hybrid RNA / DNA molecule. A guide molecule can be unimolecular (including a single molecule, also called a chimera), or modular (comprising two or more, typically two separate molecules, such as crRNA and tracrRNA, that are usually associated with each other, for example, by duplexing). Guide molecules and their constituent parts are described, for example, through Briner et al. (Molecular Cell 56(2), 333-339, October 23, 2014 (Briner)) and Cotta-Ramusino, which are incorporated herein by reference.

[0122] In bacteria and archaea, type II CRISPR systems generally consist of an RNA-guided nuclease protein, such as Cas9, a CRISPR that contains a 5' region complementary to the foreign sequence. The Cas9 guide molecule complex includes a Cas9-binding domain that is complementary to the 3' region of a Cas9-binding domain of a transactivating crRNA (crRNA), and a transactivating crRNA (tracrRNA) that includes a 5' region that is complementary to and forms a duplex with the 3' region of the Cas9-binding domain of a transactivating crRNA (tracrRNA). Without intending to be bound by any theory, it is believed that this duplex promotes the formation of the Cas9 / guide molecule complex and is required for its activity. As Type II CRISPR systems have been adapted for use in gene editing, in one non-limiting example, it has been found that the crRNA and tracrRNA can be linked into a single unimolecular or chimeric guide RNA by a four nucleotide (e.g., GAAA), "tetraloop," or "linker" sequence that bridges the complementary regions of the crRNA (at its 3' end) and the tracrRNA (at its 5' end) (Mali et al., Science. 2013 Feb 15;339(6121):823-826 ("Mali"); Jiang et al., Nat Biotechnol. 2013 Mar;31(3):233-239 ("Jiang"); and Jinek et al., 2012 Science Aug. 17;337(6096):816-821 ("Jinek"), all of which are incorporated herein by reference.

[0123] Guide molecules, whether monomolecular or modular, include a "targeting domain" that is fully or partially complementary to a targeting domain within a target sequence, such as a DNA sequence within the genome of a cell, for which editing is desired. Targeting domains have been referred to by various names in the literature, including, but not limited to, "guide sequence" (Hsu et al., Nat Biotechnol. 2013 Sep;31(9):827-832, ("Hsu"), incorporated herein by reference), "complementary region" (Cotta-Ramusino), "spacer" (Briner), and collectively, "crRNA" (Jiang). Regardless of the name given, the targeting domain is typically 10-30 nucleotides in length, and in certain embodiments 16-24 nucleotides in length (e.g., 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides in length), and is located at or near the 5' end in the case of a Cas9 guide molecule, and at or near the 3' end in the case of a Cpf1 guide molecule.

[0124] In addition to the targeting domain, a guide molecule typically (but not necessarily) contains multiple domains that can affect the formation or activity of a guide molecule / Cas9 complex, as discussed below. For example, as described above, the duplex structure formed by the first and second complementary domains of a guide molecule (also called the repeat:anti-repeat duplex) can interact with the recognition (REC) lobe of Cas9 and mediate the formation of a Cas9 / guide molecule complex (Nishimasu et al., Cell 2001, 144:131-132, both of which are incorporated herein by reference). 156, 935-949, February 27, 2014 (Nishimasu 2014) and Nishimasu et al., Cell 162, 1113-1126, August 27, 2015 (Nishimasu 2015)).

[0125] Along with the first and second complementarity domains, Cas9 guide molecules typically contain two or more additional duplex regions that are involved in nuclease activity in vivo but not necessarily in vitro (Nishimasu 2015). The first stem-loop near the 3' portion of the second complementarity domain is variously referred to as the "proximal domain", (Cotta-Ramusino) "stem-loop 1" (Nishimasu 2014 and 2015), and "nexus" (Briner). One or more additional stem-loop structures are generally present near the 3' end of the guide molecule, the number of which varies by species; S. pyogenes gRNAs typically contain two 3' stem-loops (for a total of four stem-loop structures including the repeat:anti-repeat duplex), while S. aureus and other species have only one (for a total of three stem-loop structures). A description of the species-specific classification and conserved stem-loop structures (and guide molecule structures more generally) is presented in Briner.

[0126] While the foregoing description has focused on guide molecules for use with Cas9, it should be understood that other RNA-guided nucleases have been discovered or invented (or may be discovered or invented in the future) that utilize guide molecules that differ in some respects from those described thus far. For example, Cpf1 ("CRISPR from Prevotella and Franciscella 1") is a recently discovered RNA-guided nuclease that does not require tracrRNA to function (Zetsche et al., 2015, Cell 163, 759-771 October 22, 2015 (Zetsche I) which is incorporated herein by reference). Guide molecules for use with the Cpf1 genome editing system generally include a targeting domain and a complementarity domain (alternatively referred to as a "handle"). It should also be noted that in guide molecules for use with Cpf1, the targeting domain is typically at or near the 3' end (the handle is at or near the 5' end of the Cpf1 guide molecule) rather than the 5' end as described above in connection with Cas9 guide molecules.

[0127] However, those skilled in the art will understand that although there may be structural differences between guide molecules from different prokaryotic species, or between Cpf1 and Cas9 guide molecules, the principles by which guide molecules operate are generally consistent. For this consistency of operation, guide molecules can be broadly defined by their target domain sequences, and those skilled in the art will understand that a given target domain sequence can be incorporated into any suitable guide molecule, including monomolecular or chimeric guide molecules, or guide molecules that include one or more chemical modifications and / or sequence alterations (substitutions, additional nucleotides, truncations, etc.). Therefore, for the sake of simplicity of presentation in this disclosure, guide molecules may be described only in terms of their target domain sequences.

[0128] More generally, those skilled in the art will understand that some aspects of the present disclosure relate to systems, methods and compositions that can be implemented using multiple RNA-guided nucleases.For this reason, unless otherwise stated, the term guide molecule should be understood to encompass any suitable guide molecule (e.g., gRNA) that can be used with any RNA-guided nuclease, not just guide molecules that are compatible with a particular species of Cas9 or Cpf1.By way of example, the term guide molecule can include, in certain embodiments, guide molecules for use with any RNA-guided nuclease that occurs in class 2 CRISPR systems, such as type II or type V CRISPR systems, or RNA-guided nucleases derived therefrom or made therefrom.

[0129] Cross-linking guide molecules Certain embodiments of the present disclosure relate to guide molecules that are cross-linked, for example, via non-nucleotide chemical bonds. As noted above, the location of the linkage can be within a stem-loop structure of the guide molecule. In some embodiments, the guide molecule comprises

[0130] In some embodiments, the unimolecular guide molecule comprises, 5' to 3': A first guide molecule fragment, target domain sequence; 1st lower stem sequence; First bulge arrangement; First upper stem array a first guide molecule fragment comprising: Non-nucleotide chemical bonds; and A second guide molecule fragment, second upper stem array; A second bulge arrangement; and Second Lower Stem Array a second guide molecule fragment comprising Including, wherein (a) at least one nucleotide in the first lower stem sequence base pairs with a nucleotide in the second lower stem sequence, and (b) at least one nucleotide in the first upper stem sequence base pairs with a nucleotide in the second upper stem sequence.

[0131] In some embodiments, the guide molecule does not include a tetraloop sequence between the first and second upper stem sequences. In some embodiments, the first and / or second upper stem sequences include 4 to 22 nucleotides (inclusive). In some embodiments, the first and / or second upper stem sequences include 1 to 22 nucleotides (inclusive). In some embodiments, the first and / or second upper stem sequences include 4 to 22 nucleotides (inclusive). In some embodiments, the first and second upper stem sequences include 8 to 22 nucleotides (inclusive). In some embodiments, the first and second upper stem sequences include 12 to 22 nucleotides (inclusive).

[0132] In some embodiments, the guide molecule is characterized in that the Gibbs free energy (ΔG) for the formation of a duplex between the first and second guide molecule fragments is smaller than the ΔG for the formation of a duplex between two first guide molecule fragments. In some embodiments, the ΔG for the formation of a duplex between the first and second guide molecule fragments characterized by more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or more than 95% base pairing between (i) the first and second upper stem sequences and (ii) the first and second lower stem sequences is smaller than the ΔG for the formation of a duplex characterized by less than 50%, less than 60%, less than 70%, less than 80%, less than 90%, or less than 95% base pairing between (i) and (ii).

[0133] In some embodiments, the synthetic guide molecule has the formula: [ka] is a synthetic guide molecule of During the ceremony, (N) c and (N) t wherein each N is independently a nucleotide residue, optionally modified, each independently linked to its adjacent nucleotide via a phosphodiester, phosphorothioate, phosphonoacetate, thiophosphonoacetate, or phosphoramidate linkage; (N) c is (N) t a 3' region that is complementary or partially complementary to and forms a duplex with the 5' region of c is an integer equal to or greater than 20; t is an integer equal to or greater than 20; The linker is a non-nucleotidic chemical bond; B1 and B2 are each independently a nucleobase; R2' and R3' are each independently H, OH, fluoro, chloro, bromo, NH2, SH, S-R', or O-R', where each R' is independently a protecting group or an alkyl group, where the alkyl group may be optionally substituted; and [ka] each independently represents a phosphodiester bond, a phosphorothioate bond, a phosphonoacetate bond, a thiophosphonoacetate bond, or a phosphoramidate bond.

[0134] In some embodiments, (N) t and (N) c The duplex region comprises the sequences listed in Table 4.

[0135] [Table 5]

[0136] [Table 6]

[0137] In some embodiments, the guide molecule has the formula: [ka] The During the ceremony, N, B1, B2, R 2’ , R3', a linker, and [ka] is defined above; and N----N independently represent two complementary nucleotides, optionally two complementary nucleotides that are hydrogen bonded base pairs; p and q are each independently an integer from 0 to 6 (inclusive), and p+q is an integer from 0 to 6 (inclusive); u is an integer from 2 to 22, inclusive; s is an integer from 1 to 10, inclusive; x is an integer from 1 to 3, inclusive; y is >x and is an integer from 3 to 5, inclusive; m is an integer greater than or equal to 15; and n is an integer of 30 or greater.

[0138] In some embodiments, (N--N) u and (N---N) s does not contain an identical sequence of 3 or more nucleotides. In some embodiments, (N--N) u and (N---N) s In some embodiments, (N--N) s contains the sequence N'UUU, UN'UU, UUN'U, or UUUN', (N---N) u comprises the sequence UUUU, where N' is A, G, or C. In some embodiments, (N---N) s contains the sequence UUUU, (N---N) u comprises the sequence N'UUU, UN'UU, UUN'U, or UUUN', where N' is A, G, or C. In some embodiments, N' is A. In some embodiments, N' is G. In some embodiments, N' is C.

[0139] In some aspects, the guide molecule is based on a gRNA used in the S. pyogenes or S. aureus Cas9 system. In some embodiments, the guide molecule has the formula: [ka] The During the ceremony: u' is an integer from 2 to 22 (inclusive); p' and q' are each independently an integer from 0 to 6 (inclusive), and p'+q' is an integer from 0 to 6 (inclusive).

[0140] In some embodiments, the guide molecule has the formula: [ka] or a covariant thereof. In some embodiments, (N----N) u’ is the expression: [ka] or a covariant thereof. In some embodiments, (N----N) u’ is the expression: [ka] (wherein B1 is a cytosine residue and B2 is a guanine residue) or a covariant thereof. In some embodiments, (N----N) u’ is the expression: [ka] (wherein B1 is a guanine residue and B2 is a cytosine residue) or a covariant thereof. In some embodiments, (N----N) u’ is the expression: [ka] (wherein B1 is a guanine residue and B2 is a cytosine residue) or a covariant thereof.

[0141] In some embodiments, the guide molecule has the formula: [ka] or a covariant thereof. In some embodiments, (N----N) u’ is the expression: [ka] [ka] or a covariant thereof. In some embodiments, (N----N) u’ is the expression: [ka] (wherein B1 is an adenine residue and B2 is a uracil residue) or a covariant thereof. In some embodiments, (N----N) u’ is the expression: [ka] (wherein B1 is a uracil residue and B2 is an adenine residue) or a covariant thereof. In some embodiments, (N----N) u’ is the expression: [ka] (wherein B1 is a guanine residue and B2 is a cytosine residue) or a covariant thereof.

[0142] In some embodiments, the linker has the formula: [ka] is a linker for During the ceremony: Each R2 is independently O or S; R3 each independently represents O- or COO - and L1 and R1 are each a non-nucleotide chemical linker.

[0143] In some embodiments, the chemical bond of the crosslinked guide molecule comprises a urea. In some embodiments, the urea-containing guide molecule has the formula: [ka] The wherein L and R are each independently a non-nucleotidic linker.

[0144] In some embodiments, the urea-containing guide molecule has the formula: [ka] It is of the following.

[0145] In some embodiments, the urea-containing guide molecule has the formula: [ka] It is of the following.

[0146] In some embodiments, the urea-containing guide molecule has the formula: [ka] It is of the following.

[0147] In some embodiments, the urea-containing guide molecule has the formula: [ka] It is of the following.

[0148] In some embodiments, the guide molecule comprising urea is a guide molecule of a sequence listed in Table 10 of the Examples section, where [UR] is a non-nucleotide linkage comprising urea. In some embodiments, [UR] is the following linkage between two nucleotides having nucleobases B1 and B2: [ka] Shows.

[0149] In some embodiments, the chemical bond of the crosslinked guide molecule comprises a thioether. In some embodiments, the thioether-containing guide molecule has the formula: [ka] wherein L and R are each independently a non-nucleotidic linker.

[0150] In some embodiments, the thioether-containing guide molecule has the formula: [ka] It is of the following.

[0151] In some embodiments, the thioether-containing guide molecule has the formula: [ka] It is of the following.

[0152] In some embodiments, the thioether-containing guide molecule has the formula: [ka] [ka] It is of the following.

[0153] In some embodiments, the thioether-containing guide molecule has the formula: [ka] It is of the following.

[0154] In some embodiments, the urea-containing guide molecule is a guide molecule of a sequence listed in Table 9 of the Examples section, where [L] is a thioether bond. In some embodiments, [L] is the following bond between two nucleotides having nucleobases B1 and B2: [ka] Shows.

[0155] In some embodiments, in any formula of the present application, R 2’ and R 3’ are each independently H, OH, fluoro, chloro, bromo, NH, SH, S-R', or O-R', where each R' is independently a protecting group or an optionally substituted alkyl group. In some embodiments, R 2’ and R 3’ are each independently H, OH, halogen, NH, or O-R', where each R' is independently a protecting group or an optionally substituted alkyl group. In some embodiments, R 2’ and R 3’ are each independently H, fluoro, and O-R', where R' is a protecting group or an optionally substituted alkyl group. In some embodiments, R 2’ is H. In some embodiments, R 3’ is H. In some embodiments, R 2’ is a halogen. In some embodiments, R 3’ is a halogen. In some embodiments, R 2’ is fluorine. In some embodiments, R 3’is fluorine. In some embodiments, R 2’ is O-R'. In some embodiments, R 3’ is O-R'. In some embodiments, R 2’ is O-Me. In some embodiments, R 3’ is O-Me.

[0156] In some embodiments, in any formula of the present application, p and q are each independently 0, 1, 2, 3, 4, 5, or 6. In some embodiments, p and q are each independently 2. In some embodiments, p and q are each independently 0. In some embodiments, p' and q' are each independently 0, 1, 2, 3, or 4. In some embodiments, p' and q' are each independently 2. In some embodiments, p' and q' are each independently 0.

[0157] In some embodiments, in any formula of the present application, u is an integer between 2 and 22, inclusive. In some embodiments, u is an integer between 3 and 22, inclusive. In some embodiments, u is an integer between 4 and 22, inclusive. In some embodiments, u is an integer between 8 and 22, inclusive. In some embodiments, u is an integer between 12 and 22, inclusive. In some embodiments, u is an integer between 0 and 22, inclusive. In some embodiments, u is an integer between 2 and 14, inclusive. In some embodiments, u is an integer between 4 and 14, inclusive. In some embodiments, u is an integer between 8 and 14, inclusive. In some embodiments, u is an integer between 0 and 14, inclusive. In some embodiments, u is an integer between 0 and 4, inclusive. In some embodiments, in any formula of the present application, u' is an integer between 2 and 22, inclusive. In some embodiments, u' is an integer between 3 and 22, inclusive. In some embodiments, u' is an integer between 4 and 22, inclusive. In some embodiments, u' is an integer between 8 and 22, inclusive. In some embodiments, u' is an integer between 12 and 22, inclusive. In some embodiments, u' is an integer between 0 and 22, inclusive. In some embodiments, u' is an integer between 2 and 14, inclusive. In some embodiments, u' is an integer between 4 and 14, inclusive. In some embodiments, u' is an integer between 8 and 14, inclusive. In some embodiments, u' is an integer between 0 and 14, inclusive. In some embodiments, u' is an integer between 0 and 4, inclusive.

[0158] In some embodiments, in any formula of the present application, N is independently a ribonucleotide, a deoxyribonucleotide, a modified ribonucleotide, or a modified deoxyribonucleotide. Nucleotide modifications are discussed below.

[0159] In some embodiments, in any formula herein, c is an integer equal to or greater than 20. In some embodiments, c is an integer between 20 and 60, inclusive. In some embodiments, c is an integer between 20 and 40, inclusive. In some embodiments, c is an integer between 40 and 60, inclusive. In some embodiments, c is an integer between 30 and 60, inclusive. In some embodiments, c is an integer between 20 and 50, inclusive.

[0160] In some embodiments, in any formula herein, t is an integer equal to or greater than 20. In some embodiments, t is an integer between 20 and 80, inclusive. In some embodiments, t is an integer between 20 and 50, inclusive. In some embodiments, t is an integer between 50 and 80, inclusive. In some embodiments, t is an integer between 20 and 70, inclusive. In some embodiments, t is an integer between 30 and 80, inclusive.

[0161] In some embodiments, in any formula herein, s is an integer from 1 to 10, inclusive. In some embodiments, s is an integer from 3 to 9, inclusive. In some embodiments, s is an integer from 1 to 8, inclusive. In some embodiments, s is an integer from 0 to 10, inclusive. In some embodiments, s is an integer from 2 to 6, inclusive.

[0162] In some embodiments, in any formula of the application, x is an integer from 1 to 3, inclusive. In some embodiments, x is 1. In some embodiments, x is 2. In some embodiments, x is 3. In some embodiments, in any formula of the application, y is greater than x. In some embodiments, y is an integer from 3 to 5, inclusive. In some embodiments, y is 3. In some embodiments, y is 4. In some embodiments, y is 5. In some embodiments, x is 1 and y is 3. In some embodiments, x is 2 and y is 4.

[0163] In some embodiments, in any formula herein, m is an integer of 15 or greater. In some embodiments, m is an integer between 15 and 50, inclusive. In some embodiments, m is an integer between 16 and 17. In some embodiments, m is an integer between 18 and 19. In some embodiments, m is an integer between 20 and 40, inclusive. In some embodiments, m is an integer between 30 and 50, inclusive. In some embodiments, m is an integer between 15 and 30, inclusive.

[0164] In some embodiments, in any formula herein, n is an integer equal to or greater than 30. In some embodiments, n is an integer between 30 and 70, inclusive. In some embodiments, n is an integer between 30 and 60, inclusive. In some embodiments, n is an integer between 40 and 70, inclusive.

[0165] In some embodiments, in any formula of the present application, L, R, L1, and R1 are each independently a non-nucleotidic linker. In some embodiments, L, R, L1, and R1 each independently comprise a moiety selected from the group consisting of polyethylene, polypropylene, polyethylene glycol, and polypropylene glycol. In some embodiments, L1 and R1 each independently comprise -(CH2) W -, -(CH2) W -NH-C(O)-(CH2) w -NH-, -(OCH2CH2) v -NH-C(O)-(CH2) w - or -(CH2CH2O) v - and w are each an integer from 1 to 20, inclusive, and v is each an integer from 1 to 10, inclusive. In some embodiments, L1 is -(CH2) WIn some embodiments, L1 is -(CH2). w -NH-C(O)-(CH2) w In some embodiments, L1 is -(OCH2CH2). v -NH-C(O)-(CH2) w In some embodiments, L1 is -(CH2)6-. In some embodiments, L1 is -(CH2)6-NH-C(O)-(CH2)1-NH-. In some embodiments, L1 is -(OCH2CH2)4-NH-C(O)-(CH2)2-. In some embodiments, R1 is -(CH2CH2O) v In some embodiments, R1 is -(CH2) W -NH-C(O)-(CH2) w In some embodiments, R1 is -(OCH2CH2). v -NH-C(O)-(CH2) w In some embodiments, R1 is -(CH2CH2O)4-. In some embodiments, L1 is -(CH2)6-NH-C(O)-(CH2)1-NH-. In some embodiments, R1 is -(OCH2CH2)4-NH-C(O)-(CH2)2-. In some embodiments, L1 is -(CH2)6- and R1 is -(CH2CH2O)4-. In some embodiments, L1 is -(CH2)6-NH-C(O)-(CH2)1-NH- and R1 is -(OCH2CH2)4-NH-C(O)-(CH2)2-.

[0166] In some embodiments, in any formula of the present application, R2 is O, and in some embodiments, R2 is S. In some embodiments, R3 is O - and in some embodiments, R3 is COO - In some embodiments, R2 is O and R3 is O - In some embodiments, R2 is O and R3 is COO - In some embodiments, R2 is S and R3 is O -In some embodiments, R2 is S and R3 is COO - One skilled in the art will appreciate that R3 can also exist in protonated forms (OH and COOH). Throughout this application, we refer to both the deprotonated and protonated forms of R3.

[0167] In some embodiments, in any formula of the present application, N----N each independently represents two complementary nucleotides, optionally two complementary nucleotides base-paired by hydrogen bonds. In some embodiments, all N----N represent two complementary nucleotides base-paired by hydrogen bonds. In some embodiments, some N----N represent two complementary nucleotides, and some N----N represent two complementary nucleotides base-paired by hydrogen bonds.

[0168] In some embodiments, in any formula of the present application, B1 and B2 are each independently a nucleobase. In some embodiments, B1 is guanine and B2 is cytosine. In some embodiments, B1 is cytosine and B2 is guanine. In some embodiments, B1 is adenine and B2 is uracil. In some embodiments, B1 is uracil and B2 is adenine. In some embodiments, B1 and B2 are complementary. In some embodiments, B1 and B2 are complementary and base pair via hydrogen bonds. In some embodiments, B1 and B2 are complementary and do not base pair via hydrogen bonds. In some embodiments, B1 and B2 are not complementary.

[0169] Synthesis of guide molecules Another aspect of the invention is a method of synthesizing a unimolecular guide molecule, the method comprising: annealing a first oligonucleotide and a second oligonucleotide to form a duplex between a 3' region of the first oligonucleotide and a 5' region of the second oligonucleotide, where the first oligonucleotide comprises a first reactive group that is at least one of a 2' reactive group and a 3' reactive group, and the second oligonucleotide comprises a second reactive group that is a 5' reactive group; and conjugating the annealed first and second oligonucleotides via the first and second reactive groups to form a unimolecular guide molecule that comprises a covalent bond linking the first and second oligonucleotides. Includes.

[0170] In some embodiments, the first reactive group and the second reactive group are selected from the functional groups listed in the "Summary" above. In some embodiments, the first reactive group and the second reactive group are each independently an amine moiety, a sulfhydryl moiety, a bromoacetyl moiety, a hydroxyl moiety, or a phosphate moiety. In some embodiments, the first reactive group and the second reactive group are both amine moieties. In some embodiments, the first reactive group is a sulfhydryl moiety and the second reactive group is a bromoacetyl moiety. In some embodiments, the first reactive group is a bromoacetyl moiety and the second reactive group is a sulfhydryl moiety. In some embodiments, the first reactive group is a hydroxyl moiety and the second reactive group is a phosphate moiety. In some embodiments, the first reactive group is a phosphate moiety and the second reactive group is a hydroxyl moiety.

[0171] In some embodiments, the conjugating step includes a first nucleotide at a concentration ranging from 10 μM to 1 mM. In some embodiments, the conjugating step includes a second nucleotide at a concentration ranging from 10 μM to 1 mM. In some embodiments, the concentration of either the first or second nucleotide is 10 μM, 50 μM, 100 μM, 200 μM, 400 μM, 600 μM, 800 μM, or 1 mM.

[0172] In some embodiments, the conjugating step comprises a pH in the range of 5.0 to 9.0. In some embodiments, the pH is 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or 9.0. In some embodiments, the pH is 6.0. In some embodiments, the pH is 8.0. In some embodiments, the pH is 8.5.

[0173] In some embodiments, the conjugating step is performed under argon. In some embodiments, the conjugating step is performed under ambient atmosphere.

[0174] In some embodiments, the conjugating step is carried out in water. In some embodiments, the conjugating step is carried out in water with a co-solvent. In some embodiments, the co-solvent is DMSO, DMF, NMP, DMA, morpholine, pyridine, or MeCN. In some embodiments, the co-solvent is DMSO. In some embodiments, the co-solvent is DMF.

[0175] In some embodiments, the conjugating step is performed at a temperature ranging from 0° C. to 40° C. In some embodiments, the temperature is 0° C., 4° C., 10° C., 20° C., 25° C., 30° C., 37° C., or 40° C. In some embodiments, the temperature is 25° C. In some embodiments, the temperature is 4° C.

[0176] In some embodiments, the conjugating step is carried out in the presence of a divalent metal cation. In some embodiments, the divalent metal cation is Mg 2+ , Ca 2+ , Sr 2+ , B.A. 2+ , Cr 2+ , Mn 2+ , Fe 2+ , Co 2+ , Ni 2+ , Cu 2+ , or Zn 2+In some embodiments, the divalent metal cation is Mg 2+ It is.

[0177] In some embodiments, the conjugating step includes a cross-linking reagent or agent (see Summary above). In some embodiments, the cross-linking agent is multifunctional, and in some embodiments, the cross-linking agent is bifunctional. In some embodiments, the multifunctional cross-linking agent is heterofunctional or homofunctional. In some embodiments, the cross-linking agent includes a carbonate. In some embodiments, the carbonate-containing cross-linking agent is disuccinimidyl carbonate, diimidazole carbonate, or bis-(p-nitrophenyl) carbonate. In some embodiments, the carbonate-containing cross-linking agent is disuccinimidyl carbonate.

[0178] In some embodiments, the conjugating step includes a bifunctional crosslinking reagent at a concentration ranging from 1 mM to 100 mM. In some embodiments, the concentration of the bifunctional crosslinking reagent is 1 mM, 10 mM, 20 mM, 40 mM, 60 mM, 80 mM, or 100 mM. In some embodiments, the concentration of the bifunctional crosslinking reagent is 100 to 1000 times higher than the respective concentrations of the first and second oligonucleotides. In some embodiments, the concentration of the bifunctional crosslinking reagent is 100 times, 200 times, 400 times, 600 times, 800 times, or 1000 times higher than the concentration of the first oligonucleotide. In some embodiments, the concentration of the bifunctional crosslinking reagent is 100 times, 200 times, 400 times, 600 times, 800 times, or 1000 times higher than the concentration of the second oligonucleotide.

[0179] In some embodiments, the conjugating step is performed in the presence of a chelating agent, hi some embodiments, the chelating agent is ethylenediaminetetraacetic acid (EDTA) or a salt thereof.

[0180] In some embodiments, the conjugating step is performed in the presence of an activating agent. In some embodiments, the activating agent is a carbodiimide or a salt thereof. In some embodiments, the carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N,N'-dicyclohexylcarbodiimide (DCC), or N,N'-diisopropylcarbodiimide (DIC), or a salt thereof. In some embodiments, the carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or a salt thereof.

[0181] In some embodiments, the conjugating step includes an activator at a concentration in the range of 1 mM to 100 mM. In some embodiments, the concentration of the activator is 1 mM, 10 mM, 20 mM, 40 mM, 60 mM, 80 mM, or 100 mM. In some embodiments, the concentration of the activator is 100 to 1000 times higher than the respective concentrations of the first and second oligonucleotides. In some embodiments, the concentration of the activator is 100 times, 200 times, 400 times, 600 times, 800 times, or 1000 times higher than the concentration of the first oligonucleotide. In some embodiments, the concentration of the activator is 100 times, 200 times, 400 times, 600 times, 800 times, or 1000 times higher than the concentration of the second oligonucleotide.

[0182] In some embodiments, the conjugating step is performed in the presence of a stabilizing agent. In some embodiments, the stabilizing agent is imidazole, cyanoimidazole, pyridine, or dimethylaminopyridine, or a salt thereof. In some embodiments, the stabilizing agent is imidazole. In some embodiments, the conjugating step is performed in the presence of both an activating agent and a stabilizing agent. In some embodiments, the conjugating step is performed in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and imidazole, or a salt thereof.

[0183] In some embodiments, the methods of synthesizing unimolecular guide molecules produce guide molecules of any of the formulas disclosed above.

[0184] In some embodiments, the method for synthesizing a unimolecular guide molecule results in a guide molecule having a urea linker. In some embodiments, the first reactive group and the second reactive group are both amines, and the first and second reactive groups are crosslinked with a carbonate-containing bifunctional crosslinking reagent to form a urea linker. In some embodiments, the carbonate-containing bifunctional crosslinking reagent is disuccinimidyl carbonate. In some embodiments, the method comprises synthesizing a amine having the formula: [ka] or a salt thereof. In some embodiments, the method comprises a first oligonucleotide having the formula: [ka] or a salt thereof.

[0185] In some embodiments, the method for synthesizing a unimolecular guide molecule results in a guide molecule having a thioether linker. In some embodiments, the first reactive group is a sulfhydryl group and the second reactive group is a bromoacetyl group, or the first reactive group is a bromoacetyl group and the second reactive group is a sulfhydryl group. In some embodiments, the first reactive group and the second reactive group react in the presence of a chelating agent to form a thioether bond. In some embodiments, the first reactive group and the second reactive group undergo a substitution reaction to form a thioether bond. In some embodiments, the method comprises the step of synthesizing a thioether bond having the formula: [ka] or a salt thereof, and a first oligonucleotide of the formula: [ka] or a salt thereof; or the method includes a second oligonucleotide of the formula: [ka] or a salt thereof, and a first oligonucleotide of the formula: [ka] or a salt thereof.

[0186] In some embodiments, the method for synthesizing a unimolecular guide molecule results in a guide molecule having a phosphodiester linker. In some embodiments, the first reactive group comprises a 2' or 3' hydroxyl group and the second reactive group comprises a 5' phosphate moiety. In some embodiments, the first and second reactive groups are conjugated in the presence of an activating agent to form a phosphodiester linker. In some embodiments, the activating agent is EDC. In some embodiments, the method comprises the step of synthesizing a phosphodiester linker having the formula: [ka] or a salt thereof; and formula: [ka] or a salt thereof.

[0187] In some embodiments, the method of synthesizing a unimolecular guide molecule produces a unimolecular guide molecule having at least one 2'-5' phosphodiester bond in the duplex region.

[0188] Oligonucleotide Intermediates Certain embodiments of the present disclosure relate to oligonucleotide intermediates that are useful for synthesizing cross-linked synthetic guide molecules. In some embodiments, the oligonucleotide intermediates are useful for synthesizing guide molecules that contain urea bonds, thioether bonds, or phosphodiester bonds. In some embodiments, the oligonucleotide intermediates contain annealed duplexes.

[0189] In certain embodiments, the oligonucleotide intermediate is useful for the synthesis of a guide molecule that includes a urea linkage. In some embodiments, the oligonucleotide intermediate has the formula: [ka] In some embodiments, the oligonucleotide intermediate has the formula: [ka] In some embodiments, the oligonucleotide intermediate has the formula: [ka] In some embodiments, the oligonucleotide intermediate has the formula: [ka] In some embodiments, the oligonucleotide intermediate has the formula: [ka] In some embodiments, the oligonucleotide intermediate has the formula: [ka] It is an oligonucleotide intermediate of the formula:

[0190] In some embodiments, the oligonucleotide intermediate is useful for the synthesis of a guide molecule that contains a thioether bond. In some embodiments, the oligonucleotide intermediate has the formula: [ka] In some embodiments, the oligonucleotide intermediate has the formula: [ka] In some embodiments, the oligonucleotide intermediate has the formula: [ka] In some embodiments, the oligonucleotide intermediate has the formula: [ka] It is an oligonucleotide intermediate of the formula:

[0191] In some embodiments, the oligonucleotide intermediate is useful for the synthesis of a guide molecule that includes a phosphodiester bond. In some embodiments, the oligonucleotide intermediate has the formula: [ka] wherein R6 and R7 are each independently substituted or unsubstituted alkyl, or substituted or unsubstituted carbocyclyl. In some embodiments, the oligonucleotide intermediate has the formula: [ka] where Z represents a nucleotide loop that is 4 to 6 nucleotides in length, optionally 4 or 6 nucleotides in length.

[0192] Certain embodiments of the present disclosure relate to oligonucleotide compounds that are formed as by-products in cross-linking reactions. These oligonucleotide compounds may or may not be useful as guide molecules. In some embodiments, the oligonucleotide compounds have the formula: [ka] It is an oligonucleotide compound of the formula:

[0193] Compositions of Chemically Conjugated Guide Molecules Certain embodiments of the present disclosure relate to compositions comprising the above synthetic guide molecules and compositions produced by the above methods. In some embodiments, the compositions are characterized in that more than 90% of the guide molecules therein are full-length guide molecules. In some embodiments, the compositions are characterized in that more than 85% of the guide molecules therein comprise the same target domain sequence.

[0194] In some embodiments, the composition has not been subjected to a purification step. In some embodiments, the composition of guide molecules for the CRISPR system has the formula: [ka] In some embodiments, the composition consists essentially of a guide molecule of the formula: [ka] or a salt thereof. In some embodiments, the composition consists essentially of a guide molecule of the formula: [ka] or a salt thereof. In some embodiments, the composition consists essentially of a guide molecule of the formula: [ka] [ka] or a salt thereof.

[0195] In some embodiments, the composition comprises an oligonucleotide intermediate (described above) in the presence or absence of a synthetic guide molecule. In some embodiments, the oligonucleotide intermediate of the composition has the formula: [ka] and the synthetic guide molecule is an oligonucleotide intermediate of the formula: [ka] or a pharma- ceutically acceptable salt thereof. In some embodiments, the composition has the formula: [ka] In the presence or absence of a synthetic guide molecule of the formula: [ka] [ka] or a salt thereof.

[0196] In some embodiments, the oligonucleotide intermediate in the composition has the formula: [ka] or a salt thereof, and the synthetic guide molecule has the formula: [ka] In some embodiments, the composition is a synthetic guide molecule of the formula: [ka] or a salt thereof, of the formula: [ka] [ka] [ka] [ka] The oligonucleotide intermediate includes an annealed duplex of

[0197] In some embodiments, the oligonucleotide intermediate of the composition has the formula: [ka] or and the synthetic guide molecule is an oligonucleotide intermediate of the formula: [ka] In some embodiments, the composition is a synthetic guide molecule of the formula: [ka] or a salt thereof.

[0198] In some embodiments, the composition is substantially free of homodimers. In some embodiments, the composition is substantially free of homodimers and / or by-products comprises a guide molecule synthesized using a method that includes a homobifunctional cross-linking reagent. In some embodiments, the composition is substantially free of homodimers and / or by-products comprises a guide molecule having a urea bond. In some embodiments, the guide molecule has the formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein the composition has the formula: [ka] or a pharma- ceutically acceptable salt thereof. In some embodiments, the guide molecule is substantially free of a molecule of the formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein the composition has the formula: [ka] or a pharma- ceutically acceptable salt thereof.

[0199] In some embodiments, the composition is substantially free of by-products. In some embodiments, the composition comprises a guide molecule comprising a urea bond. In some embodiments, the composition comprises a guide molecule having the formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein the composition comprises a guide molecule of the formula: [ka] In some embodiments, the composition is substantially free of molecules of the formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein the composition comprises a guide molecule of the formula: [ka] is substantially free of molecules of

[0200] In some embodiments, the composition substantially comprises a by-product. In some embodiments, the composition substantially comprises a by-product of formula (a): [ka] or a pharma- ceutically acceptable salt thereof; and (b) one or more of the following: (i) a carbodiimide or a salt thereof; (ii) an imidazole, a cyanoimidazole, a pyridine, and a dimethylaminopyridine or a salt thereof; and (iii) a compound of the formula: [ka] (wherein R4 and R5 are each independently substituted or unsubstituted alkyl, or substituted or unsubstituted carbocyclyl), or a salt thereof. In some embodiments, the carbodiimide is EDC, DCC, or DIC. In some embodiments, the composition comprises EDC. In some embodiments, the composition comprises imidazole.

[0201] In some embodiments, the composition is substantially free of the n+1 and / or n-1 species. In some embodiments, the composition comprises less than about 10%, 5%, 2%, 1%, or 0.1% guide molecules that comprise a truncation relative to the reference guide molecule sequence. In some embodiments, at least about 85%, 90%, 95%, 98%, or 99% of the guide molecules comprise a 5' sequence that includes nucleotides 1-20 of the guide molecule that is 100% identical to the corresponding 5' sequence of the reference guide molecule sequence.

[0202] In some embodiments, the composition has the formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein the composition has the formula: [ka] or a pharma- ceutically acceptable salt thereof; wherein a is not equal to c; and / or b is not equal to t. In some embodiments, the composition has the formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein the composition has the formula: [ka] or a pharma- ceutically acceptable salt thereof; where a is not equal to c; and / or b is not equal to t.

[0203] In some embodiments, the composition has the formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein the composition has the formula: [ka] or a pharma- ceutically acceptable salt thereof; wherein a is not equal to c; and / or b is not equal to t. In some embodiments, the composition has the formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein the composition has the formula: [ka] or a pharma- ceutically acceptable salt thereof; wherein a is not equal to c; and / or b is not equal to t. In some embodiments, the composition has the formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein the composition has the formula: [ka] or a pharma- ceutically acceptable salt thereof; wherein a is not equal to c; and / or b is not equal to t. In some embodiments, the composition has the formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein the composition has the formula: [ka] or a pharma- ceutically acceptable salt thereof; where a is not equal to c; and / or b is not equal to t. In some embodiments, a is less than c, and / or b is less than t.

[0204] In some embodiments, the composition has the formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein the composition comprises a guide molecule of the formula: [ka] or a pharma- ceutically acceptable salt thereof; Here, a+b=c+tk, where k is an integer from 1 to 10 inclusive.

[0205] In some embodiments, the composition comprises a synthetic unimolecular guide molecule for a CRISPR system, the guide molecule having the formula: [ka] or a pharma- ceutically acceptable salt thereof, where the 2'-5' phosphodiester bond shown in the formula is between two nucleotides in the duplex. In some embodiments, the guide molecule has the formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein at least one phosphodiester bond between two nucleotides in the duplex region shown in the formula is a 2'-5' phosphodiester bond. In some embodiments, the 2'-5' phosphodiester bond is between two nucleotides located 5' of the bulge. In some embodiments, the 2'-5' phosphodiester bond is between two nucleotides located 5' of nucleotide loop Z and 3' of the bulge. In some embodiments, the 2'-5' phosphodiester bond is between two nucleotides located 3' of nucleotide loop Z and 5' of the bulge. In some embodiments, the 2'-5' phosphodiester bond is between two nucleotides located 3' of nucleotide loop Z and 5' of the bulge.

[0206] Guide molecule design Methods for the selection and validation of target sequences and off-target analysis have been described, for example, in Mali; Hsu; Fu et al., 2014 Nat biotechnol 32(3):279-84, Heigwer et al., 2014 Nat methods 11(2):122-3; Bae et al. (2014) Bioinformatics 30(10):1473-5; and Xiao A et al. (2014) Bioinformatics 30(8):1180-1182. Each of these references is incorporated herein by reference. As a non-limiting example, the design of guide molecules can involve the use of software tools to optimize the selection of potential target sequences corresponding to the user's target sequence, for example, to minimize the overall off-target activity across the genome. Off-target activity is not limited to cleavage, but the cleavage efficiency at each off-target sequence can be predicted, for example, using an empirically derived weighting scheme. These and other guide selection methods are described in detail in Maeder and Cotta-Ramusino.

[0207] The stem-loop structure and the location of chemical bonds in the synthetic single molecule guide molecule can also be designed. The inventors have recognized the value of using Gibbs free energy difference (ΔG) to predict the ligation efficiency of chemical conjugation reactions. ΔG calculation is performed using OligoAnalyzer (available at www.idtdna.com / calc / analyzer) or a similar tool. The experimental results of chemical conjugation can be predicted by comparing the ΔG of heterodimerization to the ΔG of homodimerization of two identical oligonucleotides to form the desired annealed duplex. If the ΔG of heterodimerization is smaller than the ΔG of homodimerization, the ligation efficiency is predicted to be high. This prediction method is further described in Example XX.

[0208] Modification of guide molecules The activity, stability, or other characteristics of a guide molecule can be altered by incorporating certain modifications. As an example, a transiently expressed or delivered nucleic acid can be made susceptible to degradation, for example, by cellular nucleases. Thus, the guide molecules described herein can include one or more modified nucleosides or nucleotides that introduce stability against nucleases. Without wishing to be bound by theory, it is also believed that certain modified guide molecules described herein can exhibit a reduced innate immune response when introduced into a cell. Those skilled in the art will recognize certain cellular responses commonly observed in cells, e.g., mammalian cells, in response to exogenous nucleic acids, particularly exogenous nucleic acids of viral or bacterial origin. Such responses, which can include cytokine expression and release and induction of cell death, can all be reduced or eliminated by the modifications presented herein.

[0209] Certain exemplary modifications discussed in this section can be included anywhere within the guide molecule sequence, including, but not limited to, at or near the 5' end (e.g., within 1-10, 1-5, 1-3, or 1-2 nucleotides of the 5' end) and / or at or near the 3' end (e.g., within 1-10, 1-5, 1-3, or 1-2 nucleotides of the 3' end). In some cases, modifications are located within functional motifs, such as the repeat-anti-repeat duplex of a Cas9 guide molecule, the stem-loop structure of a Cas9 or Cpf1 guide molecule, and / or the targeting domain of a guide molecule.

[0210] In one example, the 5' end of the guide molecule may be: [ka] As shown in, the cap may include a eukaryotic mRNA cap structure or cap analog (e.g., a G(5')ppp(5')G cap analog, a m7G(5')ppp(5')G cap analog, or a 3'-O-Me-m7G(5')ppp(5')G anti-reverse cap analog (ARCA)). The cap or cap analog can be included during chemical or enzymatic synthesis of the guide molecule.

[0211] Along similar lines, the 5' end of the guide molecule can lack a 5' triphosphate group, for example, an in vitro transcribed guide molecule can be phosphatased (e.g., using calf intestinal alkaline phosphatase) to remove the 5' triphosphate group.

[0212] Another common modification involves adding multiple (e.g., 1-10, 10-20, or 25-200) adenine (A) residues, called a polyA tract, to the 3' end of the guide molecule. PolyA tracts can be added to guide molecules during chemical or enzymatic synthesis using a polyadenosine polymerase (e.g., E. coli poly(A) polymerase).

[0213] Guide RNAs can be modified with a 3'-terminal U-ribose. For example, the two terminal hydroxyl groups of U-ribose can be oxidized to aldehyde groups with concomitant opening of the ribose ring to yield a modified nucleoside, as shown below: [ka] In this formula, "U" can be an unmodified or modified uridine.

[0214] The 3' terminal U ribose can be modified with a 2'3' cyclic phosphate as shown below: [ka] In this formula, "U" can be an unmodified or modified uridine.

[0215] Guide RNAs may include 3' nucleotides that may be stabilized against degradation, for example by incorporating one or more modified nucleotides as described herein. In certain embodiments, uridine may be substituted with modified uridines, such as 5-(2-amino)propyluridine, and 5-bromouridine, or any of the modified uridines described herein; adenosine and guanosine may be substituted with modified adenosine and guanosine, for example with a modification at position 8, such as 8-bromoguanosine, or any of the modified adenosine or guanosine described herein.

[0216] In certain embodiments, sugar-modified ribonucleotides can be incorporated into the guide molecule, e.g., the 2'OH-group is replaced with a group selected from H, -OR, -R (where R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar), halo, -SH, -SR (where R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar), amino (where amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); or cyano (-CN). In certain embodiments, the phosphate backbone can be modified as described herein, e.g., with phosphorothioate (PhTx) groups. In certain embodiments, one or more nucleotides of a guide molecule may each independently be a modified or unmodified nucleotide, including, but not limited to, a 2'-sugar modification, such as, for example, 2'-O-methyl, 2'-O-methoxyethyl, or 2'-fluoro modification, including 2'-F or 2'-O-methyl, adenosine (A), 2'-F or 2'-O-methyl, cytidine (C), 2'-F or 2'-O-methyl, uridine (U), 2'-F or 2'-O-methyl, thymidine (T), 2'-F or 2'-O-methyl, guanosine (G), 2'-O-methoxyethyl-5-methyluridine (Teo), 2'-O-methoxyethyl adenosine (Aeo), 2'-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combination thereof.

[0217] Guide RNAs may also include "locked" nucleic acids (LNAs) in which the 2'OH group may be linked to the 4' carbon of the same ribose sugar, for example, by a C1-6 alkylene bridge or a C1-6 heteroalkylene bridge. Any suitable moiety may be used to provide such a bridge, such suitable moieties being methylene, propylene, ether, or amino bridges; O-amino (where amino may be, for example, NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino), and aminoalkoxy or O(CH2). n -amino (wherein amino can be, for example, NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino).

[0218] In certain embodiments, guide molecules can include modified nucleotides that are polycyclic (e.g., tricyclic; and "unlocked" forms such as glycol nucleic acids (GNAs) (e.g., R-GNAs or S-GNAs in which the ribose is replaced by a glycol unit attached to a phosphodiester bond), or threose nucleic acids (TNAs in which the ribose is replaced with α-L-threofuranosyl-(3'→2')).

[0219] In general, the guide molecule comprises a sugar group ribose, which is a five-membered ring with oxygen. Exemplary modified guide molecules can include, but are not limited to, the replacement of oxygen in ribose (e.g., with sulfur (S), selenium (Se), or alkylene, such as methylene or ethylene); the addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl); the ring contraction of ribose (e.g., to form a four-membered ring of cyclobutane or oxetane); the ring expansion of ribose (e.g., to form a six- or seven-membered ring with additional carbon or heteroatoms, such as anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino, which also has a phosphoramidate backbone). Most of the changes in sugar analogues are localized at the 2' position, but other sites, including the 4' position, are amenable to modification. In certain embodiments, the guide molecule comprises a 4'-S, 4'-Se, or 4'-C-aminomethyl-2'-O-Me modification.

[0220] In certain embodiments, deazanucleotides, such as 7-deaza-adenosine, can be incorporated into guide molecules.In certain embodiments, O- and N-alkylated nucleotides, such as N6-methyladenosine, can be incorporated into guide molecules.In certain embodiments, one or more or all nucleotides in guide molecules are deoxynucleotides.

[0221] The nucleotides of the guide molecule may also be modified at the phosphodiester bond. Such modifications may include phosphonoacetate, phosphorothioate, thiophosphonoacetate, or phosphoramidate bonds. In some embodiments, a nucleotide may be linked to its adjacent nucleotide via a phosphorothioate bond. Furthermore, the modification to the phosphodiester bond may be the only modification to the nucleotide or may be combined with other nucleotide modifications described above. For example, the modified phosphodiester bond may be combined with a modification to the sugar group of the nucleotide. In some embodiments, the 5' or 3' nucleotide comprises a 2'-OMe modified ribonucleotide residue linked to its adjacent nucleotide via a phosphorothioate bond.

[0222] RNA-guided nucleases RNA-guided nucleases according to the present disclosure include, but are not limited to, naturally occurring class 2 CRISPR nucleases such as Cas9, and Cpf1, as well as other nucleases derived or obtained therefrom. In terms of function, RNA-guided nucleases are defined as (a) nucleases that interact with (e.g., form complexes with) guide molecules (e.g., gRNA); and (b) nucleases that, together with guide molecules (e.g., gRNA), associate with and optionally cleave or modify a target region of DNA that includes (i) a sequence that is complementary to the target domain of the guide molecule (e.g., gRNA), and optionally (ii) an additional sequence called "protospacer adjacent motif" or "PAM", which will be described in more detail below. As the following examples illustrate, RNA-guided nucleases can be broadly defined by their PAM specificity and cleavage activity, even though differences exist between individual RNA-guided nucleases that share the same PAM specificity or cleavage activity. Those skilled in the art will understand that some aspects of the present disclosure relate to systems, methods, and compositions that can be implemented using any suitable RNA-guided nuclease with a particular PAM specificity and / or cleavage activity. Thus, unless otherwise specified, the term RNA-guided nuclease should be understood as a general term and is not limited to a particular type (e.g., Cas9 vs. Cpfl), species (e.g., S. pyogenes vs. S. aureus), or variation (e.g., full-length vs. cleavage or split; naturally occurring PAM specificity vs. engineered PAM specificity, etc.) of RNA-guided nuclease.

[0223] The PAM sequence derives its name from its sequence relationship to a "protospacer" sequence (or "spacer") that is complementary to the guide molecule targeting domain. Together with the protospacer sequence, the PAM sequence defines the target region or sequence for a particular RNA-guided nuclease / guide molecule combination.

[0224] Various RNA-guided nucleases may require different sequence relationships between the PAM and the protospacer. Generally, Cas9 recognizes the PAM sequence that is 3' of the visualized protospacer relative to the guide molecule.

[0225] Cpf1, on the other hand, generally recognizes a PAM sequence that is 5' of the protospacer visualized relative to the guide molecule.

[0226] In addition to recognizing specific sequence orientations of the PAM and protospacer, RNA-guided nucleases can also recognize specific PAM sequences. For example, S. aureus Cas9 recognizes the NNGRRT or NNGRRV PAM sequence, where the N residue is immediately 3' to the region recognized by the guide molecule targeting domain. S. pyogenes Cas9 recognizes the NGG PAM sequence. And F. novicida Cpfl recognizes the TTN PAM sequence. PAM sequences have been identified in various RNA-guided nucleases, and strategies for identifying novel PAM sequences are described in Shmakov et al., 2015, Molecular Cell 60, 385-397, November 5, 2015. It should also be noted that an engineered RNA-guided nuclease may have a PAM specificity that differs from that of the reference molecule (e.g., in the case of an engineered RNA-guided nuclease, the reference molecule may be a variant of the naturally occurring molecule from which the RNA-guided nuclease is derived, or a variant of the naturally occurring molecule that has the highest amino acid sequence homology to the engineered RNA-guided nuclease).

[0227] In addition to their PAM specificity, RNA-guided nucleases can be characterized by their DNA cleavage activity: naturally occurring RNA-guided nucleases typically form DSBs in target nucleic acids, but engineered mutants have been created that generate only SSBs (supra) Ran & Hsu, et al., Cell 154(6), 1380-1389, September 12, 2013 (Ran), incorporated herein by reference) or do not cleave at all.

[0228] Cas9 Crystal structures have been determined for S. pyogenes Cas9 (Jinek 2014) and for S. aureus Cas9 in complex with a single guide RNA and target DNA (Nishimasu 2014; Anders 2014; and Nishimasu 2015).

[0229] Naturally occurring Cas9 protein contains two lobes, each of which contains a specific structural and / or functional domain: the recognition (REC) lobe and the nuclease (NUC) lobe. The REC lobe contains an arginine-rich bridge helix (BH) domain, and at least one REC domain (e.g., a REC1 domain and an optional REC2 domain). The REC lobe does not share structural similarity with other known proteins, indicating that this REC lobe is a unique functional domain. Without wishing to be bound by any theory, mutational analysis suggests specific functional roles for the BH and REC domains: the BH domain plays a role in guide molecule:DNA recognition, and the REC domain appears to interact with the repeat:antirepeat duplex of the guide molecule and mediate the formation of the Cas9 / guide molecule complex.

[0230] The NUC lobe contains a RuvC domain, an HNH domain, and a PAM interaction (PI) domain. The RuvC domain shares structural similarity with retroviral integrase superfamily members and cleaves the non-complementary (i.e., bottom) strand of the target nucleic acid. The RuvC domain can be formed from two or more split RuvC motifs (such as RuvC I, RuvCII, and RuvCIII in S. pyogenes and S. aureus). Meanwhile, the HNH domain is structurally similar to the HNN endonuclease motif and cleaves the complementary (i.e., top) strand of the target nucleic acid. The PI domain, as its name suggests, contributes to PAM specificity.

[0231] Although certain functions of Cas9 are linked to the specific domains mentioned above (although not necessarily entirely determined by them), these and other functions can be mediated or influenced by other Cas9 domains or by multiple domains on either lobe. For example, in S. pyogenes Cas9, as described in Nishimasu 2014, the repeat:antirepeat duplex of the guide molecule enters the groove between the REC lobe and the NUC lobe, and nucleotides in the duplex interact with amino acids in the BH, PI, and REC domains. Some nucleotides in the first stem-loop structure also interact with amino acids in multiple domains (PI, BH, and REC1), as do some nucleotides in the second and third stem-loops (RuvC and PI domains).

[0232] Cpfl The crystal structure of Acidaminococcus sp. Cpf1 in complex with crRNA and double-stranded (ds) DNA target (containing TTTN PAM sequence) was solved by Yamano et al. (Cell. 2016 May 5;165(4):949-962 (Yamano), incorporated herein by reference). Cpf1, like Cas9, has two lobes: the REC (recognition) lobe and the NUC (nuclease) lobe. The REC lobe contains the REC1 and REC2 domains and has no similarity to any known protein structure. Meanwhile, the NUC lobe contains three RuvC domains (RuvC-1, RuvC-II, and RuvC-III) and a BH domain. However, in contrast to Cas9, the Cpfl REC lobe lacks the HNH domain and contains other domains that similarly lack similarity to known protein structures: a structurally unique PI domain, three Wedge (WED) domains (WED-1, WED-II, and WED-III), and a nuclease (Nuc) domain.

[0233] Although Cas9 and Cpfl share structural and functional similarities, it should be understood that certain Cpfl activities are mediated by structural domains that are not similar to any Cas9 domain. For example, the cleavage of the complementary strand of target DNA appears to be mediated by the Nuc domain, which is sequence- and spatially distinct from the HNH domain of Cas9. In addition, the non-target portion (handle) of the Cpfl guide molecule adopts a pseudoknot structure, rather than the stem-loop structure formed by the repeat:antirepeat duplex in the Cas9 guide molecule.

[0234] Modification of RNA-guided nucleases While the RNA-guided nucleases described above have activities and properties that may be useful in a variety of applications, one of skill in the art will understand that in some cases, RNA-guided nucleases can also be modified to change cleavage activity, PAM specificity, or other structural or functional characteristics.

[0235] First, for modifications that change cleavage activity, mutations that reduce or eliminate the activity of domains in the NUC lobe are described above. Exemplary mutations that can be made in the RuvC domain, Cas9 HNH domain, or Cpfl Nuc domain are described in Ran and Yamano, and Cotta-Ramusino. In general, mutations that reduce or eliminate the activity of one of the two nuclease domains will generate an RNA-guided nuclease with nickase activity, but it should be noted that the type of nickase activity will vary depending on which domain is inactivated. As an example, inactivation of the RuvC domain of Cas9 will generate a nickase that cuts the complementary strand or the upper strand.

[0236] On the other hand, inactivation of the Cas9 HNH domain generates a nickase that cleaves the bottom or non-complementary strand.

[0237]

[00136] Alterations in PAM specificity compared to naturally occurring Cas9 reference molecules have been described by Kleinstiver et al. for both S. pyogenes (Kleinstiver et al., Nature. 2015 Jul 23;523(7561):481-5 (Kleinstiver I) and S. aureus (Kleinstiver et al., Nat Biotechnol. 2015 Dec;33(12):1293-1298 (Kleinstiver II)). Kleinstiver et al. also describe alterations that improve the targeting fidelity of Cas9 (Nature, 2016 Jan 28;529,490-495 (Kleinstiver III)). Each of these references is incorporated herein by reference.

[0238] RNA-guided nucleases have been described by Zetsche et al. (Nat Biotechnol. 2015 Feb;33(2):139-42 (Zetsche II) which is incorporated by reference) and Fine et al. (Sci Rep. 2015 Jul;33(2):139-42 (Zetsche II) which is incorporated by reference). 1;5:10777 (Fine)) into two or more parts.

[0239] In certain embodiments, the RNA-guided nuclease can be size-optimized or truncated, for example, by one or more deletions that reduce the size of the nuclease while maintaining guide molecule association, target and PAM recognition, and cleavage activity. In certain embodiments, the RNA-guided nuclease is covalently or non-covalently linked to another polypeptide, nucleotide, or other structure, optionally using a linker. Exemplary binding nucleases and linkers are described in Guilinger et al., Nature Biotechnology 32, 577-582 (2014), which is incorporated by reference for all purposes herein.

[0240] RNA-guided nuclease also optionally comprises a tag, for example, but not limited to, a nuclear localization signal, which facilitates the movement of RNA-guided nuclease protein into the nucleus.In certain embodiments, RNA-guided nuclease can incorporate C-terminal and / or N-terminal nuclear localization signals.Nuclear localization sequences are known in the art and are described in Maeder and elsewhere.

[0241] The list of modifications above is intended to be exemplary in nature, and those skilled in the art will understand from the present disclosure that other modifications may be possible or desirable in certain applications.Thus, for the sake of brevity, the exemplary systems, methods and compositions of the present disclosure are presented with reference to specific RNA-guided nucleases, but it should be understood that the RNA-guided nucleases used can be modified without changing their working principle.Such modifications are within the scope of the present disclosure.

[0242] Nucleic acid encoding an RNA-guided nuclease Provided herein are nucleic acids encoding RNA-guided nucleases, such as Cas9, Cpfl, or functional fragments thereof. Exemplary nucleic acids encoding RNA-guided nucleases have been described previously (e.g., Cong 2013; Wang 2013; Mali 2013). 2013; see Jinek 2012).

[0243] In some cases, the nucleic acid encoding the RNA-guided nuclease can be a synthetic nucleic acid sequence.For example, synthetic nucleic acid molecules can be chemically modified.In certain embodiments, the mRNA encoding the RNA-guided nuclease can have one or more (for example, all) of the following characteristics: the mRNA can be capped; polyadenylated; and substituted with 5-methylcytidine and / or pseudouridine.

[0244] Synthetic nucleic acid sequence can also be codon-optimized, for example, at least one non-common or less common codon is replaced by common codon.For example, synthetic nucleic acid can be optimized for expression in mammalian expression system as described herein, for example, to induce synthesis of optimized messenger mRNA.An example of codon-optimized Cas9 coding sequence is shown in Cotta-Ramusino.

[0245] Additionally or alternatively, the nucleic acid encoding the RNA-guided nuclease may contain a nuclear localization sequence (NLS). Nuclear localization sequences are known in the art.

[0246] Functional analysis of candidate molecules Candidate RNA-guided nuclease, guide molecule, and their complexes can be evaluated by standard methods known in the art.See, for example, Cotta-Ramusino.The stability of RNP complexes can be evaluated by differential scanning fluorimetry, as described below.

[0247] Differential Scanning Fluorometry (DSF) The thermal stability of a ribonucleoprotein (RNP) complex containing a guide molecule and an RNA-guided nuclease can be measured via DSF. The DSF technique measures the thermal stability of a protein, which can be increased under favorable conditions, such as the addition of a bound RNA molecule, e.g., a guide molecule.

[0248] The DSF assay can be performed according to any suitable protocol and can be utilized in any suitable setting, including but not limited to (a) testing various conditions (e.g., various stoichiometric ratios of guide molecule:RNA-guided nuclease protein, different buffers, etc.) to identify optimal conditions for the formation of RNPs; and (b) testing modifications (e.g., chemical modifications, sequence alterations, etc.) of the RNA-guided nuclease and / or guide molecule to identify modifications that improve the formation or stability of RNPs. One readout of the DSF assay is a shift in the melting temperature of the RNP complex; a relatively high shift suggests that the RNP complex is more stable (and thus may have greater activity or more favorable kinetics of formation, kinetics of disassembly, or other functional characteristics) compared to a reference RNP complex characterized by a lower shift. When the DSF assay is deployed as a screening tool, a threshold melting temperature shift can be identified, so that the result is one or more RNPs with a melting temperature shift equal to or greater than the threshold. For example, the threshold can be 5-10° C. (e.g., 5°, 6°, 7°, 8°, 9°, 10°) or greater, and the result can be one or more RNPs characterized by a melting temperature shift above the threshold.

[0249] Two non-limiting examples of DSF assay conditions are described below:

[0250] To determine the best solution for forming RNP complexes, a fixed concentration (e.g., 2 μM) of Cas9 in water + 10×SYPRO Orange® (Life Technologies cat#S-6650) is dispensed into a 384-well plate. Equimolar amounts of guide molecules diluted in solutions of various pH and salt are then added. After 10 minutes of incubation at room temperature and a brief centrifugation to remove any air bubbles, a gradient from 20°C to 90°C is run with a temperature increase of 1°C every 10 seconds using a Bio-Rad CFX384™ Real-Time System C1000 Touch™ thermal cycler with Bio-Rad CFX Manager software.

[0251] The second assay consists of mixing various concentrations of guide molecule with a fixed concentration (e.g., 2 μM) of Cas9 in optimal buffer from assay 1 above and incubating in a 384-well plate (e.g., for 10 minutes at room temperature). Equal volumes of optimal buffer and 10×SYPRO Orange® (Life Technologies catalog number S-6650) are added and the plate is sealed with Microseal® B adhesive (MSB-1001). Following a brief centrifugation to remove any air bubbles, a gradient from 20° C. to 90° C. is run with a temperature increase of 1° C. every 10 seconds using a Bio-Rad CFX384™ Real-Time System C1000 Touch™ thermal cycler with Bio-Rad CFX Manager software.

[0252] Genome editing strategies In various embodiments of the present disclosure, the above-mentioned genome editing system is used to edit (i.e., modify) the target region of DNA in a cell or DNA obtained from a cell.Various strategies for making specific edits are described herein, and these strategies are generally described in terms of the desired repair result, the number and position of individual edits (e.g., SSB or DSB), and the target site of such edits.

[0253] Genome editing strategies that include the formation of SSB or DSB are characterized by repair results that include: (a) deletion of all or part of the target region; (b) insertion into the target region or replacement of all or part of it; or (c) interruption of all or part of the target region. This classification is not intended to be limiting or to be bound to any particular theory or model, but is provided only for the sake of simplicity of presentation. Those skilled in the art will understand that the listed results are not mutually exclusive, and some repairs may lead to other results. The description of a particular editing strategy or method should not be understood as requiring a particular repair result unless otherwise stated.

[0254] Replacement of a target region generally involves replacing all or part of an existing sequence in the target region with a homologous sequence, for example, via gene correction or gene conversion, two repair outcomes mediated by the HDR pathway. HDR is facilitated by the use of a donor template, which can be single-stranded or double-stranded, as described in more detail below. The single-stranded or double-stranded template can be exogenous, in which case it facilitates gene correction, or endogenous (e.g., a homologous sequence in the cellular genome), in which case it facilitates gene conversion. The exogenous template can have an asymmetric overhang (i.e., the portion of the template that is complementary to the site of the DSB can be offset in the 3' or 5' direction, rather than being located in the center of the donor template), as described, for example, by Richardson et al. (Nature Biotechnology 34,339-344 (2016), (Richardson)), which is incorporated by reference. If the template is single-stranded, the template can correspond to either the complementary strand (top) or non-complementary strand (bottom) of the target region.

[0255] Gene conversion and gene correction are sometimes facilitated by forming one or more nicks in or around the target region, as described in Ran and Cotta-Ramusino. In some cases, a dual nickase strategy is used to form two offset SSBs, thereby forming a single DSB with an overhang (e.g., a 5' overhang).

[0256] The interruption and / or deletion of all or part of the target sequence can be achieved by various repair outcomes. As an example, the sequence can be deleted by simultaneously generating two or more DSBs flanking the target region, followed by excision of the target region when the DSBs are repaired, as described by Maeder for LCA10 mutations. As another example, the sequence can be interrupted by the formation of a double-stranded break with a single-stranded overhang, followed by a deletion generated by exonuclease processing of the overhang before repair.

[0257] The interruption of one particular subset of target sequences is mediated by the formation of indels within the target sequence, where the repair outcome is typically mediated by the NHEJ pathway (including Alt-NHEJ). NHEJ is referred to as an "error-prone" repair pathway due to its association with indel mutations. However, in some cases, DSBs are repaired by NHEJ without modifying the sequence around them (so-called "complete" or "scarless" repair); this generally requires that both ends of the DSB are completely ligated. On the other hand, indels are thought to result from enzymatic processing of free DNA ends prior to ligation, which adds and / or removes nucleotides to one or both free ends of one or both strands.

[0258] Because enzymatic processing of free DSB ends can be stochastic in nature, indel mutations tend to be variable, occur along a distribution, and can be influenced by a variety of factors including the specific target site, the cell type used, the genome editing strategy used, etc. Still, it is possible to obtain limited generalizations about indel formation: deletions formed by repair of single DSBs are most commonly in the range of 1-50 bp, but can exceed 100-200 bp. Insertions formed by repair of single DSBs tend to be shorter, often containing short duplications of sequences immediately surrounding the break site. However, it is possible to obtain large insertions, and in these cases, the inserted sequences were often traced to other regions of the genome or to plasmid DNA present in the cell.

[0259] Indel mutations - and genome editing systems configured to generate indels - are useful for interrupting target sequences, for example, when the generation of a specific final sequence is not required and / or frameshift mutations are tolerated. They may also be useful in settings where a specific sequence is preferred, as long as the desired specific sequence tends to preferentially result from the repair of SSBs or DSBs at a given site. Indel mutations are also useful tools for evaluating or screening the activity of specific genome editing systems and their components. In these and other settings, indels can be characterized by (a) their relative and absolute frequency in the genome of cells contacted with the genome editing system, and (b) the distribution of numerical differences, e.g., ±1, ±2, ±3, etc., relative to the unedited sequence. As an example, in a lead discovery setting, multiple guide molecules can be screened to identify the guide molecule that most efficiently induces cleavage at the target site based on indel readouts under controlled conditions. Guides that generate indels at or above a threshold frequency, or that generate a specific distribution of indels, can be selected for further research and development. The frequency and distribution of indels can also be useful as a readout to evaluate the implementation or formulation and delivery methods of different genome editing systems, for example, by holding the guide molecule constant and varying other specific reaction conditions or delivery methods.

[0260] Multiplexing Strategy Although the exemplary strategies discussed above focus on repair outcomes mediated by a single DSB, the genome editing system of the present disclosure can also be utilized to generate two or more DSBs, either at the same locus or at different loci. Editing strategies involving the formation of multiple DSBs or SSBs are described, for example, in Cotta-Ramusino.

[0261] Donor template design The design of donor template is described in detail in the literature, for example, in Cotta-Ramusino.DNA oligomer donor template (oligodeoxynucleotide or ODN), which can be single-stranded (ssODN) or double-stranded (dsODN), can be used to facilitate HDR-based repair of DSB, and is particularly useful for introducing modification into target DNA sequence, inserting new sequence into target sequence, or completely replacing target sequence.

[0262] Whether single-stranded or double-stranded, the donor template generally contains a region that is homologous to a DNA region within or near (e.g., adjacent or proximal to) the target sequence to be cleaved. These regions of homology are referred to herein as "homology arms" and are illustrated diagrammatically below: [5' homology arm]-[substitution sequence]-[3' homology arm]

[0263] The homology arms can have any suitable length (including 0 nucleotides if only one homology arm is used), and the 3' and 5' homology arms can have the same length or can be different in length. Selection of the appropriate homology arm length can be influenced by various factors, such as a desire to avoid homology or microhomology with certain sequences, such as Alu repeats or other very common elements. For example, the 5' homology arm can be shortened to avoid sequence repeat elements. In other embodiments, the 3' homology arm can be shortened to avoid sequence repeat elements. In some embodiments, both the 5' and 3' homology arms can be shortened to avoid certain sequence repeat elements. In addition, some homology arm designs can improve the efficiency of editing or increase the frequency of the desired repair outcome. See, for example, Richardson, et al., in J. Am. Soc. 1999, 143:1311-1323, which is incorporated by reference. Nature Biotechnology 34, 339-344 (2016) (Richardson) found that the relative asymmetry of the 3' and 5' homology arms of a single-stranded donor template affects the repair rate and / or outcome.

[0264] The replacement sequence in the donor template has been described elsewhere, including Cotta-Ramusino et al. The replacement sequence can be of any suitable length (including 0 nucleotides where the desired repair outcome is a deletion) and typically includes one, two, three, or more sequence modifications to a naturally occurring sequence in the cell where editing is desired. One common sequence modification includes modification of a naturally occurring sequence to repair a mutation associated with a disease or condition for which treatment is desired. Another common sequence modification includes modification of one or more sequences that are complementary to or encode the PAM sequence of an RNA-guided nuclease or the target domain of a guide molecule used to generate SSBs or DSBs to reduce or eliminate recurrent cleavage of the target site after the replacement sequence is incorporated into the target site.

[0265] When a linear ssODN is used, the linear ssODN can be configured to (i) anneal to a nicked strand of a target nucleic acid, (ii) anneal to an intact strand of a target nucleic acid, (iii) anneal to a positive strand of a target nucleic acid, and / or (iv) anneal to a negative strand of a target nucleic acid. The ssODN can have any suitable length, for example, about, at least, or up to 150-200 nucleotides (e.g., 150, 160, 170, 180, 190, or 200 nucleotides).

[0266] It should be noted that the template nucleic acid can also be a nucleic acid vector, such as a viral genome or a circular double-stranded DNA, e.g., a plasmid. The nucleic acid vector containing the donor template can contain other coding or non-coding elements. For example, the template nucleic acid can be delivered (e.g., in an AAV genome or a lentiviral genome) as part of a viral genome that contains certain genomic backbone elements (e.g., inverted terminal repeats in the case of an AAV genome), and optionally additional sequences encoding guide molecules and / or RNA-guided nucleases. In certain embodiments, the donor template can be adjacent to or adjacent to target sites recognized by one or more guide molecules to facilitate the formation of free DSBs at one or both ends of the donor template that can participate in the repair of corresponding SSBs or DSBs formed in cellular DNA using the same guide molecules. Exemplary nucleic acid vectors suitable for use as donor templates are described in Cotta-Ramusino.

[0267] Whatever format is used, the template nucleic acid can be designed to avoid undesired sequences, in certain embodiments, one or both homology arms can be shortened to avoid overlap with certain sequence repeat elements, e.g., Alu repeats, LINE elements, etc.

[0268] target cell The genome editing system according to the present disclosure can be used to manipulate or modify cells, for example to edit or modify a target nucleic acid, which in various embodiments can be performed in vivo or ex vivo.

[0269] Various cell types can be engineered or modified according to embodiments of the present disclosure, and in some cases, such as in vivo applications, multiple cell types are engineered or modified, for example, by delivering a genome editing system according to the present disclosure to the multiple cell types. However, in other cases, it will be desirable to limit the engineering or modification to one or more specific cell types. For example, in some cases, it will be desirable to edit cells with limited differentiation potential or terminally differentiated cells, such as photoreceptor cells in the case of Maeder, where modification of the genotype is expected to change the cell phenotype. However, in other cases, it will be desirable to edit less differentiated multipotent or pluripotent stem or progenitor cells. By way of example, the cells can be embryonic stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem / progenitor cells (HSPCs), or other stem or progenitor cell types that differentiate into cell types appropriate for a given application or indication.

[0270] As a natural consequence, the cells that are modified or engineered can be dividing or non-dividing cells, depending on the cell type targeted and / or the desired editing outcome.

[0271] If the cells are manipulated or modified ex vivo, they can be used immediately (e.g., administered to a subject) or can be maintained or stored for later use. Those skilled in the art will appreciate that the cells can be maintained in culture or stored (e.g., frozen in liquid nitrogen) using any suitable method known in the art.

[0272] Implementation of genome editing systems: delivery, formulation, and routes of administration As discussed above, the genome editing system of the present disclosure can be implemented in any suitable manner, which includes, but is not limited to, the components of such a system, including RNA-guided nucleases, guide molecules, and optional donor template nucleic acids, can be delivered, formulated, or administered in any suitable form or combination of forms that result in transduction, expression, or introduction of the genome editing system and / or result in the desired repair outcome in a cell, tissue, or subject. Tables 5 and 6 show some non-limiting examples of implementations of the genome editing system. However, those skilled in the art will understand that these lists are not comprehensive and other implementations are possible. With particular reference to Table 5, the table lists some exemplary implementations of a genome editing system that includes a single guide molecule and an optional donor template. However, the genome editing system according to the present disclosure can incorporate other components such as multiple guide molecules, multiple RNA-guided nucleases, and proteins, and those skilled in the art will understand various implementations based on the principles illustrated in the table. In the table, [N / A] indicates that the genome editing system does not include the indicated component.

[0273] [Table 7]

[0274] [Table 8]

[0275] Table 6 summarizes various delivery methods for the components of the genome editing system as described herein. Again, the list is intended to be illustrative rather than limiting.

[0276] [Table 9]

[0277] Nucleic acid-based delivery of genome editing systems Nucleic acids encoding various elements of the genome editing system according to the present disclosure can be administered to a subject or delivered to a cell by methods known in the art or as described herein. For example, DNA encoding an RNA-guided nuclease and / or encoding a guide molecule, as well as a donor template nucleic acid, can be delivered, for example, by a vector (e.g., a viral vector or a non-viral vector), a non-vector-based method (e.g., using naked DNA or a DNA complex), or a combination thereof.

[0278] Nucleic acids encoding the genome editing system or components thereof can be delivered directly to cells as naked DNA or RNA, for example by transfection or electroporation, or can be conjugated to molecules (e.g., N-acetylgalactosamine) that facilitate uptake by target cells (e.g., red blood cells, HSCs). Nucleic acid vectors, such as those summarized in Table 6, can also be used.

[0279] The nucleic acid vector may include one or more sequences encoding components of the genome editing system, such as an RNA-guided nuclease, a guide molecule, and / or a donor template. The vector may also include a sequence encoding a signal peptide (e.g., for nuclear localization, nucleolar localization, or mitochondrial localization) that is associated with (e.g., inserted into or fused to) the protein-encoding sequence. As an example, the nucleic acid vector may include a Cas9 coding sequence that includes one or more nuclear localization sequences (e.g., nuclear localization sequences from SV40).

[0280] The nucleic acid vector can also include any suitable number of regulatory / control elements, such as promoters, enhancers, introns, polyadenylation signals, Kozak consensus sequences, or internal ribosome entry sites (IRES), which are well known in the art and described in Cotta-Ramusino.

[0281] Nucleic acid vectors according to the present disclosure include recombinant viral vectors. Exemplary viral vectors are described in Table 6, and further suitable viral vectors and their use and production are described in Cotta-Ramusino. Other viral vectors known in the art can also be used. In addition, viral particles can be used to deliver components of the genome editing system in the form of nucleic acids and / or peptides. For example, "empty" viral particles can be assembled to include any suitable cargo. Viral vectors and viral particles can also be engineered to incorporate targeting ligands to alter target tissue specificity.

[0282] In addition to viral vectors, non-viral vectors can be used to deliver the nucleic acid encoding the genome editing system according to the present disclosure. One important category of non-viral nucleic acid vectors is nanoparticles, which can be organic or inorganic. Nanoparticles are well known in the art and are summarized in Cotta-Ramusino. Any suitable nanoparticle design can be used to deliver the components of the genome editing system or the nucleic acid encoding such components. For example, organic (e.g., lipid and / or polymer) nanoparticles can be suitable for use as a delivery vehicle in certain embodiments of the present disclosure. Table 7 shows exemplary lipids for use in nanoparticle formulation and / or gene transfer, and Table 8 lists exemplary polymers for use in gene transfer and / or nanoparticle formulation.

[0283] [Table 10]

[0284] [Table 11]

[0285] Non-viral vectors optionally include targeting modifications to improve uptake and / or selectively target specific cell types. These targeting modifications may include, for example, cell-specific antigens, monoclonal antibodies, single-chain antibodies, aptamers, polymers, sugars (e.g., N-acetylgalactosamine (GalNAc)), and cell-penetrating peptides. Such vectors also optionally use fusogenic and endosome-destabilizing peptides / polymers, undergo acid-induced conformational changes (e.g., to facilitate endosomal escape of the cargo), and / or incorporate stimuli-cleavage polymers, for example, for release into cellular compartments. For example, disulfide-based cationic polymers can be used that are cleaved in the reducing cellular environment.

[0286] In certain embodiments, one or more nucleic acid molecules (e.g., DNA molecules) are delivered other than the components of the genome editing system, such as the RNA-guided nuclease and / or guide molecule components described herein. In certain embodiments, the nucleic acid molecules are delivered simultaneously with one or more components of the genome editing system. In certain embodiments, the nucleic acid molecules are delivered before or after one or more components of the genome editing system are delivered (e.g., less than about 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 9 hours, 12 hours, 1 day, 2 days, 3 days, 1 week, 2 weeks, or 4 weeks). In certain embodiments, the nucleic acid molecules are delivered by a means different from the means by which one or more components of the genome editing system, such as the RNA-guided nuclease and / or guide molecule components, are delivered. The nucleic acid molecules can be delivered by any delivery method described herein. For example, the nucleic acid molecules can be delivered by a viral vector, such as an integration-deficient lentivirus, and the RNA-guided nuclease and / or guide molecule components can be delivered by electroporation, such as to reduce toxicity due to the nucleic acid (e.g., DNA). In certain embodiments, the nucleic acid molecule encodes a therapeutic protein, such as a protein described herein. In certain embodiments, the nucleic acid molecule encodes an RNA molecule, such as an RNA molecule described herein.

[0287] Delivery of RNPs and / or RNAs encoding components of genome editing systems RNP (complex of guide molecule and RNA-guided nuclease) and / or RNA-guided nuclease and / or RNA encoding guide molecule can be delivered into cells or administered to a subject by methods known in the art, some of which are described in Cotta-Ramusino. In vitro, RNA encoding RNA-guided nuclease and / or RNA encoding guide molecule can be delivered, for example, by microinjection, electroporation, transient cell compression or squeezing (see, for example, Lee 2012). Lipid-mediated transfection, peptide-mediated delivery, GalNAc or other conjugate-mediated delivery, and combinations thereof can also be used for delivery in vitro and in vivo.

[0288] In vitro, delivery by electroporation involves mixing cells with or without a donor template nucleic acid molecule with an RNA-guided nuclease and / or RNA encoding a guide molecule in a cartridge, chamber, or cuvette, and applying one or more electrical impulses of defined duration and amplitude. Systems and protocols for electroporation are known in the art, and any suitable electroporation tool and / or protocol can be used in connection with various embodiments of the present disclosure.

[0289] Route of administration The genome editing system, or the cells modified or engineered using such a system, can be administered to a subject by any suitable mode or route, whether local or systemic. Systemic administration modes include oral and parenteral routes. Parenteral routes include, for example, intravenous, intramedullary, intraarterial, intramuscular, intradermal, subcutaneous, intranasal, and intraperitoneal routes. The components administered systemically can be modified or formulated to target, for example, HSC, hematopoietic stem / progenitor cells, or erythroid precursor or progenitor cells.

[0290] Local administration modes include, for example, intramedullary injection into trabecular bone or intrafemoral injection into bone marrow cavity, and injection into portal vein.In certain embodiments, significantly less amount of components (compared to systemic approach) can be effective when administered locally (e.g., directly into bone marrow) compared to when administered systemically (e.g., intravenously).Local administration methods can reduce or eliminate the occurrence of potentially toxic side effects that may occur when administering therapeutically effective amounts of components systemically.

[0291] Doses may be provided as a periodic bolus (e.g., intravenously) or as a continuous infusion from an internal or external reservoir (e.g., an intravenous bag or an implanted pump). The components may also be administered locally, for example, by continuous release from a sustained drug delivery device.

[0292] Additionally, the components may be formulated to allow release over an extended period of time. The release system may include a matrix of a biodegradable material or a material that releases the incorporated components by diffusion. The components may be distributed uniformly or non-uniformly within the release system. A variety of release systems may be useful, with the appropriate system being selected depending on the release rate required for a particular application. Both non-degradable and degradable release systems may be used. Suitable release systems include polymers and polymeric matrices, non-polymeric matrices, or inorganic and organic excipients and diluents, such as, but not limited to, calcium carbonate and sugars (e.g., trehalose). The release system may be either natural or synthetic. However, synthetic release systems are generally preferred because they are more reliable, reproducible, and produce a more defined release profile. The release system material may be selected such that components with different molecular weights are released by diffusion through the material or by its degradation.

[0293] Representative synthetic biodegradable polymers include, for example, polyamides, such as poly(amino acids) and poly(peptides); polyesters, such as poly(lactic acid), poly(glycolic acid), copolymers of lactic acid and poly(caprolactone); poly(anhydrides); polyorthoesters; polycarbonates; and chemical derivatives thereof (substitution, addition, hydroxylation, oxidation of chemical groups, e.g., alkyl, alkylene, and other modifications routinely performed by one of ordinary skill in the art), copolymers and mixtures thereof. Representative synthetic non-biodegradable polymers include, for example, polyethers such as poly(ethylene oxide), poly(ethylene glycol), and poly(tetramethylene oxide); vinyl polymers such as methyl, ethyl, other alkyl, hydroxyethyl methacrylate, acrylic acid and methacrylic acid-polyacrylates and polymethacrylates, and others such as poly(vinyl alcohol), poly(vinylpyrrolidone), and poly(vinyl acetate); poly(urethanes); celluloses and their derivatives such as alkyl, hydroxyalkyl, ethers, esters, nitrocellulose, and various cellulose acetates; polysiloxanes; and any chemical derivatives thereof (substitution, addition, hydroxylation, oxidation of chemical groups, e.g., alkyl, alkylene, and other modifications routinely performed by one of ordinary skill in the art), copolymers, and mixtures thereof.

[0294] Poly(lactide-co-glycolide) microspheres may also be used. Typically, the microspheres are composed of polymers of lactic acid and glycolic acid and are structured to form hollow spheres. The spheres may be about 15-30 microns in diameter and can be loaded with the components described herein.

[0295] Multimodal or differential delivery of components In light of the present disclosure, one of skill in the art will understand that the different components of the genome editing systems disclosed herein can be delivered together or separately, and simultaneously or non-simultaneously. Separate and / or non-simultaneous delivery of the components of the genome editing system may be particularly desirable to provide temporal or spatial control over the function of the genome editing system to limit certain effects caused by their activity.

[0296] Different or differential modes, as used herein, refer to delivery modes that confer different pharmacodynamic or pharmacokinetic properties to the component molecules of interest, such as, for example, RNA-guided nuclease molecules, guide molecules, template nucleic acids, or payloads.For example, these delivery modes can result in different tissue distributions, different half-lives, or different temporal distributions, for example, in selected compartments, tissues, or organs.

[0297] Some delivery modes result in more sustained expression and presence of the components, such as delivery by a nucleic acid vector that persists in the cell or its descendants, for example by autonomous replication or insertion into the cellular nucleic acid. Examples include delivery by viruses, such as AAV or lentiviruses.

[0298] As an example, components of genome editing system, such as RNA-guided nuclease and guide molecule, can be delivered by modes that result in different half-life or persistence of components delivered to the body or to a specific compartment, tissue or organ.In certain embodiments, guide molecule can be delivered by such modes.Components of RNA-guided nuclease molecule can be delivered to the body or to a specific compartment or tissue or organ by modes that reduce persistence or exposure.

[0299] More generally, in certain embodiments, a first delivery mode is used to deliver a first component, and a second delivery mode is used to deliver a second component. The first delivery mode imparts a first pharmacodynamic or pharmacokinetic property. The first pharmacodynamic property may be, for example, the distribution, persistence, or exposure of the component, or the nucleic acid encoding the component, in the body, compartment, tissue, or organ. The second delivery mode imparts a second pharmacodynamic or pharmacokinetic property. The second pharmacodynamic property may be, for example, the distribution, persistence, or exposure of the component, or the nucleic acid encoding the component, in the body, compartment, tissue, or organ.

[0300] In certain embodiments, a first pharmacodynamic or pharmacokinetic property, eg, distribution, persistence, or exposure, is more defined than a second pharmacodynamic or pharmacokinetic property.

[0301] In certain embodiments, the first delivery mode is selected to optimize, e.g., minimize, a pharmacodynamic or pharmacokinetic property, e.g., distribution, duration, or exposure.

[0302] In certain embodiments, the second delivery mode is selected to optimize, e.g., maximize, a pharmacodynamic or pharmacokinetic property, e.g., distribution, duration, or exposure.

[0303] In certain embodiments, the first delivery mode involves the use of relatively persistent elements, such as, for example, nucleic acids, such as plasmids or viral vectors, such as AAV or lentivirus, such that because such vectors are relatively persistent, the products transcribed therefrom are also relatively persistent.

[0304] In certain embodiments, the second delivery mode comprises a relatively transient element, such as an RNA molecule or a protein.

[0305] In certain embodiments, the first component includes a guide molecule, and the delivery mode is relatively sustained, for example, the guide molecule is transcribed from a plasmid or a viral vector, such as AAV or lentivirus. These genes do not code for protein products, and the guide molecule cannot act alone, so the transcription of these genes has little physiological consequence. The second component, the RNA-guided nuclease molecule, is delivered in a transient manner, for example as an mRNA or protein, and the complete RNA-guided nuclease molecule / guide molecule complex is present and active for only a short period of time.

[0306] Furthermore, the components can be delivered in different molecular forms or using different delivery vectors that complement each other to enhance safety and tissue specificity.

[0307] The use of differential delivery modes can increase performance, safety and / or efficiency, for example, reducing the possibility of eventual off-target modification. Because peptides from bacterial Cas enzymes are displayed on the surface of cells by MHC molecules, delivery of immunogenic components, such as Cas9 molecules, by low-persistence modes can reduce immunogenicity. A two-part delivery system can alleviate these issues.

[0308] Differential delivery modes can be used to deliver components to different but overlapping target regions. The formation of active complexes is minimized outside the overlap of target regions. Thus, in certain embodiments, a first component, such as, for example, a guide molecule, is delivered by a first delivery mode that results in a first spatial (e.g., tissue, etc.) distribution. A second component, such as, for example, an RNA-guided nuclease molecule, is delivered by a second delivery mode that results in a second spatial (e.g., tissue, etc.) distribution. In certain embodiments, the first mode includes a first element selected from liposomes, nanoparticles, such as, for example, polymer nanoparticles, and nucleic acids, such as, for example, viral vectors. The second mode includes a second element selected from the above group. In certain embodiments, the first delivery mode includes a first targeting element, such as, for example, a cell-specific receptor or an antibody, and the second delivery mode does not include that element. In certain embodiments, the second delivery mode includes a second targeting element, such as, for example, a second cell-specific receptor or a second antibody.

[0309] When RNA-guided nuclease molecules are delivered in viral delivery vectors, liposomes, or polymer nanoparticles, there is the possibility of delivery to and therapeutic activity in multiple tissues when targeting only a single tissue would be desirable. A two-part delivery system can solve this problem and increase tissue specificity. When the guide molecule and the RNA-guided nuclease molecule are packaged in separate delivery vehicles with different but overlapping tissue tropisms, only fully functional complexes are formed in tissues targeted by both vectors. EXAMPLES

[0310] Certain principles of the present disclosure are illustrated by the following non-limiting examples.

[0311] Example 1: Exemplary process for conjugation of amine-functionalized guide molecule fragments using disuccinimidyl carbonate As illustrated in Figure 1A, a first 5' guide molecule fragment (e.g., a 34-mer) is synthesized with a (C6)-NH2 linker at the 3' end, and a second 3' guide molecule fragment (e.g., a 66-mer) is synthesized with a TEG-NH2 linker at the 5' end. The two guide molecule fragments are mixed in a 1:1 molar ratio in a buffer containing 10 mM sodium borate, 150 mM NaCl, and 5 mM MgCl2 at pH 8.5. The resulting guide molecule concentration is approximately 50-100 μM. The two guide molecule fragments are annealed, followed by the addition of disuccinimidyl carbonate (DSC) in DMF (final concentration of 2.5 mM). The reaction mixture is vortexed briefly and then mixed at room temperature for 1 h, followed by removal of excess disuccinimidyl carbonate and anion exchange HPLC purification.

[0312] Example 2: Exemplary process for conjugation of a thiol-functionalized guide molecule fragment to a bromoacetyl-functionalized guide molecule fragment As illustrated in FIG. 2A, a first 5' guide molecule fragment (e.g., a 34-mer) is synthesized with a (C6)-NH2 linker at the 3' end. The first 5' guide molecule fragment is suspended in 100 mM borate buffer at pH 8.5. The guide molecule concentration is approximately 100 μM to 1 mM. 0.2 volumes of succinimidyl-3-(bromoacetamido)propionate (SBAP) in DMSO (50 equivalents) are added to the guide molecule solution. After mixing for 30 minutes at room temperature, 10 volumes of 100 mM phosphate buffer (pH 7.0) are added. The mixture is concentrated 10 times or more on a 10,000 MW Amicon. The mixture is further processed by (a) adding 10 volumes of water and (b) concentrating 10 times or more on a 10,000 MW Amicon. Steps (a) and (b) are repeated three times to obtain a first 5' guide molecule fragment (e.g., a 34-mer) bearing a bromoacetyl moiety at its 3' end.

[0313] As illustrated in FIG. 2B, a second 3' guide molecule fragment (e.g., a 66-mer) is synthesized with a TEG-NH2 linker at the 5' end. The second 3' guide molecule fragment is suspended in 100 mM borate buffer at pH 8.5 containing 1 mM EDTA. The guide molecule concentration is approximately 100 μM to 1 mM. 0.2 volumes of succinimidyl-3-(2-pyridyldithio)propionate (SPDP) in DMSO (50 equivalents) are added to the guide molecule solution. After mixing for 1 hour at room temperature, 1 M dithiothreitol (DTT) in 1×PBS is added. The final concentration of DTT in the mixture is 20 mM. After mixing for 30 minutes at room temperature, 5 M NaCl is added to a final concentration of 0.3 M NaCl in the mixture, followed by 3 volumes of ethanol. The mixture is further processed by (a) cooling to -20°C for 15 min; (b) centrifuging (preferably at 4°C) at 17,000g for 5 min; (c) removing the supernatant; (d) suspending the residue in 0.3 M NaCl (sparged with argon); and (e) adding 3 volumes of ethanol. Steps (a)-(e) are repeated three times. The resulting pellet (i.e., the second 3' guide molecule fragment with a thiol at the 5' end) is dried under vacuum.

[0314] As illustrated in FIG. 2C, a second 3' guide molecule fragment (e.g., a 66-mer) with a thiol at the 5' end is suspended in 100 mM phosphate buffer at pH 8 containing 2 mM EDTA (sparged with argon). The guide molecule concentration is about 100 μM to 1 mM. A first 5' guide molecule fragment (e.g., a 34-mer) with a bromoacetyl moiety at the 3' end is suspended in water (about 0.1 volume relative to the volume of the second 3' guide molecule fragment mixture). The guide molecule concentration is about 100 μM to 1 mM. The first 5' guide molecule fragment mixture is added to the second 3' guide molecule fragment mixture (sparged with argon). The reaction mixture is mixed at room temperature overnight, followed by anion exchange HPLC purification.

[0315] Example 3: Exemplary process for conjugation of a phosphate guide molecular fragment to a 3' hydroxyl guide molecular fragment using a carbodiimide As illustrated in Figure 3A and Figure 3B, a first 5' guide molecule fragment (e.g., a 34-mer) is synthesized using standard phosphoramidate chemistry. A second 3' guide molecule fragment (e.g., a 66-mer) containing a 5'-phosphate is also synthesized. The first and second guide molecule fragments are mixed in a 1:1 molar ratio in coupling buffer (100 mM 2-(N-morpholino)ethanesulfonic acid (MES), pH 6, 150 mM NaCl, 5 mM MgCl2, and 10 mM ZnCl2). The two guide molecule fragments are annealed, followed by the addition of 100 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 90 mM imidazole. The reaction mixture is mixed at 4 °C for 1-5 days, followed by desalting and anion exchange HPLC purification.

[0316] Example 4: Evaluation of guide molecule activity in HEK293T cells The activity of the guide molecules conjugated according to the process of Example 2 was evaluated in HEK293T cells by T7E1 cleavage assay. For clarity, all guide molecules used in this example contained identical target domain sequences and substantially similar RNA backbone sequences, as shown in Table 9 below. In the table, the target domain sequences are indicated as degenerate sequences by "N", while the position of the bridge between the two guide molecule fragments is indicated by [L].

[0317] [Table 12]

[0318] Various concentrations of ribonucleoprotein complexes containing various unimolecular guide molecules generated by IVT, synthetic unimolecular guide molecules (i.e., prepared without conjugation), or synthetic unimolecular guide molecules conjugated by the bromoacetyl-thiol process of Example 2 were introduced into HEK293T cells by lipofection (CRISPR-Max, Thermo Fisher Scientific, Waltham, MA), and genomic DNA was subsequently harvested. Cleavage was assessed using a standard T7E1 cleavage assay using a commercially available kit (Surveyor™, sold by Integrated DNA Systems, Coralville, Iowa). The results are shown in Figure 4.

[0319] As the results show, the conjugated guide molecule supports cleavage in HEK293 cells in a dose-dependent manner, consistent with that observed with unimolecular guide molecules generated by IVT or synthetic unimolecular guide molecules. It should be noted that the annealed unconjugated guide molecule fragments supported a similar dose-dependent, but lower level of cleavage. These results suggest that the conjugated guide molecule according to the method of the present disclosure supports high levels of DNA cleavage in substantially the same manner as unimolecular guide molecules generated by IVT or synthetic unimolecular guide molecules.

[0320] Example 5: Assessment of guide molecule purity by gel electrophoresis and mass spectrometry The purity of the composition of the guide molecule conjugated with a urea linker according to the process of Example 1 was compared to the purity of the composition of a commercially available synthetic unimolecular guide molecule (i.e., prepared without conjugation) by total ion current chromatography and mass spectrometry. 100 pmol of analyte was injected for mass spectrometry. The analysis was performed using a Waters ACQUITY HPLC system. This was achieved by LC-MS on a Bruker microTOF-QII mass spectrometer equipped with a UPLC system. A ThermoDNAPac C 18 column was used for separation. The results are shown in Figure 5.

[0321] Figure 5A shows a representative ion chromatograph, and Figure 5B shows a deconvoluted mass spectrum of an ion-exchange purified guide molecule conjugated with a urea linker according to the process of Example 1. Figure 5C shows a representative ion chromatograph, and Figure 5D shows a deconvoluted mass spectrum of a commercially available synthetic unimolecular guide molecule. The mass spectrum was evaluated for the peaks highlighted in the ion chromatograph. Figure 5E shows a magnified version of the mass spectrum. The mass spectrum of the commercially available synthetic unimolecular guide molecule is on the left (34% purity of total mass), and the mass spectrum of the guide molecule conjugated with a urea linker according to the process of Example 1 is on the right (72% purity of total mass).

[0322] Example 6: Assessment of guide molecule purity by sequence analysis The purity of compositions of guide molecules conjugated with urea bonds, as described in Example 1, was compared to the purity of compositions of commercially available synthetic unimolecular guide molecules (i.e., prepared without conjugation) and to the purity of compositions of guide molecules conjugated with thioether bonds, as described in Example 2. All compositions of guide molecules were based on the same predefined guide molecule sequence.

[0323] FIG. 6A shows a plot representing the frequency of individual base and length changes at each position from the 5' end of complementary DNA (cDNA) generated from a synthetic unimolecular guide molecule containing a urea bond, and FIG. 6B shows a plot representing the frequency of individual base and length changes at each position from the 5' end of cDNA generated from a commercially available synthetic unimolecular guide molecule (i.e., prepared without conjugation). The box encloses the 20 bp target domain of the guide molecule. In this example, the guide molecule containing a urea bond resulted in higher sequence fidelity in the target domain (less than 1% of the guide molecules contain deletions at any given position and less than 1% of the guide molecules contain substitutions at any given position) compared to the commercially available synthetic unimolecular guide molecule (i.e., less than 10% of the guide molecules contain deletions at any given position and less than 5% contain substitutions at any given position).

[0324] Figure 6C shows plots representing the frequency of individual base and length changes at each position from the 5' end of cDNA generated from synthetic unimolecular guide molecules containing thioether bonds. As shown in Figure 6C, a high level of 5' sequence fidelity is observed, demonstrating the generation of guide molecule compositions with high levels of sequence fidelity and purity. The alignments in Figure 6A (urea bond) and Figure 6C (thioether bond) also showed a relatively high frequency of mismatch / indel regions at the binding site (position 34). These data suggest that guide molecules synthesized by the disclosed method have reduced frequencies of deletions and substitutions compared to commercially available guide molecules.

[0325] Figures 7A and 7B are graphs showing the change in internal sequence length (+5 to -5) at position 41 from the 5' end of cDNA generated from a synthetic unimolecular guide molecule containing a urea bond (Figure 7A) and a commercially available synthetic unimolecular guide molecule (i.e., prepared without conjugation) (Figure 7B). As shown, the guide molecule containing a urea bond reduced the frequency and length of insertions / deletions compared to the commercially available synthetic unimolecular guide molecule (i.e., prepared without conjugation).

[0326] Example 7: Evaluation of guide molecule activity in CD34+ cells The activity of guide molecules with urea bonds conjugated according to the process of Example 1 was evaluated in CD34+ cells by next generation sequencing technology. The guide molecules discussed in this example included three target domain sequences and one of various guide molecule backbone sequences, as shown in Table 10 below and in Figures 8A-8L, 9A-9E, and 10A-10D. The position of the urea bond between the two guide molecule fragments is indicated by [UR] in Table 10 and by (R) in Figures 8A-8L, 9A-9E, and 10A-10D. The guide molecule with the first two target domain sequences (indicated as gRNA 1 followed by a letter or gRNA 2 followed by a letter) was based on the S. pyogenes gRNA backbone, and the guide molecule with the third target domain sequence (indicated as gRNA 3 followed by a letter) was based on the S. aureus gRNA backbone.

[0327] The conjugated guide molecule was resuspended in pH 7.5 buffer, melted, and reannealed, then added to a suspension of S. pyogenes Cas9 to yield a solution containing 55 μM fully complexed ribonucleoprotein.

[0328] Human CD34+ cells were counted, centrifuged to pellet, resuspended in P3 Nucleofection Buffer, and then dispensed into each well of a 96-well Nucleocuvette Plate pre-filled with human HSC medium (StemSpan™ Serum-Free Expansion Medium, StemCell Technologies, Vancouver, British Columbia, Canada) to obtain 50,000 cells / well. The fully complexed ribonucleoprotein solution as described above was added to each well of the Nucleocuvette Plate, followed by gentle mixing. Nucleofection was performed with an Amaxa Nucleofector System (Lonza, Basel, Switzerland). Nucleofected cells were incubated at 37°C and 5% CO2 for 72 hours to allow plateau editing. Genomic DNA was then extracted from the nucleofected cells using a DNAdvance DNA isolation kit according to the manufacturer's instructions. To quantify the insertion and deletion (indel) rates relative to the wild-type human reference sequence, next-generation sequencing technology was used to assess truncations. The results of gRNAs from Table 10 tested in CD34+ cells are shown in Figure 11.

[0329] As the results in Figure 11 show, ligated guide molecules generated according to Example 1 support DNA cleavage in CD34+ cells. The indel rate was found to increase with increasing stem-loop length, while incorporation of UA exchanges adjacent to the stem-loop sequence (see gRNA 1E, gRNA 1F, and gRNA 2D) mitigates the effect. These data suggest that chemically conjugated synthetic unimolecular guide molecules with longer stem-loop features result in higher levels of DNA cleavage in cells. Furthermore, DNA cleavage activity is independent of ligation efficiency and must be determined empirically.

[0330] [Table 13]

[0331] [Table 14]

[0332] Example 8: Evaluation of computational models of ligation efficiency The ligation efficiency of the reaction described in Example 1 is one measure of the suitability of a particular guide molecule structure. Because the reactive functional groups of the first and second guide molecule fragments in Example 1 are the same (amine), competitive homocoupling is a potential by-product. This example assessed whether the ligation efficiency (i.e., the percentage of heterocoupling products in the reaction products) could be predicted by computational modeling of the free energy difference of the homocoupling reaction (ΔG1) compared to the free energy difference of the heterocoupling reaction (ΔG2) using the OligoAnalyzer 3.1 tool available at http: / / www.idtdna.com / calc / analyzer. The results of this analysis are shown in Table 11.

[0333] [Table 15]

[0334] As shown in Table 11, ligation efficiency (measured by densitometry after gel analysis) was well predicted for most sequences with more negative ΔG2-ΔG1 values ​​corresponding to more favorable ligation efficiency (e.g., compared to gRNA 2A and 2C). However, ligation efficiency to form a particular guide molecule does not always correlate with ΔG2-ΔG1 values ​​(see, e.g., gRNA 1G, where more negative ΔG2-ΔG1 values ​​did not result in higher ligation efficiency), indicating that modifications and experimentation may be required for conjugation of a particular guide molecule fragment. For example, the ligation efficiency of gRNA 1G was improved by performing a UA exchange in the sequence of the lower stem (comparing the ligation efficiency of gRNA 1G to that of gRNA 1E), which was designed to prevent alternating annealing of the two guide molecule fragments prior to ligation.

[0335] Example 9: Characterization of urea bonds by mass spectrometry Chemically conjugated guide molecules containing urea linkages and synthesized as described in Example 1 were characterized by mass spectrometry. After synthesis, chemical ligation, and purification, gRNA 1A (see Table 10) was cleaved into fragments at the 3' end of each G nucleotide in the primary sequence using T1 endonuclease. These fragments were analyzed using LC-MS. In particular, a fragment containing a urea linkage, A-[UR]-AAUAG (A34:G39), was detected at m / z=1190.7 with a retention time of 4.50 min (Figure 12A and Figure 12B). LC / MS-MS analysis of this precursor ion revealed collision-induced dissociation fragment ions consistent with the urea linkage in gRNA 1A.

[0336] Example 10: Characterization of Carbamate By-Products Figure 13A shows LC-MS data for an unpurified composition of urea-linked guide molecules in which both a major product (A-2, retention time 3.25 min) and a minor product (A-1, retention time 3.14 min) are present. We note that for illustrative purposes, the minor product (A-1) in Figure 13A was enriched by combining fractions from anion exchange purification that contained a higher percentage of the carbamate minor product. By-products are typically detected in up to 10% yield in the synthesis of guide molecules by the process of Example 1. Analysis of each peak by mass spectrometry showed that both products have the same molecular weight (see Figures 13B and 13C).

[0337] With this in mind, we hypothesized that the minor product is a carbamate by-product resulting from the reaction between the 5'-NH2 at the 5' end of the 3' guide molecule fragment and the 2'-OH at the 3' end of the 5' guide molecule fragment, as follows: [ka]

[0338] To further confirm the assignment of the carbamate by-product, chemical modification with phenoxyacetic acid N-hydroxysuccinimide ester was performed. Basic chemical principles predict that only the minor products (carbamates) have reactive nucleophilic centers (free amines) and therefore only the minor products are chemically functionalized. Thus, addition of phenoxyacetic acid N-hydroxysuccinimide ester to a crude composition of urea-linked guide molecules should result in a mixture of the major product (urea) and the chemically modified minor product (carbamate): [ka]

[0339] Figure 14A shows the LC-MS data of the guide molecule composition after chemical modification. The major product (B-1, urea) has the same retention time as in the original analysis (3.26 min, Figure 13A), while the retention time of the minor product (B-1, carbamate) shifts to 3.86 min, which is consistent with chemical functionalization of the free amine moiety. Furthermore, mass analysis of the peak at 3.86 min (M+134) indicates that the predicted functionalization has occurred (see Figure 14B). These results suggest that the minor product is indeed a carbamate by-product.

[0340] To further confirm the identity of the carbamate by-product, the mixture of the major product (urea) and the chemically modified minor product (carbamate) was digested with ribonuclease A (see Example 9), which cleaves the guide molecule at the 3' end of each G nucleotide in the primary sequence. The fragments were then analyzed by LC-MS, and both the urea linkage (G35-[UR]-C36) and the chemically modified carbamate linkage (G35-[CA+PAA]-C36) were detected. Figure 15A shows the LC-MS trace of the fragment mixture with the urea linkage at a retention time of 4.31 minutes and the chemically modified carbamate linkage at a retention time of 5.77 minutes. Figure 15B shows the mass spectrum of the peak at 4.31 minutes, with m / z=532.1 being [M-2H] 2- FIG. 15C shows the mass spectrum of the peak at 5.77 min, m / z=599.1 corresponds to [M-2H] 2- The mass spectrum was further analyzed using LC-MS / MS techniques. m / z 532.1, [M-2H] 2- The LC-MS / MS spectrum of the urea-linked product at (Figure 15D) contains the typical ad and xz ions observed in oligonucleotide collision-induced dissociation (CID) experiments. In addition, MS / MS fragment ions were observed on both sides of the UR linkage from the 5' end (m / z = 487.1 and 461.1) and 3' end (m / z = 603.1 and 577.1). In contrast, at m / z 599.1, [M-2H] 2-In the LC-MS / MS spectrum of the chemically modified carbamate-linked product in (Figure 15E), only two product ions were observed, including MS / MS fragment ions from the 5' end of the carbamate linkage (m / z = 595.2) and the 3' end of the carbamate linkage (m / z = 603.1).

[0341] Example 11: Nucleotide Modification for Single Product Formation We hypothesize that the formation of the carbamate by-product described in Example 10 can be prevented through strategic 2' modifications at the 3'-terminal nucleotide of the 5' guide molecule fragment. For example, we hypothesize that the replacement of the 2'-OH in the nucleotide at the 3'-end of the 5' guide molecule fragment with a 2'-H will cause the synthesis of a urea-linked guide molecule by the process of Example 1 to yield a single urea-linked product free of the carbamate by-product: [ka]

[0342] FIG. 16A shows LC-MS data of the crude reaction mixture (upper spectrum) from the reaction with the 2'-H modified 5' guide molecule fragment compared to the crude reaction mixture (lower spectrum) from the reaction with the unmodified version of the same 5' guide molecule. No formation of carbamate by-products is observed with the 2'-H modified 5' guide molecule fragment (upper spectrum). In contrast, the crude reaction mixture from the reaction with the unmodified version of the same 5' guide molecule fragment (lower spectrum) contained a mixture of the main urea coupling product (A-2) and a trace carbamate by-product (A1). Note that unlike Example 10, the carbamate by-products were not concentrated and were therefore detected at much lower levels than in FIG. 13A of Example 10. Furthermore, mass spectrometry of the product of reaction with the 2'-H modified 5' guide molecule fragment (B) gave M-16 (compared to A-2, the unmodified urea-linked major product) as expected for a molecule in which the 2'-OH was replaced with a 2'-H (see Figures 16B and 16C).

[0343] A similar experiment was performed using gRNA 1L from Table 10, which contains the same 2'-H modification. The formation of a 2'-H modified urea-linked guide molecule was confirmed by T1 endonuclease digestion followed by mass spectrometry (see Example 9). The fragment containing the urea linkage, (2'-HA)-[UR]-AAUAG(A34:G39), was detected at a retention time of 4.65 min (Figure 17A) with m / z=1182.7 (Figure 17B). LC-MS / MS analysis of this precursor ion revealed fragment ions consistent with the urea linkage in reaction with a 2'-H modified nucleotide.

[0344] These results suggest that the 2'-OH modification at the 3'-terminal nucleotide of the 5' guide molecule fragment can avoid the formation of carbamate by-products, resulting in the synthesis of urea-linked guide molecules with high purity, which streamlines the overall process of producing conjugated guide molecules.

[0345] Incorporation by Reference All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0346] equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein which equivalents are intended to be encompassed by the following claims.

Claims

[Claim 1] The invention described in this specification.

Images