Modified guide rnas for gene editing

Abstract

To provide modified guide RNAs having improved in vitro and in vivo activity in gene editing methods.SOLUTION: A guide RNA (gRNA) is provided which is a short-single guide RNA (short-sgRNA) comprising a conserved portion of an sgRNA comprising a hairpin region, where the hairpin region lacks at least 5-10 nucleotides, and where the short-sgRNA comprises a 5' end modification or a 3' end modification.SELECTED DRAWING: None

Description

Technical Field

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 682,838, filed on Jun. 8, 2018 and U.S. Provisional Patent Application No. 62 / 682,820, filed on Jun. 8, 2018, each of which is hereby incorporated by reference in its entirety for all purposes.

[0002] The present disclosure relates to the field of gene editing using a CRISPR / Cas system that is part of a prokaryotic immune system that recognizes and cleaves exogenous genetic elements. The CRISPR / C as system relies on a single nuclease, CRISPR-associated protein 9 ( Cas9), to induce site-specific disruption of DNA. Cas9 is guided to a specific DNA sequence by a small RNA molecule called guide RNA (gRNA ). The complete guide RNA comprises a tracrRNA (trRNA) and a crisprRNA (crRNA). The crRNA containing the guide region needs to associate covalently or non-covalently with the trRNA to form the complete gRNA, or may be understood to associate so as to be referred to as gRNA under the understanding that the trRNA and crRNA may be included within a single guide RNA (sgRNA) or within two separate RNA molecules of a dual guide RNA (dgRNA). Cas9 in combination with the trRNA and crRNA or sgRNA is referred to as a Cas9 ribonucleoprotein complex (RNP).

[0003]

[0003] Oligonucleotides, particularly RNA, may be degraded in cells and in serum by non-enzymatic cleavage, endonuclease cleavage, or exonuclease cleavage. Such Improvements to prevent unwanted degradation, improve gRNA stability, and enhance gene editing efficiency The resulting methods and compositions are particularly desirable for therapeutic applications. Summary of the Invention

[0004] In some embodiments, genome editing tools are provided that include modified guide RNAs (gRNAs). The gRNA modifications described herein are provided for the purposes of the present invention. Cas9 (e.g., SaCas9, spyC as9, and equivalents).

[0005] In some embodiments, modified crisprRNA (crRNA) and / or modified In some embodiments, modified tracrRNA (trRNA) is provided. A and / or modified trRNAs include dual guide RNAs (dgRNAs). In some embodiments, the modified crRNA and / or the modified trRNA comprises a single guide RNA. The modifications of the crRNA and / or trRNA described herein include The addition improves the stability of the gRNA and the gRNA / Cas9 complex, allowing it to cleave the target DNA. Improve the activity of Cas9 (e.g., SauCas9, spyCas9, and equivalents) that In some embodiments, the crRNA portion of the dgRNA or sgRNA is a target It is modified in the ligation domain.

[0006] In some embodiments, the genome comprises short single guide RNAs (short sgRNAs). Editing tools are provided. In some embodiments, the short sgRNA is modified. The short sgRNAs described herein improve the stability of short sgRNAs and short sgRNA / Cas9 complexes and may improve the activity of Cas9 (e.g., SauCas9, sp yCas9, and equivalents) that cleave target DNA.

[0007] In some embodiments, the gRNA (e.g., sgRNA, short sgRNA, dgRN A, or crRNA) comprises modifications at one or more YA sites, such as those described in the following embodiments, Table 1, and Examples and associated figures. To avoid doubt, sgRNAs include, but are not limited to, short sgRNAs. As discussed in the Examples section, gRNAs can be found to be highly sensitive to RN ase A-like degradation patterns, including cleavage at unmodified YA sites. Furthermore, YA site modifications can reduce or eliminate such cleavage, and many YA site modifications appear to be tolerated without adversely affecting the ability to induce cleavage by nucleases such as C as9. Certain gRN A positions, including but not limited to those containing YA sites, have also been found to be modifiable despite other descriptions that they should not be modified due to concerns that they will be contacted by Cas9 and lose activity ( Yin et al., Nature Biotechnol. 35:1179-118 7 (2017)). Such modifications can further reduce unwanted gRNA degradation without impairing activity. Yin et al., Nature Biotechnol. 35:1179-118 7 (2017)). Such modifications can further reduce unwanted gRNA degradation without impairing activity.

[0008] The following embodiments are included. Embodiment 01 includes a short single guide comprising a conserved portion of an sgRNA that includes a hairpin region ​​​​​A guide RNA (gRNA) that is RNA (short sgRNA), wherein the hairpin region lacks at least 5 to 10 nucleotides, and the short sgRNA includes a 5'-end modification or a 3' '-end modification. Embodiment 02 is the gRNA described in Embodiment 1, wherein the short sgRNA includes a 5'-end modification. Embodiment 03 is the gRNA described in any one of Embodiments 1 to 2, wherein the short sgRNA includes a 3'-end modification. Embodiment 04 is the gRNA described in any one of Embodiments 1 to 3, wherein the short sgRNA includes a 5'-end modification and a 3'-end modification. Embodiment 05 is the gRNA described in any one of Embodiments 1 to 4, wherein the short sgRNA includes a 3'-tail. Embodiment 06 is the gRNA described in Embodiment 5, wherein the 3'-tail includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. Embodiment 07 is the gRNA described in Embodiment 5, wherein the 3'-tail includes about 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 7, 1 to 1 0, at least 1 to 5, at least 1 to 3, at least 1 to 4, at least 1 to 5, at least 1 to 5, at least 1 to 7, or at least 1 to 10 nucleotides. Embodiment 08 is the gRNA described in any one of Embodiments 1 to 7, wherein the short sgRNA does not include a 3'-tail. Embodiment 09 is the gRNA described in any one of Embodiments 1 to 8, which includes a modification in the hairpin region. Embodiment 10 is the gRNA described in any one of Embodiments 1 to 9, which includes a 3'-end modification and a modification in the hairpin region. Embodiment 10 is the gRNA described in any one of Embodiments 1 to 9, which includes a 3'-end modification and a modification in the hairpin region. Embodiment 10 is the gRNA described in any one of Embodiments 1 to 9, which includes a 3'-end modification and a modification in the hairpin region. Embodiment 10 is the gRNA described in any one of Embodiments 1 to 9, which includes a 3'-end modification and a modification in the hairpin region. Embodiment 10 is the gRNA described in any one of Embodiments 1 to 9, which includes a 3'-end modification and a modification in the hairpin region. Embodiment 10 is the gRNA described in any one of Embodiments 1 to 9, which includes a 3'-end modification and a modification in the hairpin region. Embodiment 10 is the gRNA described in any one of Embodiments 1 to 9, which includes a 3'-end modification and a modification in the hairpin region. Embodiment 09 is the gRNA described in any one of Embodiments 1 to 8, which includes a modification in the hairpin region. Embodiment 09 is the gRNA described in any one of Embodiments 1 to 8, which includes a modification in the hairpin region. Embodiment 10 is the gRNA described in any one of Embodiments 1 to 9, which includes a 3'-end modification and a modification in the hairpin region. Embodiment 11 includes 3'-end modification, modification in the hairpin region, and 5'-end modification, and is the gRNA according to any one of Embodiments 1 to 10. Embodiment 12 includes 5'-end modification and modification in the hairpin region, and is the gRNA according to any one of Embodiments 1 to 11. Embodiment 13 is the gRNA according to any one of Embodiments 1 to 12, in which at least 5 to 10 missing nucleotides are consecutive. Embodiment 14 is such that at least 5 to 10 missing nucleotides are i. within Hairpin 1, ii. within Hairpin 1 and within the "N" between Hairpin 1 and Hairpin 2, iii. within Hairpin 1 and within the 2 nucleotides immediately 3' to Hairpin 1, iv. including at least a part of Hairpin 1, v. within Hairpin 2, vi. including at least a part of Hairpin 2, vii. within Hairpin 1 and Hairpin 2, viii. including at least a part of Hairpin 1 and including the " N" between Hairpin 1 and Hairpin 2, ix. including at least a part of Hairpin 2 and including the "N" between Hairpin 1 and Hairpin 2 x. including at least a part of Hairpin 1, including the "N" between Hairpin 1 and Hairpin 2, and including at least a part of Hairpin 2, xi. within Hairpin 1 or Hairpin 2, and optionally including the "N" between Hairpin 1 and Hairpin n 2, xii. consecutive, xiii. consecutive and including the "N" between Hairpin 1 and Hairpin 2, xiv. consecutive and spanning at least a part of Hairpin 1 and a part of Hairpin 2, xv. Continuous, covering at least a part of Hairpin 1 and extending to "N", or xvi. Continuous, covering at least a part of Hairpin 1 and two nucleotides immediately 3' to Hairpin 1, the gRNA according to any one of Embodiments 1 to 13. Embodiment 15 is the gRNA according to any one of Embodiments 1 to 14, further comprising a guide region. Embodiment 16 is such that the 3' and / or 5' end modification is a protective end modification, for example, a 2'- O-methyl (2'-OMe) modified nucleotide, a 2'-O-(2-methoxyethyl) (2 '-O-moe) modified nucleotide, a 2'-fluoro (2'-F) modified nucleotide, an internucleotide phosphorothioate (PS) bond, an inverted abasic modified nucleotide, or a modified nucleotide selected from combinations thereof, the gRNA according to any one of Embodiments 1 to 15. Embodiment 17 is such that the modification in the hairpin region is a 2'-O-methyl (2'-OMe) modified nucleotide, a 2'-fluoro (2'-F) modified nucleotide, an internucleotide phosphorothioate (PS) bond, or a modified nucleotide selected from combinations thereof, the gRNA according to any one of Embodiments 1 to 16. Embodiment 18 is such that the 3' and / or 5' end modification comprises or further comprises a 2'-O-methyl (2'-OM e) modified nucleotide, the gRNA according to any one of Embodiments 1 to 17. Embodiment 19 is such that the 3' and / or 5' end modification comprises or further comprises a 2'-fluoro (2'-F) modified nucleotide, the gRNA according to any one of Embodiments 1 to 18. ​​​​​Embodiment 20 is the gRNA according to any one of Embodiments 1 to 19, wherein the 3' and / or 5' end modification contains or further contains a phosphorothioate (PS) bond between nucleotides. Embodiment 21 is the gRNA according to any one of Embodiments 1 to 20, wherein the 3' and / or 5' end modification contains or further contains an inverted abasic modified nucleotide. Embodiment 22 is the gRNA according to any one of Embodiments 1 to 21, wherein the modification in the hairpin region contains or further contains a 2'-O-methyl (2'-OMe) modified nucleotide. Embodiment 23 is the gRNA according to any one of Embodiments 1 to 22, wherein the modification in the hairpin region contains or further contains a 2'-fluoro (2'-F) modified nucleotide. Embodiment 24 is that the 3' end modification is i. modification of any one or more of the last 7, 6, 5, 4, 3, 2, or 1 nucleotides, ii. 1 modified nucleotide, iii. 2 modified nucleotides, iv. 3 modified nucleotides, v. 4 modified nucleotides, vi. 5 modified nucleotides, vii. 6 modified nucleotides, and viii. any one of 7 modified nucleotides, and is the gRNA according to any one of Embodiments 1 to 23. Embodiment 25 is that at least 5 to 10 nucleotides contain nucleotides 54 to 61 of SEQ ID NO: 400, nucleotides 53 to 60 of SEQ ID NO: 400, or nucleotides 54 to 58 of SEQ ID NO: 400, and optionally, the short sgRNA contains at least H1-1 ​​​​​​​​​​​​Any one of Embodiments 1 to 24, including the modifications of H1-5 and H2-1 to H2-12 is the gRNA described in Embodiment 26 is such that at least 5 to 10 nucleotides are i. consisting of 5 to 10 nucleotides, ii. consisting of 6 to 10 nucleotides, iii. consisting of 5 nucleotides, iv. consisting of 6 nucleotides, v. consisting of 7 nucleotides, vi. consisting of 8 nucleotides, vii. consisting of 9 nucleotides, viii. consisting of 10 nucleotides, ix. consisting of 5 to 10 consecutive nucleotides, x. consisting of 6 to 10 consecutive nucleotides, xi. consisting of 5 consecutive nucleotides, xii. consisting of 6 consecutive nucleotides, xiii. consisting of 7 consecutive nucleotides, xiv. consisting of 8 consecutive nucleotides, xv. consisting of 9 consecutive nucleotides, or xvi. consisting of 10 consecutive nucleotides, and is the gRNA described in any one of Embodiments 1 to 25 as described. Embodiment 27 is such that the 3'-end modification is i. internucleotide phosphorothioate (PS) bond, ii. 2'-OMe modified nucleotide, iii. 2'-O-moe modified nucleotide, iv. 2'-F modified nucleotide, v. inverted abasic modified nucleotide, and vi. is the gRNA described in any one of Embodiments 1 to 26, including one or more of (i) to (v). as described. Embodiment 28 is such that the short sgRNA is i. Phosphorothioate (PS) bond between nucleotides, ii. 2'-OMe modified nucleotide, iii. 2'-O-moe modified nucleotide, iv. 2'-F modified nucleotide, v. Inverted abasic modified nucleotide, and vi. A gRNA according to any one of Embodiments 1 to 27, comprising one or more of one or more combinations of (i) to (v), and a 3' tail rule. Embodiment 29 is such that the short sgRNA i. has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 PS bonds between nucleotides , ii. has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or 18 PS bonds between nucleotides, iii. has about 1 to 3, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, or 1 to 10 PS bonds between nucleotides, iv. has about 1 to 3, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 12, 1 to 14, 1 to 16, 1 to 18, or 1 to 20 PS bonds between nucleotides, and v. is a gRNA according to any one of Embodiments 1 to 28, comprising one or more of the PS bonds between each nucleotide. Embodiment 30 is such that the 3' end modification comprises at least one PS bond, i. one PS bond is present, and the bond is between the last nucleotide and the second last nucleotide, ii. two PS bonds are present between the last three nucleotides, iii. a PS bond is present between any one or more of the last four nucleotides, iv. a PS bond is present between any one or more of the last five nucleotides, ​​​​v. among the last 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides one or more of which have a PS bond between any one or more of Embodiments 1-29 is the gRNA described in any one of Embodiment 31 is such that the 3'-end modification further comprises at least one 2'-OMe, 2'-O-moe , inverted abasic, or 2'-F modified nucleotide, as described in Embodiment 31 is the gRNA of Embodiment 32 is such that the 3'-end modification is i. a modification of one or more of the last 1-7 nucleotides, wherein the modification is a PS bond, inverted abasic nucleotide, 2'-OMe, 2'-O-moe, 2'-F, or a combination thereof ii. a modification of the last nucleotide by 2'-OMe, 2'-O-moe, 2'-F, or a combination thereof, and an optional one or two PS bonds to the adjacent nucleotide and / or the first nucleotide of the 3'-tail iii. a modification of the last and / or the second last nucleotide by 2'-OMe, 2'-O-moe, 2'-F, or a combination thereof, and optionally one or more PS bonds iv. a modification of the last, second last, and / or third last nucleotide by 2'-OMe, 2'-O-moe, 2'-F, or a combination thereof, and optionally one or more PS bonds v. a modification of the last, second last, third last, and / or fourth last nucleotide by 2'-OMe, 2'-O-moe, 2'-F, or a combination thereof, and optionally one or more PS bonds vi. a modification of the last, second last, third last, fourth last, and / or fifth last nucleotide by 2'-OMe, 2'-O-moe, 2'-F, or a combination thereof, and optionally one or more PS bonds vii. a modification of the last, second last, third last, fourth last, fifth last, and / or sixth last nucleotide by 2'-OMe, 2'-O-moe, 2'-F, or a combination thereof, and optionally one or more PS bonds viii. a modification of the last, second last, third last, fourth last, fifth last, sixth last, and / or seventh last nucleotide by 2'-OMe, 2'-O-moe, 2'-F, or a combination thereof, and optionally one or more PS bonds, or vi. Modified at the last, second last, third last, fourth last, and / or fifth last nucleotide by 2'-OMe, 2'-O-moe, 2'-F, or a combination thereof and, optionally, comprising one or more PS linkages, a gRNA according to any one of embodiments 1 to 31 is provided. Embodiment 33 is a gRNA according to any one of embodiments 1 to 32, wherein the sgRNA comprises a 3' tail and the 3' tail comprises one or more modifications of any of the nucleotides present in the 3' tail is provided. Embodiment 34 is the gRNA according to embodiment 33, wherein the 3' tail is fully modified is provided. Embodiment 35 is a gRNA according to embodiment 33, wherein at least 5 to 10 nucleotides comprise nucleotides 54 to 61 of SEQ ID NO: 400, nucleotides 53 to 60 of SEQ ID NO: 400, or nucleotides 54 to 58 of SEQ ID NO: 400, and optionally, the short sgRNA comprises at least modifications H1-1 to H1-5 and H2-1 to H2-12 is provided. Embodiment 36 is a short sgRNA comprising i. a 3' end modification shown in any one of SEQ IDs 1 to 54 ii. (i) a 2'-OMe modified nucleotide at the last nucleotide of the conserved region of the sgRNA or short sgRNA, (ii) three consecutive 2'O-moe modified nucleotides immediately 5' to the 2'-OMe modified nucleotide, and (iii) three consecutive PS linkages between the last three nucleotides iii. (i) five consecutive 2'-OMe modified nucleotides from the 3' end of the 3' end portion, and (ii) three PS linkages between the last three nucleotides is provided. is provided. is provided. is provided. is provided. is provided. is provided. is provided. iv. Inverted abasic modified nucleotides at the last nucleotide of the conserved region of sgRNA or short sgRNA, v. (i) Inverted abasic modified nucleotides at the last nucleotide of the conserved region of sgRNA or short sgRNA, and (ii) three consecutive 2'-OMe modified nucleotides at the last three nucleotides of the conserved region of sgRNA or short sgRNA, vi. (i) Fifteen consecutive 2'-OMe modified nucleotides from the 3' end of the 3' end, (ii) five consecutive 2'-F modified nucleotides immediately 5' to the 2'-OMe modified nucleotide, and (iii) three PS linkages between the last three nucleotides, vii. (i) Alternating 2'-OMe modified nucleotides and 2'-F modified nucleotides in the last 20 nucleotides of the conserved region of sgRNA or short sgRNA, and (ii) three PS linkages between the last three nucleotides, viii. (i) Two or three consecutive 2'-OMe modified nucleotides, and (ii) three PS linkages between the last three nucleotides, ix. One PS linkage between the last nucleotide and the nucleotide adjacent to the last one, x. (i) Fifteen or twenty consecutive 2'-OMe modified nucleotides, and (ii) one or more of the three PS linkages between the last three nucleotides, which is a gRNA according to any one of Embodiments 1 to 35. Embodiment 37 is such that the 5' end modification is i. Modification of any one or more of nucleotides 1 to 7 in the guide region, ii. One modified nucleotide, iii. Two modified nucleotides, iv. Three modified nucleotides,​​​​​​​​​​​​ v. four modified nucleotides, vi. five modified nucleotides, vii. six modified nucleotides, and viii. any one of seven modified nucleotides, of the gRNA according to any one of Embodiments 1 to 36 which is the gRNA described in any one of them. Embodiment 38 is the gRNA according to any one of Embodiments 1 to 37, wherein the 5'-terminal modification comprises modification of 1 to 7, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 nucleotides. . Embodiment 39 is such that at least 5 to 10 nucleotides i. include nucleotides 54 to 61 of SEQ ID NO: 400, ii. include nucleotides 53 to 60 of SEQ ID NO: 400, iii. include nucleotides 54 to 58 of SEQ ID NO: 400, iv. consist of nucleotides 54 to 61 of SEQ ID NO: 400, v. consist of nucleotides 53 to 60 of SEQ ID NO: 400, or vi. consist of nucleotides 54 to 58 of SEQ ID NO: 400, which is the gRNA described in Embodiments 1 to 38 . Embodiment 40 is such that the 5'-terminal modification is i. internucleotide phosphorothioate (PS) bond, ii. 2'-OMe modified nucleotide, iii. 2'-O-moe modified nucleotide, iv. 2'-F modified nucleotide, v. inverted abasic modified nucleotide, vi. deoxyribonucleotide, vii. inosine, and viii. includes one or more of one or more combinations of (i.) to (vii.), which is the gRNA according to any one of Embodiments 1 to 39. Embodiment 41 is such that the 5'-terminal modification is i. PS linkages between 1, 2, 3, 4, 5, 6, and / or 7 nucleotides, or ii. about 1 - 2, 1 - 3, 1 - 4, 1 - 5, 1 - 6, or 1 - 7 nucleotides The gRNA according to any one of Embodiments 1 - 40, comprising a PS linkage therebetween. Embodiment 42 is such that the 5'-end modification comprises at least one PS linkage, i. There is 1 PS linkage, and the linkage is between nucleotides 1 and 2 of the guide region , ii. There are 2 PS linkages, and the linkages are between nucleotides 1 and 2 and between 2 and 3 of the guide region, iii. There is at least one PS linkage between any one or more of between nucleotides 1 and 2, 2 and 3, and 3 and 4, iv. There is at least one PS linkage between any one or more of between nucleotides 1 and 2, 2 and 3, 3 and 4, and 4 and 5 of the guide region, v. There is at least one PS linkage between any one or more of between nucleotides 1 and 2, 2 and 3, 3 and 4, 4 and 5, and 5 and 6 of the guide region, vi. There is at least one PS linkage between any one or more of between nucleotides 1 and 2, 2 and 3, 3 and 4, 4 and 5, 5 and 6, and 6 and 7 of the guide region, or vii. There is at least one PS linkage between any one or more of between nucleotides 1 and 2, 2 and 3, 3 and 4, 4 and 5, 5 and 6, 6 and 7, and 7 and 8 of the guide region, The gRNA according to any one of Embodiments 1 - 41. Embodiment 43 is such that the 5'-end modification further comprises at least one 2'-OMe, 2'-O-moe, inverted abasic, or 2'-F modified nucleotide, as described in Embodiment 42. or vii. There is at least one PS linkage between any one or more of between nucleotides 1 and 2, 2 and 3, 3 and 4, 4 and 5, 5 and 6, 6 and 7, and 7 and 8 of the guide region, The gRNA according to any one of Embodiments 1 - 41. Embodiment 43 is such that the 5'-end modification further comprises at least one 2'-OMe, 2'-O-moe, inverted abasic, or 2'-F modified nucleotide, as described in Embodiment 42. It is the gRNA of Embodiment 44 is such that the short sgRNA i. One or more modifications among nucleotides 1 to 7 of the variable region, where the modification is PS bonding, inverted abasic nucleotide, 2'-OMe, 2'-O-moe, 2'-F, 2' -H (deoxyribonucleotide), inosine, and / or a combination thereof a certain modification, ii. Modification of the first nucleotide of the guide region by 2'-OMe, 2'-O-moe, 2'-F, 2'-H, inosine, or a combination thereof, and optionally PS bonding to the adjacent nucleotide iii. Modification of the first and / or second nucleotide of the variable region by 2'-OMe, 2'-O-Moe, 2'-F, 2'-H, inosine, or a combination thereof, and optionally one or more PS bonds iv. Modification of the first, second, and / or third nucleotide of the variable region by 2'-OMe, 2'-O-moe, 2'-F, 2'-H, inosine, or a combination thereof, and optionally one or more PS bonds v. Modification of the first, second, third, and / or fourth nucleotide of the variable region by 2'-OMe, 2'-O-moe, 2'-F, 2'-H, inosine, or a combination thereof, and optionally one or more PS bonds vi. Modification of the first, second, third, fourth, and / or fifth nucleotide of the variable region by 2'-OMe, 2'-O-moe, 2'-F, 2'-H, inosine, or a combination thereof, and optionally one or more PS bonds, or vii. Modification of the first, second, third, fourth, and / or fifth nucleotide of the variable region by 2'-OMe, 2'-O-moe, 2'-F, 2'-H, inosine, or a combination thereof, and optionally one or more PS bonds, or viii. Modification of the first, second, third, fourth, and / or fifth nucleotide of the variable region by 2'-OMe, 2'-O-moe, 2'-F, 2'-H, inosine, or a combination thereof, and optionally one or more PS bonds, or ix. Modification of the first, second, third, fourth, and / or fifth nucleotide of the variable region by 2'-OMe, 2'-O-moe, 2'-F, 2'-H, inosine, or a combination thereof, and optionally one or more PS bonds, or Embodiment 45 is that a short sgRNA has i. a 5'-end modification shown by any one of SEQ ID NOs: 1 to 54, ii. 2'-OMe-modified nucleotides at nucleotides 1, 2, and 3 in the guide region nucleotides, iii. 2'-OMe-modified nucleotides at nucleotides 1, 2, and 3 in the guide region nucleotides, and PS bonds between nucleotides 1 and 2, 2 and 3, and 3 and 4 in the guide region, iv. 2'-OMe-modified nucleotides at 1, 2, 3, 4, and 5 in the guide region nucleotides, v. 2'-OMe-modified nucleotides at 1, 2, 3, 4, and nucleotide 5 in the guide region nucleotides, and PS bonds between nucleotides 1 and 2, 2 and 3, 3 and 4 in the guide region, 4 and 5, and 5 and 6, vi. 2'O-moe-modified nucleotides at nucleotides 1, 2, and 3 in the guide region nucleotides, vii. 2'O-moe-modified nucleotides at nucleotides 1, 2, and 3 in the guide region nucleotides, and PS bonds between nucleotides 1 and 2, 2 and 3, and 3 and 4 in the guide region, viii. an inverted abasic-modified nucleotide at nucleotide 1 in the guide region, ix. an inverted abasic-modified nucleotide at nucleotide 1 in the guide region, and 2'-OMe-modified nucleotides at nucleotides 1, 2, and 3 in the guide region , x. an inverted abasic-modified nucleotide at nucleotide 1 in the guide region, the guide region's 2'-OMe-modified nucleotides at nucleotides 1, 2, and 3, and PS bonds between nucleotides 1 and 2, 2 and 3, 3 and 4, 4 and 5, and Any one or more of the PS linkages between 5 and 6, and is the gRNA according to any one of Embodiments 1 to 44. It is the gRNA according to any one of Embodiments 1 to 44. Embodiment 46 is the gRNA according to any one of Embodiments 1 to 45, wherein the upper stem region contains at least one modification. It is the gRNA according to any one of Embodiments 1 to 45. Embodiment 47 is that the upper stem modification is i. Modification of any one or more of US1 to US12 in the upper stem region, ii. Modification of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 0, 11, or all 12 nucleotides, and iii. Any one or more of the modifications of about 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, or 1 to 12 nucleotides in the upper stem region, and is the gRNA according to any one of Embodiments 1 to 46. It is the gRNA according to any one of Embodiments 1 to 46. Embodiment 48 is that the upper stem modification is i. 2'-OMe modified nucleotide, ii. 2'-O-moe modified nucleotide, iii. 2'-F modified nucleotide, and iv. Any one or more of one or more combinations of (i.) to (iii.), and is the gRNA according to Embodiment 47. It is the gRNA according to Embodiment 47. Embodiment 49 is a guide RNA which is a short sgRNA containing a conserved part of the sgRNA containing a hairpin region, wherein the hairpin region lacks at least 5 to 10 nucleotides, and the short sgRNA has a 5'-end modification, and contains one or more modifications in one or more of i. the upper stem region, ii. the hairpin 1 region, and iii. the hairpin 2 region, and the 5'-end modification is a 5'-protected end modification, for example, at least within the first 7 nucleotides. It contains one or more modifications in one or more of i. the upper stem region, A guide RNA comprising two phosphorothioate (PS) linkages. Embodiment 50 is the gRNA according to Embodiment 49, wherein at least one modification comprises a 2'-O-methyl (2'-OMe) modified nucleotide. Embodiment 51 is the gRNA according to Embodiment 49 or 50, wherein at least one modification comprises a 2'-fluoro (2'-F) modified nucleotide. Embodiment 52 is the gRNA according to any one of Embodiments 49 to 51, wherein at least one modification comprises an internucleotide phosphorothioate (PS) linkage. Embodiment 53 is the gRNA according to any one of Embodiments 49 to 52, wherein the short sgRNA comprises one or more modifications in the upper stem region. Embodiment 54 is the gRNA according to Embodiment 53, which comprises a modification in any one of US1-US12. Embodiment 55 is the gRNA according to any one of Embodiments 49 to 54, wherein the short sgRNA comprises one or more modifications in the hairpin 1 region. Embodiment 56 is the gRNA according to Embodiment 55, wherein the short sgRNA comprises a modification in H1-1. Embodiment 57 is the gRNA according to any one of Embodiments 49 to 56, wherein the short sgRNA comprises one or more modifications in the hairpin 2 region. Embodiment 58 is the gRNA according to Embodiment 57, wherein the short sgRNA comprises a modification in H2-1. Embodiment 59 is the gRNA according to any one of Embodiments 49 to 58, wherein the short sgRNA comprises modifications in H1-1 to H1-12. Embodiment 60 is the gRNA according to any one of Embodiments 49 to 59, wherein the short sgRNA comprises modifications in H2-1 to H2-15. Embodiment 61 is the gRNA according to any one of Embodiments 49 to 60, wherein the short sgRNA comprises modifications in the loop region. Embodiment 62 is the gRNA according to Embodiment 61, wherein the short sgRNA comprises a modification in L-1. Embodiment 63 is the gRNA according to any one of Embodiments 49 to 62, wherein the short sgRNA comprises modifications in the loop region and the upper stem region. Embodiment 64 is the gRNA according to Embodiment 63, wherein the short sgRNA comprises a modification in the loop region and the upper stem region and a modification in H1-1. Embodiment 65 is the gRNA according to any one of Embodiments 49 to 64, wherein the short sgRNA comprises modifications in the loop region and the upper stem region and a modification in H2-1. Embodiment 66 is the gRNA according to any one of Embodiments 49 to 65, wherein the short sgRNA comprises modifications in the loop region and the upper stem region and modifications in H1-1 to H1-12. Embodiment 67 is the gRNA according to any one of Embodiments 49 to 66, wherein the short sgRNA comprises modifications in the loop region and the upper stem region and modifications in H2-1 to H2-15. Embodiment 68 is the gRNA according to any one of Embodiments 49 to 67, wherein the short sgRNA comprises modifications in the loop region and the upper stem region and modifications in H1-1 to H1-12 and H2-1 to H2-15. Embodiment 69 is the gRNA according to any one of Embodiments 49 to 68, wherein the short sgRNA comprises modifications in the loop region and the upper stem region and modifications in H1-1 to H1-12 and H2-1 to H2-15 and a modification in L-1. Embodiment 70 is the gRNA according to any one of Embodiments 49 to 69, wherein the short sgRNA comprises modifications in the loop region and the upper stem region and modifications in H1-1 to H1-12 and H2-1 to H2-15 and a modification in L-1 and a modification in the upper stem region and a modification in H1-1. The gRNA according to any one of Forms 49 to 59. Embodiment 61 is the gRNA according to any one of Embodiments 49 to 60, wherein the short sgRNA contains one or more modifications in each of the upper stem region, the hairpin 1 region, and the hairpin 2 region. RNA. Embodiment 62 is the gRNA according to any one of Embodiments 49 to 61, wherein the short sgRNA contains modified nucleotides between the hairpin 1 region and the hairpin 2 region. gRNA. Embodiment 63 is the gRNA according to any one of Embodiments 49 to 62, further comprising a lower stem region containing modifications. gRNA. Embodiment 64 is the gRNA according to any one of Embodiments 49 to 63, further comprising a 3'-end modification. gRNA. Embodiment 65 is the gRNA according to Embodiment 64, wherein at least two of the last four nucleotides at the 3'-end of the 3'-end portion are modified. gRNA. Embodiment 66 is the gRNA according to Embodiment 64, wherein at least two of the last four nucleotides at the 3'-end of the 3'-end portion are modified with 2'-OMe, 2'-F, or 2'-O-moe. gRNA. Embodiment 67 is the gRNA according to any one of Embodiments 64 to 66, further comprising phosphorothioate (PS) bonds between one or more of the last four nucleotides at the 3'-end of the 3'-end portion. gRNA. Embodiment 68 is the gRNA according to any one of Embodiments 49 to 67, further comprising a bulge region containing modifications. gRNA. Embodiment 69 is the gRNA according to any one of Embodiments 49 to 68, further comprising a nexus region containing modifications. gRNA. Embodiment 70 is at least the first three nucleotides at the 5'-end of the variable region and gRNA. gRNA. The gRNA according to any one of Embodiments 49 to 69, wherein the last three nucleotides at the 3'-end of the 3'-terminal portion are modified. Embodiment 71 is the gRNA according to any one of Embodiments 49 to 70, wherein the first four nucleotides at the 5'-end of the variable region and the last four nucleotides at the 3'-end of the 3'-terminal portion are linked by phosphorothioate (PS) bonds. Embodiment 72 is the gRNA according to any one of Embodiments 70 to 71, wherein the terminal modification contains 2'-OMe. Embodiment 73 is the gRNA according to any one of Embodiments 70 to 71, wherein the terminal modification contains 2'-F. Embodiment 74 is the gRNA according to any one of Embodiments 49 to 73, wherein the first four nucleotides at the 5'-end of the variable region and the last four nucleotides at the 3'-end of the 3'-terminal portion are linked by PS bonds, and the first three nucleotides at the 5'-end of the variable region and the last three nucleotides at the 3'-end of the 3'-terminal portion contain 2'-OMe modification. Embodiment 75 is the gRNA according to any one of Embodiments 49 to 74, wherein the first four nucleotides at the 5'-terminal portion and the last four nucleotides at the 3'-terminal portion are linked by PS bonds, and the first three nucleotides at the 5'-terminal portion and the last three nucleotides at the 3'-terminal portion contain 2'-OMe, 2'-F, and / or 2'-O-moe modification. Embodiment 76 is the gRNA according to any one of Embodiments 49 to 75, wherein LS1, LS6, LS7, LS8, LS11, and / or LS12 are modified with 2'-OMe. In Embodiment 77, each nucleotide in the bulge region is modified with 2'-OMe and is the gRNA according to any one of Embodiments 49 to 76. In Embodiment 78, at least 50% of the nucleotides in the bulge region are modified with 2'-OM e and is the gRNA according to any one of Embodiments 49 to 77. In Embodiment 79, each nucleotide in the upper stem region is modified with 2'-OMe and is the gRNA according to any one of Embodiments 49 to 78. In Embodiment 80, N16, N17, and / or N18 in the nexus region are modified with 2 '-OMe and is the gRNA according to any one of Embodiments 49 to 79 . In Embodiment 81, N15, N16, N17, and / or N1 8 in the nexus region are modified and is the gRNA according to any one of Embodiments 49 to 80. In Embodiment 82, the modification in the nexus region is selected from 2'-OMe and 2'F and is the gRNA according to Embodiment 80 or 81. In Embodiment 83, N16, N17, and N18 are linked by a PS bond and is the gRNA according to any one of Embodiments 80 to 82 . In Embodiment 84, each nucleotide remaining in the hairpin 1 region is modified with 2'-OMe and is the gRNA according to any one of Embodiments 49 to 83. In Embodiment 85, each nucleotide in the hairpin 2 region is modified with 2'-OMe and is the gRNA according to any one of Embodiments 49 to 84. In Embodiment 86, it is a short sgRNA containing a conserved portion of the sgRNA including the hairpin region It is a guide RNA, and the hairpin region lacks at least 5 to 10 nucleotides , the short sgRNA includes a 5'-end modification or a 3'-end modification, and the short sgRNA is i. At least one modification in the upper stem region, and ii. A guide RNA that further includes any one or more of the 3' tails Embodiment 87 is such that the upper stem modification is i. Modification of each nucleotide (US1-US12) in the upper stem region ii. Modification of any one or more of US1-US12 in the upper stem region iii. Modification of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or all 12 nucleotides, and iv. Any one or more of the modifications of about 1-2, 1-3, 1-4, 1-5, 1-6, 1-7 , 1-8, 1-9, 1-10, or 1-12 nucleotides, which is the gRNA described in Embodiment 86 including one or more of them Embodiment 88 is such that the 5'-end modification is i. Modification of any one or more of nucleotides 1-7 in the variable region ii. One modified nucleotide iii. Two modified nucleotides iv. Three modified nucleotides v. Four modified nucleotides vi. Five modified nucleotides vii. Six modified nucleotides, and viii. Any one or more of seven modified nucleotides, which is the gRNA described in any one of Embodiments 86 ~87 Embodiment 89 is such that the 5'-end modification is i. SEQ ID NOs: 1-54, 401-532, 1001, 1007-1132, 1205 ~1212, 1322~1406, 1417~1501, 1511~1596, 3018 ~3059, 3063~3104, 3108~3149, 3153~3194, 3198 ~3239, 3243~3284, 3295~3341, 3343~3385, 3388 ~3430, or a 5'-end modification shown in any one of 3549~3552 ii. 2'-OMe modified nucleotides at nucleotides 1, 2, and 3 of the variable region nucleotides iii. 2'-OMe modified nucleotides at nucleotides 1, 2, and 3 of the variable region, and PS linkages between nucleotides 1 and 2, 2 and 3, and 3 and 4 of the variable region iv. 2'-OMe modified nucleotides at nucleotides 1, 2, 3, 4, and 5 of the variable region nucleotides v. 2'-OMe modified nucleotides at nucleotides 1, 2, 3, 4, and 5 of the variable region, and PS linkages between nucleotides 1 and 2, 2 and 3, 3 and 4, 4 and 5 and 5 and 6 of the variable region vi. 2'O-moe modified nucleotides at nucleotides 1, 2, and 3 of the variable region nucleotides vii. 2'O-moe modified nucleotides at nucleotides 1, 2, and 3 of the variable region, and PS linkages between nucleotides 1 and 2, 2 and 3, and 3 and 4 of the variable region viii. Inverted abasic modified nucleotide at nucleotide 1 of the variable region ix. Inverted abasic modified nucleotide at nucleotide 1 of the variable region, and 2'-OMe modified nucleotides at nucleotides 1, 2, and 3 of the variable region as well as ​​​​x. An inverted abasic modified nucleotide at nucleotide 1 of the variable region, variable region 2'-OMe modified nucleotides at nucleotides 1, 2, and 3 of the , and any one or more of the PS linkages between nucleotides 1 and 2, 2 and 3, 3 and 4, 4 and 5, and 5 and 6 of the variable region, which is the gRNA described in any one of Embodiments 86 to 88. Embodiment 90 is the gRNA described in Embodiment 89, which includes 2'- OMe modified nucleotides at least at nucleotides 1, 2, and 3 of the variable region, and PS linkages between nucleotides 1 and 2, 2 and 3 , and 3 and 4 of the variable region. Embodiment 91 is the gRNA described in Embodiment 89, which includes 2 '-OMe modified nucleotides at least at nucleotides 1, 2, 3, and 4 of the variable region, and PS linkages between nucleotides 1 and 2, 2 and 3 , and 3 and 4 of the variable region. Embodiment 92 is i. One or more modifications of any one of the last 7, 6, 5, 4, 3, 2, or 1 nucleotides , ii. One modified nucleotide, iii. Two modified nucleotides, iv. Three modified nucleotides, v. Four modified nucleotides, vi. Five modified nucleotides, vii. Six modified nucleotides, and viii. A 3'-end modification including one or more of seven modified nucleotides, which is the gRNA described in any one of Embodiments 86 to 91. Embodiment 93 is such that the short sgRNA i. SEQ ID NOs: 1 to 54, 401 to 532, 1001, 1007 to 1132, 1205 ​​~1212, 1322~1406, 1417~1501, 1511~1596, 3018 ~3059, 3063~3104, 3108~3149, 3153~3194, 3198 ~3239, 3243~3284, 3295~3341, 3343~3385, 3388 ~3430, or a 3' end modification shown in any one of 3549~3552 ii. (i) a 2'-OMe modified nucleotide at the last nucleotide of the conserved region of the sgRNA or short sgRNA, (ii) three consecutive 2'O-moe modified nucleotides immediately 5' to the 2'-OMe modified nucleotide, and (iii) three consecutive PS linkages between the last three nucleotides iii. (i) five consecutive 2'-OMe modified nucleotides, and (ii) three PS linkages between the last three nucleotides iv. an inverted abasic modified nucleotide at the last nucleotide of the conserved region of the sgRNA or short sgRNA v. (i) an inverted abasic modified nucleotide at the last nucleotide of the conserved region of the sgRNA or short sgRNA, and (ii) three consecutive 2'-OMe modified nucleotides at the last three nucleotides of the conserved region of the sgRNA or short sgRNA vi. (i) fifteen consecutive 2'-OMe modified nucleotides, (ii) five consecutive 2'-F modified nucleotides immediately 5' to the 2'-OMe modified nucleotide, and (iii) three PS linkages between the last three nucleotides vii. (i) alternating 2'-OMe modified nucleotides and 2'-F modified nucleotides in the last 20 nucleotides of the conserved region of the sgRNA or short sgRNA ​​​​​​​​​​​and (ii) three PS linkages between the last three nucleotides viii. (i) two or three consecutive 2'-OMe modified nucleotides, and ( ii) three PS linkages between the last three nucleotides ix. one PS linkage between the last nucleotide and the penultimate nucleotide and x. fifteen or twenty consecutive 2'-OMe modified nucleotides, and (ii) the gRNA according to any one of embodiments 86 ~92, comprising any one of the three PS linkages between the last three nucleotides. Embodiment 94 is the gRNA according to any one of embodiments 86-93, wherein the sgRNA comprises a 3' tail, and the 3' tail comprises one or more modifications of the nucleotides present in the 3' tail. Embodiment 95 is the gRNA according to embodiment 94, wherein the 3' tail is fully modified. Embodiment 96 is the gRNA according to embodiment 94, wherein the 3' tail comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1-2 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, or 1-10 nucleotides, and optionally one or more of these nucleotides are modified. Embodiment 97 is a guide RNA, a short sgRNA comprising any one of SEQ ID NOs: 1-54, 201-254, and 301-354, comprising the modifications in Table 1. Embodiment 98 is a short sgRNA comprising a nucleic acid having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, or 70% identity to any one of the nucleic acids of SEQ ID NOs: 1-54, 201-254, and 301-354. 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, or 1-10 nucleotides, and optionally one or more of these nucleotides are modified. Embodiment 96 is the gRNA according to embodiment 94, wherein the 3' tail comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1-2 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, or 1-10 nucleotides, and optionally one or more of these nucleotides are modified. Embodiment 97 is a guide RNA, a short sgRNA comprising any one of SEQ ID NOs: 1-54, 201-254, and 301-354, comprising the modifications in Table 1. Embodiment 98 is a short sgRNA comprising a nucleic acid having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, or 70% identity to any one of the nucleic acids of SEQ ID NOs: 1-54, 201-254, and 301-354. Embodiment 98 is a short sgRNA comprising a nucleic acid having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, or 70% identity to any one of the nucleic acids of SEQ ID NOs: 1-54, 201-254, and 301-354. any one of SEQ ID NOs: 1-54, 201-254, and 301-354, and a nucleic acid having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, or 70% identity to any one of the nucleic acids of SEQ ID NOs: 1-54, 201-254, and 301-354. 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, or 1-10 nucleotides, and optionally one or more of these nucleotides are modified. It is a guide RNA, and the modification at each nucleotide of the short sgRNA corresponding to the nucleotide of the reference sequence identifier in Table 1 is the same as or equivalent to the modification shown in the reference sequence identifier in Table 1. The modification at each nucleotide of the short sgRNA corresponding to the nucleotide of the reference sequence identifier in Table 1 is the same as or equivalent to the modification shown in the reference sequence identifier in Table 1. is equivalent. Embodiment 99 is the gRNA according to any one of Embodiments 1 to 98, including the YA modification at the YA site of at least one guide region. ~98. Embodiment 100 is the gRNA according to any one of Embodiments 1 to 99, including the YA modification at the YA site of at least one guide region that is not a 5'-terminal modification. ~99. Embodiment 101 is the gRNA according to any one of Embodiments 1 to 100, including the YA modification at one or more guide region YA sites, and the guide region YA site is at or after nucleotide 8 from the 5'-end of the 5'-terminal portion. ~100. is the gRNA according to any one of Embodiments 1 to 100, including the YA modification at one or more guide region YA sites, and the guide region YA site is at or after nucleotide 8 from the 5'-end of the 5'-terminal portion. Embodiment 102 includes the YA modification at one or more guide region YA sites, and the short sgRNA is a. Modification in one or more of H1-1 and H2-1, b. YA modification at 1, 2, 3, 4, or 5 guide region YA sites, c. YA modification at 1, 2, 3, 4, or 5 guide region YA sites, where the modification of at least one guide region YA site is different from any 5'-terminal modification of the sgRNA, ~32. YA modification, d. YA modification at one or more guide region YA sites, where the guide region YA site is at or after nucleotide 8 from the 5'-end of the 5'-terminal portion, YA modification, e. YA modification at one or more guide region YA sites, where the guide region YA site is within the 5'-terminal, 6'-terminal, 7'-terminal, 8'-terminal, 9'-terminal, or 10'-terminal from the 5'-end of the 5'-terminal portion, f. YA modification at one or more guide region YA sites, where the guide region YA site is within nucleotides 17, 16, 15, 14, 13, 12, 11, 10, or 9 of the nucleotide at the 3'-end of the guide region, the YA modification g. YA modification at a guide region YA site other than the 5'-end modification h. YA modification at two or more guide region YA sites, where the guide region YA site is at or after nucleotide 8 from the 5'-end of the 5'-end portion, the YA modification i. YA modification at two or more guide region YA sites, where two guide region Y A sites are within the 5'-end, 6'-end, 7'-end, 8'-end, 9'-end, or 10'-end of the nucleotide from the 5'-end of the 5'-end portion, the YA modification j. YA modification at two or more guide region YA sites, where the guide region YA site is within nucleotides 17, 16, 15, 14, 13, 12, 11, 10, or 9 of the nucleotide at the 3'-end of the guide region, the YA modification k. YA modification at two or more guide region YA sites other than the 5'-end modification l. YA modification at two or more guide region YA sites, where the modification of the guide region YA site includes one or more of the YA modifications including a modification that does not include at least one nucleotide located on the 5'-side of the guide region YA site is the gRNA according to any one of Embodiments 1 to 101. Embodiment 103 includes a YA modification, and the modification includes 2'-fluoro, 2'-H, 2'-O Me, ENA, UNA, inosine, or PS, and is the gRNA according to any one of Embodiments 1 to 102. Embodiment 104 includes a YA modification, and the modification changes the structure of the dinucleotide motif ​​​​​​and is the gRNA according to any one of Embodiments 1 to 103, which reduces RNA endonuclease activity. Embodiment 105 includes a YA modification that prevents recognition or cleavage of the YA site by RNase and / or stabilizes the RNA structure, and is the gRNA according to any one of Embodiments 1 to 104. Embodiment 106 includes a YA modification that a. a ribose modification selected from 2'-O-alkyl, 2'-F, 2'-moe, 2'-F arabinose, and 2'-H (deoxyribose), b. bicyclic ribose analogs such as LNA, BNA, and ENA, c. unlocked nucleic acid modifications, d. base modifications such as inosine, pseudouridine, and 5'-methylcytosine, and and e. any one or more of internucleoside bond modifications such as phosphorothioate, and is the gRNA according to any one of Embodiments 1 to 105. Embodiment 107 includes a YA modification at one or more conserved region YA sites and is the gRNA according to any one of Embodiments 1 to 106. Embodiment 108 includes a YA modification at one or more of conserved region YA sites 2, 3, 4, and 10 and is the gRNA according to any one of Embodiments 1 to 107. Embodiment 109 includes a YA modification at one or more of conserved region YA sites 1 and 8 and is the gRNA according to any one of Embodiments 1 to 108. Embodiment 110 includes a YA modification at conserved region YA site 1 and is the gRNA according to any one of Embodiments 1 to 109. Embodiment 111 includes a YA modification at conserved region YA site 2 and is the gRNA according to any one of Embodiments 1 to 110. ​​​​​​​​​ Embodiment 112 is the gRNA according to any one of Embodiments 1 to 111, including the YA modification of the YA site 3 in the storage region YA. Embodiment 113 is the gRNA according to any one of Embodiments 1 to 112, including the YA modification of the YA site 4 in the storage region YA. Embodiment 114 is the gRNA according to any one of Embodiments 1 to 113, including the YA modification of the YA site 5 in the storage region YA. Embodiment 115 is the gRNA according to any one of Embodiments 1 to 114, including the YA modification of the YA site 6 in the storage region YA. Embodiment 116 is the gRNA according to any one of Embodiments 1 to 115, including the YA modification of the YA site 7 in the storage region YA. Embodiment 117 is the gRNA according to any one of Embodiments 1 to 116, including the YA modification of the YA site 8 in the storage region YA. Embodiment 118 is the gRNA according to any one of Embodiments 1 to 117, including the YA modification of the YA site 9 in the storage region YA. Embodiment 119 is the gRNA according to any one of Embodiments 1 to 118, including the YA modification of the YA site 10 in the storage region YA. Embodiment 120 is a. YA modifications of the YA sites 2, 3, 4, and 10 in the storage region YA, b. YA modifications of the YA sites 2, 3, and 4 in the storage region YA, c. YA modifications of the YA sites 2, 3, and 10 in the storage region YA, d. YA modifications of the YA sites 2, 4, and 10 in the storage region YA, e. YA modifications of the YA sites 3, 4, and 10 in the storage region YA, f. YA modifications of the YA sites 2 and 10 in the storage region YA, g. YA modifications of the YA sites 2 and 4 in the storage region YA, h. YA modifications of the YA sites 2 and 3 in the storage region YA, i. YA modifications of the YA sites 3 and 4 in the storage region YA,​​​​​​​​ j. YA modification of storage regions YA sites 3 and 10, k. YA modification of storage regions YA sites 4 and 10, l. YA modification of storage regions YA sites 1 and 5, m. YA modification of storage regions YA sites 1 and 6, n. YA modification of storage regions YA sites 1 and 7, o. YA modification of storage regions YA sites 1 and 8, p. YA modification of storage regions YA sites 1 and 9, q. YA modification of storage regions YA sites 8 and 5, r. YA modification of storage regions YA sites 8 and 6, s. YA modification of storage regions YA sites 8 and 7, and t. includes one or more of YA modifications of storage regions YA sites 8 and 9, Optionally, the sgRNA further includes a Y A modification of storage regions YA sites 2, 3, 4, and / or 10, and is the gRNA according to any one of Embodiments 1 to 119. Embodiment 121 is the gRNA according to any one of Embodiments 1 to 120, wherein at least one modified YA site optionally includes a 2'-OMe modification at the pyrimidine of the YA site. Embodiment 122 is the gRNA according to any one of Embodiments 1 to 121, wherein at least one modified YA site optionally includes a 2'-fluoro modification at the pyrimidine of the YA site. Embodiment 123 is the gRNA according to any one of Embodiments 1 to 122, wherein at least one modified YA site optionally includes a PS modification at the pyrimidine of the YA site. Embodiment 124 is such that the gRNA includes at least 1, 2, 3, 4, 5 of nucleotides 1, 2, 3, 4, 6, 7, 8, 9 , 10, 11, 13, 14, 17, and 18, A guide region comprising modifications in 6, 7, 8, 9, 10, 11, 12, 13, or all thereof, optionally wherein the modification is 2'-OMe, 2'-fluoro, 2'-H, inosine , or a phosphorothioate modification, the g RNA according to any one of embodiments 1 to 123 Embodiment 125 is a gRNA in which the short sgRNA comprises a guide region comprising modifications at nucleotides 1, 2, 3, 4, 6, 7, 8, 9 , 10, 11, 13, 14, 17, and 18, optionally wherein the modification is 2'-OMe, 2'-fluoro, 2'-H, inosine, or a pho sphorothioate modification, the gRNA according to any one of embodiments 1 to 124 thereof Embodiment 126 is a gRNA according to embodiments 124 - 125, wherein the 2'-OMe modification is absent from the guide region at nucleotides 6 - 11 and the 13 terminus Embodiment 127 is a gRNA according to embodiments 124 - 126, wherein the 2'-fluoro modification is absent from the guide region at nucleotides 1 - 7, 15, 16, and 19 terminus Embodiment 128 is a gRNA according to embodiments 124 - 127, wherein the phosphorothioate modification is absent from the guide region at nucleotides 4, 5, 11 - 14, 1 7, and 18 Embodiment 129 is a gRNA according to embodiments 124 - 128, wherein the guide region comprises unmodified nucleotide 20 Embodiment 130 is a gRNA according to embodiments 124 - 129, wherein the guide region consists of 20 nucleotides Embodiment 131 is a gRNA according to embodiments 124 - 130, wherein the guide region comprises a YA site at nucleotides 5 - 6 and a modification at nucleotide 5 ​​​​​ Embodiment 132 is the gRNA described in Embodiments 124 to 131, wherein the guide region contains the YA site at nucleotides 12-13 and the modification at nucleotide 12. Embodiment 133 is the gRNA described in Embodiments 124 to 132, wherein the guide region contains the YA site at nucleotides 15-16 and the modification at nucleotide 15. Embodiment 134 is the gRNA described in Embodiments 124 to 133, wherein the guide region contains the YA site at nucleotides 16-17 and the modification at nucleotide 16. Embodiment 135 is the gRNA described in Embodiments 124 to 134, wherein the guide region contains the YA site at nucleotides 19-20 and the modification at nucleotide 19. Embodiment 136 is the gRNA described in Embodiments 124 to 130 or 132 to 135, wherein the guide region does not contain the YA site at nucleotides 5-6 and nucleotide 5 is unmodified. Embodiment 137 is the gRNA described in Embodiments 124 to 131 or 133 to 136, wherein the guide region does not contain the YA site at nucleotides 12-13 and nucleotide 12 is unmodified. Embodiment 138 is the gRNA described in Embodiments 124 to 132 or 134 to 137, wherein the guide region does not contain the YA site at nucleotides 15-16 and nucleotide 15 is unmodified. Embodiment 139 is the gRNA described in Embodiments 124 to 133 or 135 to 138, wherein the guide region does not contain the YA site at nucleotides 16-17 and nucleotide 16 is unmodified. Embodiment 140 is the gRNA described in Embodiments 124 to 133 or 135 to 139, wherein the guide region does not contain the YA site at nucleotides 19-20. , Embodiment 124-134 or 136-139, wherein nucleotide 19 is unmodified The gRNA described in Embodiment 141 is a short sgRNA that is as follows: (a) 2'-OMe and phosphorothioate modifications at nucleotide 1, (b) 2'-OMe and phosphorothioate modifications at nucleotide 2, (c) 2'-OMe and phosphorothioate modifications at nucleotide 3, (d) 2'-OMe modification at nucleotide 4, (e) Phosphorothioate modification at nucleotide 6, (f) Phosphorothioate modification at nucleotide 7, (g) 2'-fluoro and phosphorothioate modifications at nucleotide 8, (h) 2'-fluoro and phosphorothioate modifications at nucleotide 9, (i) 2'-fluoro and phosphorothioate modifications at nucleotide 10, (j) 2'-fluoro modification at nucleotide 11, (k) 2'-fluoro modification at nucleotide 13, (l) 2'-fluoro modification at nucleotide 14, (m) 2'-fluoro modification at nucleotide 17, and (n) A guide region containing one or more of the 2'-fluoro modifications at nucleotide 18 The gRNA according to Embodiments 124-140. Embodiment 142 is a gRNA according to Embodiments 124-141, wherein the guide region contains each of the modifications described in the previous embodiments. Embodiment 143 is a gRNA wherein the guide region is as follows: (a) When nucleotides 5 and 6 form a YA site, 2'-OMe modification at nucleotide 5 (b) When nucleotides 12 and 13 form a YA site, at nucleotide 12 2'-OMe modification in (c) When nucleotides 15 and 16 form a YA site, in nucleotide 15 phosphorothioate or 2'-H modification in (d) When nucleotides 16 and 17 form a YA site, in nucleotide 16 phosphorothioate modification in (e) When nucleotides 19 and 20 form a YA site, in nucleotide 19 at least 1, 2, 3, or 4 of phosphorothioate or 2'-fluoro modification, the gRNA of Embodiments 124 - 142. Embodiment 144 is the gRNA of Embodiments 124 - 143, wherein the guide region contains a YA site at nucleotides 5 - 6 and a 2'-OMe modification at nucleotide 5. Embodiment 145 is the gRNA according to Embodiments 124 - 144, wherein the guide region contains a YA site at nucleotides 12 - 13 and a 2'-OMe modification at nucleotide 12. NA. Embodiment 146 is the gRNA according to Embodiments 124 - 145, wherein the guide region contains a YA site at nucleotides 15 - 16 and a phosphorothioate modification at nucleotide 15. Embodiment 147 is the gRNA according to Embodiments 124 - 146, wherein the guide region contains a YA site at nucleotides 16 - 17 and a phosphorothioate modification at nucleotide 16. Embodiment 148 is the gRNA according to Embodiments 124 - 147, wherein the guide region contains a YA site at nucleotides 19 - 20 and a phosphorothioate modification at nucleotide 19. Embodiment 149 is the gRNA wherein the guide region contains a 2'-fluoro modification at nucleotide 19 mu, the gRNA described in Embodiments 124 to 148. Embodiment 150 is a gRNA described in Embodiments 124 to 149, wherein the guide region contains unmodified nucleotide 15 or contains only phosphorothioate modification at nucleotide 15. mu, the gRNA described in Embodiments 124 to 149, wherein the guide region contains unmodified nucleotide 15 or contains only phosphorothioate modification at nucleotide 15. gRNA. Embodiment 151 is a gRNA described in Embodiments 124 to 150, wherein the guide region contains unmodified nucleotide 16 or contains only phosphorothioate modification at nucleotide 16. mu, the gRNA described in Embodiments 124 to 150, wherein the guide region contains unmodified nucleotide 16 or contains only phosphorothioate modification at nucleotide 16. gRNA. Embodiment 152 is a. YA modification at the YA site of two or more guide regions, b. YA modification at one or more of conserved region YA sites 2, 3, 4, and 10, and c. a single guide RNA (sgRNA) containing YA modification at one or more of conserved region YA sites 1 and 8. guide RNA. Embodiment 153 is a. YA modification at the YA site of one or more guide regions starting from nucleotide 8 or later from the 5'-end of the 5'-terminal portion, b. YA modification at one or more of conserved region YA sites 2, 3, 4, and 10, and optionally c. a single guide RNA (sgRNA) containing YA modification at one or more of conserved region YA sites 1 and 8. guide RNA. Embodiment 154 is a. YA modification at the YA site of one or more guide regions within 13 nucleotides of the 3'-terminal nucleotide of the guide region, b. YA modification at one or more of conserved region YA sites 2, 3, 4, and 10, c. a single guide RNA (sgRNA) containing YA modification at one or more of conserved region YA sites 1 and 8. b. YA modification at one or more of conserved region YA sites 2, 3, 4, and 10, and c. a single guide RNA (sgRNA) containing YA modification at one or more of conserved region YA sites 1 and 8. A guide RNA (sgRNA), which is a guide RNA. Embodiment 155 is a. 5'-end modification and 3'-end modification, b. YA modification in one or more of conserved region YA sites 2, 3, 4, and 10, and c. A single guide RNA (sgRNA) containing YA modification in one or more of conserved region YA sites 1 and 8 A guide RNA, which is a guide RNA. Embodiment 156 is a. A YA modification in at least one guide region YA site, wherein the modification of the guide region YA site does not include at least one nucleotide located on the 5'-side of the guide region YA site and contains the modification, b. YA modification in one or more of conserved region YA sites 2, 3, 4, and 10, and c. A single guide RNA (sgRNA) containing YA modification in one or more of conserved region YA sites 1 and 8 A guide RNA, which is a guide RNA. Embodiment 157 is a. YA modification in one or more of conserved region YA sites 2, 3, 4, and 10, and b. A single guide RNA (sgRNA) containing YA modification in conserved region YA sites 1 and 8 A guide RNA, which is a guide RNA. Embodiment 158 is a. YA modification in one or more guide region YA sites starting from nucleotide 8 or later from the 5'-end of the 5'-terminal portion, b. YA modification in one or more of conserved region YA sites 2, 3, 4, and 10, and c. A single guide RNA (sgRNA) containing modification in one or more of H1-1 and H2-1 A guide RNA, which is a guide RNA. ​​ Embodiment 159 is a. YA modification in one or more of storage regions YA sites 2, 3, 4, and 10 b. YA modification in one or more of storage regions YA sites 1, 5, 6, 7, 8, and 9 and c. A single guide R NA (sgRNA) that includes a modification in one or more of H1-1 and H2-1 is a guide RNA. Embodiment 160 is a. Modification in 6 nucleotides from the 5' end of the 5' end portion or 1 or more nucleotides located thereafter, such as YA modification b. YA modification in one or more guide sequence YA sites c. A modification in one or more of B3, B4, and B5, where B6 does not include a 2'-OMe modification or includes a modification other than 2'-OMe d. A modification in LS10, where LS10 includes a modification other than 2'-fluoro , a modification, and / or e. A modification in one or more of N2, N3, N4, N5, N6, N7, N10, or N11 is a sgRNA that includes one or more of the following: i. At least one of nucleotides 8-11, 13, 14, 17, or 18 from the 5' end of the 5' end portion does not include a 2'-fluoro modification ii. At least one of nucleotides 6-10 from the 5' end of the 5' end portion does not include a phosphorothioate bond iii. At least one of B2, B3, B4, or B5 does not include a 2'-OMe modification iv. At least one of LS1, LS8, or LS10 does not include a 2'-OMe modification v.N2, N3, N4, N5, N6, N7, N10, N11, N16, or N1 at least one of 7 does not contain a 2'-OMe modification, vi.H1-1 contains a modification, vii.H2-1 contains a modification, or viii.at least one of H1-2, H1-3, H1-4, H1-5, H1-6, H1-7, H1- 8, H1-9, H1-10, H2-1, H2-2, H2-3, H2-4, H2-5, H2 -6, H2-7, H2-8, H2-9, H2-10, H2-11, H2-12, H2-1 3, H2-14, or H2-15 does not contain a phosphorothioate bond, is a guide RNA to which at least one of the above applies. Embodiment 161 is i.a modification in nucleotide 6 from the 5'-end of the 5'-terminal portion or one or more subsequent nucleotides, such as a YA modification, or ii.a YA modification at one or more guide sequence YA sites is a guide RNA containing any one or more of the following: a.at least one of nucleotides 8-11, 13, 14, 17, or 18 from the 5'-end of the 5'-terminal portion does not contain a 2'-fluoro modification, or b.at least one of nucleotides 6-10 from the 5'-end of the 5'-terminal portion does not contain a phosphorothioate bond, at least one of the above applies, as follows: c.at least one of nucleotides 7-10 from the 5'-end of the 5'-terminal portion does not contain a 2'-OMe modification, d.nucleotide 20 from the 5'-end of the 5'-terminal portion does not contain a 2'-OMe modification , or e.the guide RNA contains any one of nucleotides 1-20 from the 5'-end of the 5'-terminal portion comprising a 2'-fluoro modification in any one or more thereof, and at least one of nucleotides 11, 12, 13, 14, 17, or 18 from the 5'-end of the 5'-terminal portion does not contain a 2'- fluoro modification, and optionally, nucleotide 12 from the 5'-end of the 5'-terminal portion does not contain a 2'-fluoro modification, and at least one of which also applies, is a guide RNA. Embodiment 162 is guanosine at N14 and / or the following: a. a YA modification at the YA site in one or more nucleotides 8 or subsequent to nucleotide 8 from the 5'-end of the 5'-terminal portion of the guide region, b. a YA modification in one or more of conserved regions YA sites 1, 5, and 6, where when YA site 6 is modified and LS9 is unmodified, the modification of LS12 is other than 2'-OMe, a YA modification, c. a modification at LS9, where when LS9 is modified and LS5, LS7, and LS 12 are unmodified, the modification of LS9 is other than 2'-fluoro, a modification, d. a modification at LS12, where when LS12 is modified and LS9 is unmodified the modification of LS12 is other than 2'-OMe, a modification, e. a modification at LS8 or LS11, where at least one of LS8 and LS11 contains a modification other than 2'-OMe, a modification, and / or f. a modification at N6, N14, or N17, where when N17 is modified and N6 and N14 are unmodified, the modification of N17 is other than 2'-fluoro and other than 2'- OMe, a modification A guide RNA that is a sgRNA containing one or more of the following: i. At least one of nucleotides 8 - 11, 13 - 14, 17, or 18 from the 5' end of the 5' terminal portion does not contain a 2'-fluoro modification, ii. At least one of nucleotides 6 - 10 from the 5' end of the 5' terminal portion does not contain a phosphorothioate bond, iii. At least one of B2, B3, B4, or B5 does not contain a 2'-OMe modification, iv. At least one of LS1, LS8, or LS10 does not contain a 2'-OMe modification, v. At least one of N2, N3, N4, N5, N6, N7, N10, N11, N16, or N17 does not contain a 2'-OMe modification, vi. H1-1 contains a modification, vii. H2-1 contains a modification, or viii. At least one of H1-2, H1-3, H1-4, H1-5, H1-6, H1-7, H1-8, H1-9, H1-10, H2-1, H2-2, H2-3, H2-4, H2-5, H2-6, H2-7, H2-8, H2-9, H2-10, H2-11, H2-12, H2-13, H2-14, or H2-15 does not contain a phosphorothioate bond, and it is a guide RNA for which at least one of the above applies. Embodiment 163 is a. YA modification at 1, 2, 3, 4, or 5 guide region YA sites, b. YA modification at 1, 2, 3, 4, or 5 guide region YA sites, where the modification of at least one guide region YA site is different from any 5' terminal modification of the sgRNA, c. YA modification at one or more guide region YA sites starting from nucleotide 8 or later from the 5' end of the 5' terminal portion. viii. At least one of H1-2, H1-3, H1-4, H1-5, H1-6, H1-7, H1- 8, H1-9, H1-10, H2-1, H2-2, H2-3, H2-4, H2-5, H2 -6, H2-7, H2-8, H2-9, H2-10, H2-11, H2-12, H2-1 3, H2-14, or H2-15 does not contain a phosphorothioate bond, and it is a guide RNA for which at least one of the above applies. is a guide RNA. Embodiment 163 is a. YA modification at 1, 2, 3, 4, or 5 guide region YA sites, b. YA modification at 1, 2, 3, 4, or 5 guide region YA sites, where at least one guide region YA site modification is different from any 5' terminal modification of the sgRNA, c. YA modification at one or more guide region YA sites starting from nucleotide 8 or later from the 5' end of the 5' terminal portion. c. One or more YA modifications at the guide region YA sites starting from nucleotide 8 or later from the 5' end of the 5' terminal portion. YA modifications at the guide region YA sites. d. YA modifications in one or more guide region YA sites within nucleotide 5, 6, 7, 8, 9 ends, or 10 ends, counted from the 5'-end of the 5'-terminal portion, e. YA modifications in one or more guide region YA sites within 17, 16, 15, 14, 13, 12, 11, 10, or 9 nucleotides of the 3'-terminal nucleotide of the guide region wherein, f. YA modifications in guide region YA sites other than 5'-terminal modifications, or g. YA modifications in guide region YA sites, wherein the modification of the guide region YA site is a modification that does not include at least one nucleotide located on the 5'-side of the guide region YA site containing the YA modification, and is the gRNA according to Embodiment 161 or 162. Embodiment 164 is a. YA modifications in two or more guide region YA sites at nucleotide 8 or later, counted from the 5'-end of the 5'-terminal portion, wherein, b. YA modifications in two or more guide region YA sites within nucleotide 5, 6, 7, 8, 9 ends, or 10 ends, counted from the 5'-end of the 5'-terminal portion, c. YA modifications in two or more guide region YA sites within 17, 16, 15, 14, 13, 12, 11, 10, or 9 nucleotides of the 3'-terminal nucleotide of the guide region wherein, d. YA modifications in two or more guide region YA sites other than 5'-terminal modifications, or e. YA modifications in two or more guide region YA sites, wherein the modification of the guide region YA site is a modification that does not include at least one nucleotide located on the 5'-side of the guide region YA site containing the YA modification, and is the gRNA according to Embodiment 163. Embodiment 165 is a. Three or more of the nucleotides 8 from the 5'-end of the 5'-terminal portion or those following it YA modification in the YA site of the guide region b. YA modification in three or more YA sites of the guide region within the nucleotide 5-end, 6-end, 7-end, 8-end, 9 end, or 10-end from the 5'-end of the 5'-terminal portion c. YA modification in three or more YA sites of the guide region within 17, 16, 15, 14, 13, 12, 11, 10, or 9 nucleotides of the 3'-terminal nucleotide of the guide region YA modification occurring therein d. YA modification in three or more YA sites of the guide region other than the 5'-terminal modification, or e. YA modification in three or more YA sites of the guide region, wherein the modification of the guide region YA site includes a modification that does not include at least one nucleotide located on the 5'-side of the guide region YA site The gRNA according to Embodiment 163, which includes the YA modification. Embodiment 166 is the gRNA according to any one of Embodiments 161 to 165, which includes at least one YA modification at nucleotide 6 from the 5'-end of the 5'-terminal portion. Embodiment 167 is the gRNA according to any one of Embodiments 161 to 166, which includes at least one YA modification at nucleotide 7 from the 5'-end of the 5'-terminal portion. Embodiment 168 is the gRNA according to any one of Embodiments 161 to 167, which includes at least one YA modification at nucleotide 8 from the 5'-end of the 5'-terminal portion. Embodiment 169 is the gRNA according to any one of Embodiments 161 to 168, which includes at least one YA modification at nucleotide 9 from the 5'-end of the 5'-terminal portion. Embodiment 170 is at least The gRNA according to any one of Embodiments 161 to 169, comprising one YA modification . Embodiment 171 is the gRNA according to any one of Embodiments 161 to 170, comprising at least one YA modification at nucleotide 11 from the 5'-end of the 5'-terminal portion . Embodiment 172 is the gRNA according to any one of Embodiments 161 to 171, comprising at least one YA modification at nucleotide 12 from the 5'-end of the 5'-terminal portion . Embodiment 173 is the gRNA according to any one of Embodiments 161 to 172, comprising at least one YA modification at nucleotide 13 from the 5'-end of the 5'-terminal portion . Embodiment 174 is the gRNA according to any one of Embodiments 161 to 173, comprising at least one YA modification at nucleotide 14 from the 5'-end of the 5'-terminal portion . Embodiment 175 is the gRNA according to any one of Embodiments 161 to 174, comprising at least one YA modification at nucleotide 15 from the 5'-end of the 5'-terminal portion . Embodiment 176 is the gRNA according to any one of Embodiments 161 to 175, comprising at least one YA modification at nucleotide 16 from the 5'-end of the 5'-terminal portion . Embodiment 177 is the gRNA according to any one of Embodiments 161 to 176, comprising at least one YA modification at nucleotide 17 from the 5'-end of the 5'-terminal portion . Embodiment 178 is the gRNA according to any one of Embodiments 161 to 177, comprising at least one YA modification at nucleotide 18 from the 5'-end of the 5'-terminal portion . Embodiment 179 is the gRNA according to any one of Embodiments 161 to 178, which includes at least one YA modification at nucleotide 19 from the 5'-end of the 5'-terminal portion . Embodiment 180 is the gRNA according to any one of Embodiments 161 to 179, which includes at least one YA modification at nucleotide 20 from the 5'-end of the 5'-terminal portion . Embodiment 181 is the gRNA according to any one of Embodiments 161 to 180, wherein at least 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides out of nucleotides 8 to 11, 13 to 14, and 17 to 18 from the 5'-end of the 5'-terminal portion include a YA modification, and optionally, the modification includes 2'-fluoro, 2'-H, 2' -OMe, or PS . Embodiment 182 is the gRNA according to Embodiment 181, wherein the modification is 2'-fluoro . Embodiment 183 is the gRNA according to Embodiment 181, wherein the modification is 2'-OMe or 2'-H . Embodiment 184 is the gRNA according to Embodiment 181, wherein the modification is PS Embodiment 185 is the gRNA according to any one of Embodiments 161 to 184, wherein at least 1, 2, 3 , 4, or 5 nucleotides out of nucleotides 6 to 10 from the 5'-end include a YA modification, and optionally, the modification includes 2'-fluoro, 2' -H, 2'-OMe, inosine, or PS . Embodiment 186 is the gRNA according to Embodiment 185, wherein the modification is PS Embodiment 187 is the gRNA according to Embodiment 185, wherein the modification is 2'-fluoro or 2'-H . Embodiment 188 is the gRNA described in Embodiment 185, wherein the modification is 2'-OMe. There is. Embodiment 189 is as follows: a. One, two, three, four, five, six, seven, or eight YA modifications from nucleotides 8-11, 13-14, and 17-18 from the 5' end of the 5' end, where the YA modification is optionally a 2'-fluoro modification and a modification other than 2'-fluoro in one or more of nucleotides 6-10 from the 5' end of the 5' end, the YA modification, b. A YA modification other than PS in one or more of nucleotides 8-11, 13-14, and 17-18 from the 5' end of the 5' end, and one, two, three, four, or five YA modifications in nucleotides 6-10 from the 5' end of the 5' end wherein the modification is optionally a PS modification, the YA modification, c. One, two, three, four, five, six, seven, or eight YA modifications in nucleotides 8-11, 13-14, and 17-18 from the 5' end of the 5' end, where the YA modification is optionally a 2'-fluoro modification, the YA modification, and a modification other than 2'-fluoro in nucleotides 6-10 from the 5' end of the 5' end d. A YA modification other than PS in each of nucleotides 8-11, 13-14, and 17-18 from the 5' end of the 5' end of the 5' end, and one, two, three, four, or five YA modifications in nucleotides 6-10 from the 5' end of the 5' end wherein the modification is optionally a PS modification, the YA modification, e. One, two, three, four, five, six, seven, or eight YA modifications in nucleotides 8-11, 13-14, and 17-18 from the 5' end of the 5' end, where the YA modification is optionally a 2'-fluoro modification, the YA modification, f. A YA modification other than PS in each of nucleotides 8-11, 13-14, and 17-18 from the 5' end of the 5' end, and one, two, three, four, or five YA modifications in nucleotides 6-10 from the 5' end of the 5' end wherein the modification is optionally a PS modification, the YA modification, g. One, two, three, four, five, six, seven, or eight YA modifications in nucleotides 8-11, 13-14, and 17-18 from the 5' end of the 5' end, where the YA modification is optionally a 2'-fluoro modification, the YA modification, and a modification other than 2'-fluoro in nucleotides 6-10 from the 5' end of the 5' end h. A YA modification other than PS in each of nucleotides 8-11, 13-14, and 17-18 from the 5' end of the 5' end of the 5' end, and one, two, three, four, or five YA modifications in nucleotides 6-10 from the 5' end of the 5' end wherein the modification is optionally a PS modification, the YA modification, i. One, two, three, four, five, six, seven, or eight YA modifications in nucleotides 8-11, 13-14, and 17-18 from the 5' end of the 5' end, where the YA modification is optionally a 2'-fluoro modification, the YA modification, is a YA modification that is optionally a 2'-fluoro modification, and one or more PS modifications at any one of nucleotides 6 to 10 from the 5'-end of the 5'-terminal portion c. one or more PS modifications at any one of nucleotides 6 to 10 from the 5'-end of the 5'-terminal portion f. at least one 2'-fluoro modification at any one of nucleotides 8 to 11, 13 to 14, and 17 to 18 from the 5'-end of the 5'-terminal portion, and one, two, three, four, or five YA modifications at any one of nucleotides 6 to 10 from the 5'-end of the 5'-terminal portion, wherein the modification is a YA modification that is optionally a PS modification g. one, two, three, four, five, six, seven, or eight YA modifications at any one of nucleotides 8 to 11, 13 to 14, and 17 to 18 from the 5'-end of the 5'-terminal portion, wherein the YA modification is a YA modification that is optionally a 2'-fluoro modification, and a PS modification at each of nucleotides 6 to 10 from the 5'-end of the 5'-terminal portion h. a 2'-fluoro modification at each of nucleotides 8 to 11, 13 to 14, and 17 to 18 from the 5'-end of the 5'-terminal portion, and one, two, three, four, or five YA modifications at any one of nucleotides 6 to 10 from the 5'-end of the 5'-terminal portion, wherein the modification is a YA modification that is optionally a PS modification, and includes one or more of any one of the modifications, a gRNA according to any one of embodiments 161 to 1 88 i. a 2'-fluoro modification at each of nucleotides 8 to 11, 13 to 14, and 17 to 18 from the 5'-end of the 5'-terminal portion, and one, two, three, four, or five YA modifications at any one of nucleotides 6 to 10 from the 5'-end of the 5'-terminal portion, wherein the modification is a YA modification that is optionally a PS modification, and includes one or more of any one of the modifications, a gRNA according to any one of embodiments 161 to 1 88 j. a 2'-fluoro modification at each of nucleotides 8 to 11, 13 to 14, and 17 to 18 from the 5'-end of the 5'-terminal portion, and one, two, three, four, or five YA modifications at any one of nucleotides 6 to 10 from the 5'-end of the 5'-terminal portion, wherein the modification is a YA modification that is optionally a PS modification, and includes one or more of any one of the modifications, a gRNA according to any one of embodiments 161 to 1 Embodiment 190 is a. at least two, three, or four modified YA sites including a first modified YA site where nucleotides 4 to 20 from the 5'-end of the 5'-terminal portion include a 2'-OMe modification and a second modified YA site including a 2'-fluoro modification or a PS modification b. nucleotides 4 to 20 from the 5'-end of the 5'-terminal portion include a 2'-fluoro modification and include at least two, three, or four modified YA sites c. nucleotides 4 to 20 from the 5'-end of the 5'-terminal portion include a 2'-fluoro modification The first modified YA site and the second modified YA site containing 2'-OMe modification or PS modification containing at least two, three, or four modified YA sites including the position c. Nucleotides 4 to 20 from the 5'-end of the 5'-terminal part contain the first modified YA site containing PS modification and the second modified YA site containing 2'-OMe modification or 2'-fluoro modification and contain at least two, three, or four modified YA sites including the position d. Nucleotides 4 to 20 from the 5'-end of the 5'-terminal part contain at least two, three, or four modified YA sites including YA modification e. Nucleotides 4 to 20 from the 5'-end of the 5'-terminal part contain the first modified YA site containing 2'-OMe modification, the second modified YA site containing 2'-fluoro modification, and the third modified YA site containing PS modification and contain at least three or four modified YA sites including the position f. Nucleotides 4 to 20 from the 5'-end of the 5'-terminal part contain the first modified YA site containing 2'-OMe modification, the second modified YA site containing 2'-fluoro modification, the third modified YA site containing 2'-fluoro modification, and the fourth modified YA site containing PS modification and contain at least three or four modified YA sites including the position g. Nucleotides 4 to 20 from the 5'-end of the 5'-terminal part contain at least three or four modified YA sites including YA modification h. Nucleotides 4 to 20 from the 5'-end of the 5'-terminal part contain the first modified YA site containing 2'-OMe modification, the second modified YA site containing 2'-fluoro modification, the third modified YA site containing PS modification, and the fourth modified YA site containing PS modification and contain at least four modified YA sites including the position i. Nucleotides 4 to 40 from the 5'-end of the 5'-terminal part contain at least two modified YA sites including YA modification or j. Nucleotides 4 to 40 from the 5'-end of the 5'-terminal part contain the first modified YA site containing 2'-OMe modification, the second modified YA site containing 2'-fluoro modification, the third modified YA site containing PS modification, and the fourth modified YA site containing PS modification and contain at least four modified YA sites including the position k. Nucleotides 4 to 40 from the 5'-end of the 5'-terminal part contain at least three modified YA sites including YA modification or l. Nucleotides 4 to 40 from the 5'-end of the 5'-terminal part contain the first modified YA site containing 2'-OMe modification, the second modified YA site containing 2'-fluoro modification, the third modified YA site containing PS modification, and the fourth modified YA site containing PS modification and contain at least four modified YA sites including the position m. Nucleotides 4 to 40 from the 5'-end of the 5'-terminal part contain at least three modified YA sites including YA modification The gRNA according to any one of Embodiments 161 to 189, which also contains four modified YA sites is as follows. In Embodiment 191, nucleotides 4 to 20 from the 5'-end of the 5'-terminal portion are at least the gRNA according to any one of Embodiments 161 to 190, which contains five modified YA sites is as follows. In Embodiment 192, at least five modified YA sites include a fifth modified YA site containing a PS modification, and optionally, the third modified YA site contains a 2'-fluoro modification. The gRNA is according to any one of Embodiments 161 to 191 is as follows. is as follows. In Embodiment 193, the first, second, and (where applicable) third, fourth, and fifth of at least five modified YA sites are arranged in the 5' to 3' direction. The gRNA is according to any one of Embodiments 161 to 192 is as follows. In Embodiment 194, the first, second, and (where applicable) third, fourth, and fifth of at least five modified YA sites are not arranged in the 5' to 3' direction. The gRNA is according to any one of Embodiments 161 to 193 is as follows. In Embodiment 195, nucleotides 4 to 20 from the 5'-end of the 5'-terminal portion contain at least two, three, four, or five modified YA sites containing deoxyribonucleotides, and optionally, the deoxyribonucleotides are pyrimidines of the YA site. The gRNA is according to any one of Embodiments 16 1 to 194 is as follows. is as follows. In Embodiment 196 a. At least one, two, three, or four of nucleotides 8 to 11 from the 5'-end of the 5'-terminal portion are optionally 2'-fluoro modified, including YA modification b. Nucleotides 8 to 11, 13, 14, 17, and from the 5'-end of the 5'-terminal portion At least 1, 2, 3, 4, 5, 6, 7, or 8 of 18 contain a YA modification , optionally, when the YA modification is present at nucleotides 8 - 11, it is 2'-OMe and when it is present at nucleotides 13, 14, 17, or 18, it is 2'-fluoro , c. At least one or both of nucleotides 17 and 18 from the 5'-end of the 5'-terminal portion contain a YA modification, optionally being a 2'-fluoro modification d. At least one or both of nucleotides 17 and 18 from the 5'-end of the 5'-terminal portion contain a YA modification, optionally being a 2'-fluoro modification, or e. At least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of nucleotides 4 - 14, 17, and 18 from the 5'-end of the 5'-terminal portion contain a YA modification, optionally being a 2'-fluoro modification, which is the gRNA according to any one of Embodiments 161 - 195 f. At least one or both of nucleotides 17 and 18 from the 5'-end of the 5'-terminal portion contain a YA modification, optionally being a 2'-fluoro modification, which is the gRNA according to any one of Embodiments 161 - 195 e. At least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of nucleotides 4 - 14, 17, and 18 from the 5'-end of the 5'-terminal portion contain a YA modification, optionally being a 2'-fluoro modification, which is the gRNA according to any one of Embodiments 161 - 195 At least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of nucleotides 4 - 14, 17, and 18 from the 5'-end of the 5'-terminal portion contain a YA modification, optionally being a 2'-fluoro modification, which is the gRNA according to any one of Embodiments 161 - 195 At least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of nucleotides 4 - 14, 17, and 18 from the 5'-end of the 5'-terminal portion contain a YA modification, optionally being a 2'-fluoro modification, which is the gRNA according to any one of Embodiments 161 - 195 The gRNA according to any one of Embodiments 161 - 195 Embodiment 197 is the gRNA according to any one of Embodiments 161 - 196, wherein at least 1, 2, 3, 4, 5, or 6 of nucleotides 4 - 10 from the 5'-end of the 5'-terminal portion contain a YA modification, optionally being a 2'-OMe modification Embodiment 197 is the gRNA according to any one of Embodiments 161 - 196, wherein at least 1, 2, 3, 4, 5, or 6 of nucleotides 4 - 10 from the 5'-end of the 5'-terminal portion contain a YA modification, optionally being a 2'-OMe modification The gRNA according to any one of Embodiments 161 - 196 Embodiment 198 is the gRNA according to any one of Embodiments 161 - 197, wherein nucleotides 4 - 10 from the 5'-end of the 5'-terminal portion contain a YA modification, optionally being a 2'-OMe modification Embodiment 198 is the gRNA according to any one of Embodiments 161 - 197, wherein nucleotides 4 - 10 from the 5'-end of the 5'-terminal portion contain a YA modification, optionally being a 2'-OMe modification The gRNA according to any one of Embodiments 161 - 197 Embodiment 199 is a. The 5'-protected terminal modification contains at least 1 of nucleotides 1 - 3 from the 5'-end of the 5'-terminal portion, optionally being a 2'-OMe modification a. The 5'-protected terminal modification contains at least 1 of nucleotides 1 - 3 from the 5'-end of the 5'-terminal portion, optionally being a 2'-OMe modification b. At least two of nucleotides 1 to 3 from the 5'-end of the 5'-terminal portion are optionally 2'-OMe modified, including a 5'-protected terminal modification, or c. Each of nucleotides 1 to 3 from the 5'-end of the 5'-terminal portion is optionally 2' -OMe modified, including a 5'-protected terminal modification, and is the gRNA according to any one of Embodiments 161 to 198. Embodiment 200 is a gRNA including a 5'-terminal modification, wherein at least 1, 2, 3, 4, or 5 of nucleotides 11, 13, 14, 17, and 18 from the 5'-end of the 5'-terminal portion are optionally 2'-fluoro modified, and is the gRNA according to any one of Embodiments 161 to 199. Embodiment 201 is a gRNA according to any one of Embodiments 161 to 200, wherein nucleotide 15 from the 5'-end of the 5'-terminal portion is unmodified or modified only with phosphorothioate. Embodiment 202 is a gRNA according to any one of Embodiments 161 to 200, wherein nucleotide 16 from the 5'-end of the 5'-terminal portion is unmodified or modified only with phosphorothioate. Embodiment 203 is a gRNA according to any one of Embodiments 1 to 202, wherein nucleotide 3 from the 5'-end of the 5'-terminal portion is unmodified or modified only with phosphorothioate. Embodiment 204 is a crRNA or dgRNA and is the gRNA according to any one of Embodiments 161 to 203. Embodiment 205 is an sgRNA and is the gRNA according to any one of Embodiments 161 to 203. Embodiment 206 is a short sgRNA and is the gRNA according to any one of Embodiments 161 to 203. ​​​​​​​​​​​​​ It is the gRNA described. Embodiment 207 is the gRNA according to any one of Embodiment 205 or 20 6, which includes the YA modification of the conserved region YA site 1. Embodiment 208 is the gRNA according to any one of Embodiments 205 to 207, which includes the YA modification of the conserved region YA site 2. Embodiment 209 is the gRNA according to any one of Embodiments 205 to 208, which includes the YA modification of the conserved region YA site 3. Embodiment 210 is the gRNA according to any one of Embodiments 205 to 209, which includes the YA modification of the conserved region YA site 4. Embodiment 211 is the gRNA according to any one of Embodiments 205 to 210, which includes the YA modification of the conserved region YA site 5. Embodiment 212 is the gRNA according to any one of Embodiments 205 to 211, which includes the YA modification of the conserved region YA site 6. Embodiment 213 is the gRNA according to any one of Embodiments 205 to 212, which includes the YA modification of the conserved region YA site 7. Embodiment 214 is the gRNA according to any one of Embodiments 205 to 213, which includes the YA modification of the conserved region YA site 8. Embodiment 215 is the gRNA according to any one of Embodiments 205 to 214, which includes the YA modification of the conserved region YA site 9. Embodiment 216 is the gRNA according to any one of Embodiments 205 to 215, which includes the YA modification of the conserved region YA site 10. Embodiment 217 is a. YA modifications of conserved region YA sites 2, 3, 4, and 10, b. YA modifications of conserved region YA sites 2, 3, and 4, c. YA modifications of conserved region YA sites 2, 3, and 10, d. YA modification of conserved region YA sites 2, 4, and 10; e. YA modification of conserved region YA sites 3, 4, and 10; f. YA modification of conserved region YA sites 2 and 10; g. YA modification of conserved region YA sites 2 and 4; h. YA modification of conserved region YA sites 2 and 3; i. YA modification of conserved region YA sites 3 and 4, j. YA modification of conserved region YA sites 3 and 10, or k. Any of embodiments 205-216, comprising YA modifications at conserved region YA sites 4 and 10. The gRNA is one described in any one of the above. Embodiment 218 is a. YA modification of conserved region YA sites 1 and 5, b. YA modification of conserved region YA sites 1 and 6, c. YA modification of conserved region YA sites 1 and 7; d. YA modification of conserved region YA sites 1 and 8; e. YA modification of conserved region YA sites 1 and 9, f. YA modification of conserved region YA sites 8 and 5; g. YA modification of conserved region YA sites 8 and 6; h. YA modification of conserved region YA sites 8 and 7, or i. comprising YA modifications at conserved region YA sites 8 and 9; Optionally, the sgRNA comprises a YA modification at conserved YA sites 2, 3, 4, and 10. The gRNA according to any one of embodiments 205 to 217, further comprising: Embodiment 219 relates to at least one modified YA moiety, optionally including a nucleotide sequence that aligns with the pyrimidine dinucleotide of the YA moiety. 219. The method according to any one of embodiments 205 to 218, comprising a 2'-OMe modification in the amide. The gRNA is described below. Embodiment 220 relates to at least one modified YA moiety, optionally including a YA moiety pyrimidine. Any one of embodiments 205 to 219, comprising a 2'-fluoro modification in the amide. The gRNA is as described above. Embodiment 221 relates to at least one modified YA moiety, optionally including a nucleotide sequence that aligns with the pyrimidine dinucleotide of the YA moiety. 221. The gR according to any one of embodiments 205 to 220, comprising a PS modification in a cyclodextrin. It is NA. Embodiment 222 is a method for the preparation of a compound comprising at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 modifications. the YA site optionally comprises a 2'-OMe modification on the pyrimidine of the YA site; The gRNA according to any one of embodiments 205 to 221. Embodiment 223 is a method for the preparation of a compound comprising at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 modifications. The YA site optionally includes a 2'-fluoro modification on the pyrimidine of the YA site. , any one of gRNAs of embodiments 205 to 222. Embodiment 224 is a method for the preparation of a compound comprising at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 modifications.

[0023] Embodiments in which the YA site optionally contains a PS modification on the pyrimidine of the YA site. The gRNA is any one of 205 to 223. Embodiment 225 is a method for the preparation of a compound having at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 modifications. The YA site comprises a ribozyme at the 2' position, optionally at the pyrimidine of the YA site. and optionally 2'-O alkyl, 2'-H, and 2'-fluoro modifications. The gRNA of any one of embodiments 205 to 224 is selected from the group consisting of gRNAs. Embodiment 226 is a. Conserved regions YA sites 1 and 8, optionally with , including a 2'-fluoro modification; b. Conserved region YA sites 5 and 6, 5 and 7, 5 and 9, 6 and 7, 6 and 9, 5, 6, and 7; 5, 6, and 9; 6, 7, and 9; or 5, 6, 7, and Oligonucleotide 9 optionally includes a 2'-OMe modification at the pyrimidine of the YA site. c. Conserved region YA site 1 includes a 2'-fluoro modification, and conserved region YA sites 5 and 6, 5 and 7, 5 and 9, 6 and 7, 6 and 9, 5, 6, and 7, 5, 6, and 9, 6, 7, and 9, or 5, 6, 7, and 9 optionally include a 2'-OMe modification at the pyrimidine of the YA site. d. Conserved region YA site 8 includes a 2'-fluoro modification, and conserved region YA sites 5 and 6, 5 and 7, 5 and 9, 6 and 7, 6 and 9, 5, 6, and 7, 5, 6, and 9, 6, 7, and 9, or 5, 6, 7, and 9 optionally include a 2'-OMe modification at the pyrimidine of the YA site. e. Conserved region YA site 1 includes a 2'-fluoro modification at the pyrimidine of the YA site, and conserved region YA sites 5 and 6, 5 and 7, 5 and 9, 6 and 7, 6 and 9, 5, 6, and 7, 5, 6, and 9, 6, 7, and 9; or 5, 6, 7, and 9 optionally include a 2'-OMe modification at the pyrimidine of the YA site. f. Conserved region YA site 8 includes a 2'-fluoro modification at the pyrimidine of the YA site, and conserved region YA sites 5 and 6, 5 and 7, 5 and 9, 6 and 7, 6 and 9, 5, 6, and 7, 5, 6, and 9, 6, 7, and 9; or 5, 6, 7, and 9 optionally include a 2'-OMe modification at the pyrimidine of the YA site. g. Conserved region YA sites 1 and 8 include a 2'-fluoro modification, and conserved region YA sites 5 and 6, 5 and 7, 5 and 9, 6 and 7, 6 and 9, 5, 6, and 7, 5 , 6, and 9, 6, 7, and 9, or 5, 6, 7, and 9 optionally include a 2'-OMe modification by In the pyrimidine of the YA region, including 2'-OMe modification, or h. The conserved regions YA sites 1 and 8 contain 2'-fluoro modification in the pyrimidine of the YA region and the conserved regions YA sites 5 and 6, 5 and 7, 5 and 9, 6 and 7, 6 and 9, 5, 6, and 7, 5, 6, and 9, 6, 7, and 9, or 5, 6, 7 and 9 optionally contain 2'-OMe modification in the pyrimidine of the YA region, and it is the gRNA according to any one of Embodiments 205 to 225. Embodiment 227 is the gRNA according to any one of Embodiments 205 to 226, which contains YA modification where the conserved regions YA sites 7 and 9 are optionally 2'-OMe modification Embodiment 228 is the gRNA according to any one of Embodiments 205 to 227, which contains YA modification where the conserved regions YA sites 5, 6, 7, and 9 are optionally 2'- OMe modification Embodiment 229 is the gRNA according to any one of Embodiments 205 to 228, where the conserved region YA site 8 contains 2'-fluoro modification Embodiment 230 is the gRNA according to any one of Embodiments 205 to 229, where the conserved region YA site 8 contains deoxyribonucleotide modification Embodiment 231 is the gRNA according to any one of Embodiments 205 to 230, where the conserved region YA site 8 is excluded by base substitution, and optionally, the base substitution excludes uracil at YA site 8, and further optionally, the base substitution is substitution of uracil with guanine Embodiment 232 is the gRNA according to any one of Embodiments 205 to 231, where the conserved region YA site 1 contains 2'-fluoro modification ​​​​​​​​​Embodiment 233 is the gRNA according to any one of Embodiments 205 to 232, wherein the storage region YA site 1 contains a PS modification. Embodiment 234 is the gRNA according to any one of Embodiments 205 to 233, wherein one, two, three, four, five, six, or seven of LS5, LS7, LS8, LS9, LS10, LS11, and LS12 contain a modification, and optionally, the modification is a 2'-fluoro and / or 2'-OMe modification. Embodiment 235 is the gRNA according to any one of Embodiments 205 to 234, wherein when the modifications in LS5, LS7, LS9, and LS11 are present, they contain a 2'-fluoro modification, and optionally, each of LS5, LS7, LS9, and LS11 contains a 2'-fluoro modification. Embodiment 236 is the gRNA according to any one of Embodiments 205 to 235, wherein when the modifications in LS8, LS10, and LS12 are present, they contain a 2'-OMe modification, and each of LS8, LS10, and LS12 contains a 2'-OMe modification. Embodiment 237 is the gRNA according to any one of Embodiments 205 to 236, wherein one, two, three, four, five, six, seven, eight, nine, or ten of N2, N3, N4, N5, N6, N7, N10, N11, N16, and N17 optionally contain a 2'-OMe modification. Embodiment 238 is the gRNA according to any one of Embodiments 205 to 237, wherein H2-2 contains a modification, and optionally, H2 is unmodified otherwise. Embodiment 239 is the gRNA according to any one of Embodiments 205 to 238, wherein H2-2 contains a 2'-OMe modification. Embodiment 240 is such that US3, US9, and US12 include modifications and, optionally, , US is otherwise unmodified, and the g RNA is as described in any one of Embodiments 205 to 239. Embodiment 241 is such that US3, US9, and US12 include 2'-OMe modifications, and the gRNA is as described in any one of Embodiments 205 to 240. Embodiment 242 is such that nucleotides 6 to 10 from the 5'-end of the 5'-terminal portion include PS modifications, and nucleotides 8 to 11, 13, 14, 17, and 1 8 from the 5'-end of the 5'-terminal portion include 2'-fluoro modifications, and the gR NA is as described in any one of Embodiments 205 to 241. Embodiment 243 is such that each guide region YA site includes 2'-fluoro modifications and, optionally, excludes nucleotides 15 and / or 16 from the 5'-end of the 5'-terminal portion, and the gRNA is as described in any one of Embodiments 205 to 242. Embodiment 244 is such that nucleotides 4, 8, and 11 from the 5'-end of the 5'-terminal portion include YA modifications, and, optionally, nucleotide 4 includes 2'-OMe modifications, and nucleotides 8 and 11 include 2'-fluoro modifications, and the gRNA is as described in any one of Embodiments 205 to 243. Embodiment 245 is such that 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more modified YA sites include YA modifications at the pyrimidine positions of the YA sites, and the gRNA is as described in any one of Embodiments 205 to 244. Embodiment 246 is such that 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modified conserved region YA sites include YA modifications at the pyrimidine positions of the YA sites, and the gRNA is as described in Embodiment 245. ​​​​​​​​ Embodiment 247 is the gRNA according to any one of Embodiments 205 to 246, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more modified YA sites include YA modifications at the adenine position of the YA site. Embodiment 248 is the gRNA according to Embodiment 247, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modified conserved region YA sites include YA site modifications at the adenine position of the YA site. Embodiment 249 is a. modification of H1-1, b. modification of H2-1, or c. modification of H1-1 and H2-1, and is the gRNA according to any one of Embodiments 205 to 248. Embodiment 250 is the gRNA according to Embodiment 249, wherein H1-1 and / or H2-1 include 2'-OMe modifications. Embodiment 251 is the gRNA according to Embodiment 250, wherein H1-1 and / or H2-1 include 2'-fluoro modifications. Embodiment 252 is the gRNA according to Embodiment 251, wherein H1-1 and / or H2-1 include PS modifications. Embodiment 253 includes modifications at B3, and optionally, B6 does not include 2'-OMe modifications or includes modifications other than 2'-OMe, and is the gRNA according to any one of Embodiments 205 to 252. Embodiment 254 includes modifications at B4, and optionally, B6 does not include 2'-OMe modifications or includes modifications other than 2'-OMe, and is the gRNA according to any one of Embodiments 205 to 253. Embodiment 255 includes modifications at B5, and optionally, B6 does not include 2'-OMe modifications or includes modifications other than 2'-OMe.​​​​​​​​​​​ A gRNA according to any one of embodiments 205 to 254, which contains no modification or contains a modification other than 2'-OMe. Embodiment 256 contains a modification at LS10, and optionally, LS10 contains a modification other than 2'-fluoro, and is a gRNA according to any one of embodiments 205 to 255. Embodiment 257 contains a modification at N2 and is a gRNA according to any one of embodiments 205 to 256. Embodiment 258 contains a modification at N3 and is a gRNA according to any one of embodiments 205 to 257. Embodiment 259 contains a modification at N4 and is a gRNA according to any one of embodiments 205 to 258. Embodiment 260 contains a modification at N5 and is a gRNA according to any one of embodiments 205 to 259. Embodiment 261 contains a modification at N6 and is a gRNA according to any one of embodiments 205 to 260. Embodiment 262 contains a modification at N7 and is a gRNA according to any one of embodiments 205 to 261. Embodiment 263 contains a modification at N10 and is a gRNA according to any one of embodiments 205 to 262. Embodiment 264 contains a modification at N11 and is a gRNA according to any one of embodiments 205 to 263. Embodiment 265 a. Nucleotide 8 from the 5'-end of the 5'-terminal portion does not contain a 2'-fluoro modification , b. Nucleotide 9 from the 5'-end of the 5'-terminal portion does not contain a 2'-fluoro modification , c. The nucleotide 10 from the 5'-end of the 5'-terminal portion does not contain a 2'-fluoro modification no d. The nucleotide 11 from the 5'-end of the 5'-terminal portion does not contain a 2'-fluoro modification no e. The nucleotide 13 from the 5'-end of the 5'-terminal portion does not contain a 2'-fluoro modification no f. The nucleotide 14 from the 5'-end of the 5'-terminal portion does not contain a 2'-fluoro modification no g. The nucleotide 17 from the 5'-end of the 5'-terminal portion does not contain a 2'-fluoro modification no, and / or h. The nucleotide 18 from the 5'-end of the 5'-terminal portion does not contain a 2'-fluoro modification no, which is the gRNA according to any one of Embodiments 205 to 264 Embodiment 266 is a. The nucleotide 6 from the 5'-end of the 5'-terminal portion does not contain a 2'-fluoro modification , b. The nucleotide 7 from the 5'-end of the 5'-terminal portion does not contain a 2'-fluoro modification , c. The nucleotide 8 from the 5'-end of the 5'-terminal portion does not contain a 2'-fluoro modification , d. The nucleotide 9 from the 5'-end of the 5'-terminal portion does not contain a 2'-fluoro modification , and / or e. The nucleotide 10 from the 5'-end of the 5'-terminal portion does not contain a 2'-fluoro modification no, which is the gRNA according to any one of Embodiments 205 to 265 Embodiment 267 is a. The nucleotide 6 from the 5'-end of the 5'-terminal portion does not contain a phosphorothioate bond no b. The nucleotide 7 from the 5'-end of the 5'-terminal portion does not contain a phosphorothioate bond no c. The nucleotide 8 from the 5'-end of the 5'-terminal portion does not contain a phosphorothioate bond and d. The nucleotide 9 from the 5'-end of the 5'-terminal portion does not contain a phosphorothioate bond and / or e. The nucleotide 10 from the 5'-end of the 5'-terminal portion does not contain a phosphorothioate bond, and is the gRNA according to any one of Embodiments 205 to 266 Embodiment 268 is a. The nucleotide 7 from the 5'-end of the 5'-terminal portion does not contain a 2'-OMe modification b. The nucleotide 8 from the 5'-end of the 5'-terminal portion does not contain a 2'-OMe modification c. The nucleotide 9 from the 5'-end of the 5'-terminal portion does not contain a 2'-OMe modification and / or d. The nucleotide 10 from the 5'-end of the 5'-terminal portion does not contain a 2'-OMe modification and is the gRNA according to any one of Embodiments 205 to 267 Embodiment 269 is the gRNA according to any one of Embodiments 205 to 268, wherein the nucleotide 20 from the 5'-end of the 5'-terminal portion does not contain a 2'-OMe modification Embodiment 270 is the gRNA according to any one of Embodiments 205 to 269, wherein the guide RNA contains a 2'-fluoro modification at any one or more of nucleotides 1 to 11 and 13 to 20 from the 5'-end of the 5'-terminal portion, and the nucleotide 12 from the 5'-end of the 5'-terminal portion does not contain a 2'-fluoro modification Embodiment 271 is the gRNA according to any one of Embodiments 205 to 269, wherein the guide RNA contains a 2'-fluoro modification at any one or more of nucleotides 1 to 20 from the 5'-end of the 5'-terminal portion and a. The nucleotide 11 from the 5'-end of the 5'-terminal portion does not contain a 2'-fluoro modification and b. The nucleotide 12 from the 5'-end of the 5'-terminal portion does not contain a 2'-fluoro modification no c. The nucleotide 13 from the 5'-end of the 5'-terminal portion does not contain a 2'-fluoro modification no d. The nucleotide 14 from the 5'-end of the 5'-terminal portion does not contain a 2'-fluoro modification no e. The nucleotide 17 from the 5'-end of the 5'-terminal portion does not contain a 2'-fluoro modification no, and / or f. The nucleotide 18 from the 5'-end of the 5'-terminal portion does not contain a 2'-fluoro modification no, and is the gRNA according to any one of Embodiments 205 to 270. Embodiment 272 is a. B2 does not contain a 2'-OMe modification b. B3 does not contain a 2'-OMe modification c. B4 does not contain a 2'-OMe modification, and / or d. B5 does not contain a 2'-OMe modification, and is the gRNA according to any one of Embodiments 205 to 271 described therein. Embodiment 273 is a. LS1 does not contain a 2'-OMe modification b. LS8 does not contain a 2'-OMe modification, and / or c. LS10 does not contain a 2'-OMe modification, and is the gRNA according to any one of Embodiments 205 to 272 described therein. Embodiment 274 is a. N2 does not contain a 2'-OMe modification b. N3 does not contain a 2'-OMe modification c. N4 does not contain a 2'-OMe modification d. N5 does not contain a 2'-OMe modification e. N6 does not contain a 2'-OMe modification f. N7 does not contain a 2'-OMe modification g. N10 does not contain a 2'-OMe modification h. N11 does not contain a 2'-OMe modification, i. N16 does not contain a 2'-OMe modification, and / or j. N17 does not contain a 2'-OMe modification, any one of Embodiments 205 to 273 is the gRNA described in item 1. Embodiment 275 is a. H1-2 does not contain a phosphorothioate bond, b. H1-3 does not contain a phosphorothioate bond, c. H1-4 does not contain a phosphorothioate bond, d. H1-5 does not contain a phosphorothioate bond, e. H1-6 does not contain a phosphorothioate bond, f. H1-7 does not contain a phosphorothioate bond, g. H1-8 does not contain a phosphorothioate bond, h. H1-9 does not contain a phosphorothioate bond, i. H1-10 does not contain a phosphorothioate bond, j. H2-1 does not contain a phosphorothioate bond, k. H2-2 does not contain a phosphorothioate bond, l. H2-3 does not contain a phosphorothioate bond, m. H2-4 does not contain a phosphorothioate bond, n. H2-5 does not contain a phosphorothioate bond, o. H2-6 does not contain a phosphorothioate bond, p. H2-7 does not contain a phosphorothioate bond, q. H2-8 does not contain a phosphorothioate bond, r. H2-9 does not contain a phosphorothioate bond, s. H2-10 does not contain a phosphorothioate bond, t. H2-11 does not contain a phosphorothioate bond, u. H2-12 does not contain a phosphorothioate bond, v. H2-13 does not contain phosphorothioate linkages, w. H2-14 does not contain phosphorothioate linkages, and / or x. H2-15 does not contain phosphorothioate linkages, of Embodiments 205-274 is the gRNA described in any one of them. Embodiment 276 is a. Optionally PS-modified, nucleotides 6- 10 from the 5'-end of the 5'-terminal portion, b. Optionally 2'-fluoro-modified, nucleotides from the 5'-end of the 5'-terminal portion 8-11, 13, 14, 17, and 18, and c. Optionally 2'-OMe modification, or the conserved region YA site 1 or 8 , is a sgRNA including the modifications in H1-1 and H2-1, is a gRNA. Embodiment 277 is a. Optionally 2'-OMe modified, conserved region YA sites 1, 5, 6, 7, and 9, and b. Optionally 2'-fluoro modified, including the YA modification at conserved region YA site 8 , is a sgRNA, is a gRNA. Embodiment 278 is a gRNA including the YA modification at four guide region YA sites wherein at least one of the YA sites is at or after nucleotide 8 from the 5'-end of the 5'-terminal portion, a. The first YA site includes 2'-OMe modification, b. The second YA site includes 2'-fluoro modification, c. The third YA site includes 2'-fluoro or PS modification, d. The fourth YA site includes PS modification, Optionally, the first, second, third, and fourth YA sites are arranged in the 5' to 3' direction , is a gRNA. ​Embodiment 279 is the gRNA of Embodiment 278, wherein the third YA site contains a PS modification. There is. Embodiment 280 is the gRNA according to Embodiment 278 or 279, wherein the third YA site contains a 2'-fluoro modification. Or there is. Embodiment 281 is the gRNA according to any one of Embodiments 278 to 280, further comprising a fifth YA site containing a PS modification, which is optionally located on the 3' side of the fourth YA site. There is. There is. Embodiment 282 is the gRNA according to any one of Embodiments 205 to 281, wherein the conserved region YA sites 1, 5, 6, 7, and 9 contain a YA modification that is optionally a 2'-OMe modification, and the conserved region YA site 8 contains a modification that is optionally a 2'-fluoro modification. There is. There is. There is. Embodiment 283 is a. Nucleotide 4 from the 5' end of the 5' end, wherein the YA modification is optionally a 2'-OMe modification, nucleotide 4, b. Nucleotides 6 to 10 from the 5' end of the 5' end, which is optionally a PS modification, 10, c. Nucleotides 8 to 11, 13, 14, 17, and 18 from the 5' end of the 5' end, which is optionally a 2'-fluoro modification, d. LS5, LS7, LS9, and L S11, which is optionally a 2'-fluoro modification, e. LS8, LS10, and LS12, which are optionally 2'-OMe modifications, f. N2, N3, N4, N5, N6, N7 N10, N11, N16, and N17, which are optionally 2'-OMe modifications, and g. An sgRNA containing a YA modification at N14, which is optionally a 2'-fluoro modification, There is. Embodiment 284 is a. The nucleotide 4 from the 5'-end of the 5'-terminal part contains a 2'-OMe modification, b. The nucleotides 6 - 10 from the 5'-end of the 5'-terminal part contain a PS modification, c. The nucleotides 8 - 11, 13, 14, 17, and 18 from the 5'-end of the 5'-terminal part contain a 2'-fluoro modification, d. LS5, LS7, LS9, and LS11 contain a 2'-fluoro modification, e. LS8, LS10, and LS12 contain a 2'-OMe modification, f. N2, N3, N4, N5, N6, N7, N10, N11, N16, and N17 contain a 2'-OMe modification, and g. N14 contains a 2'-fluoro modification, and one or more of the above apply to Embodiment 1 61~ is the gRNA described in any one of them. Embodiment 285 is the gRNA according to any one of Embodiments 161 - 284, wherein at least one YA modification contains a modification at the pyrimidine position of the YA site Embodiment 286 is the gRNA according to any one of Embodiments 161 - 285, wherein at least one YA modification contains a modification at the adenine position of the YA site Embodiment 287 is the gRNA according to any one of Embodiments 161 - 286, wherein at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 YA sites contain a YA modification at the pyrimidine position of the YA site Embodiment 288 is the gRNA according to any one of Embodiments 161 - 287, wherein at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 YA sites contain a YA modification at the adenine position of the YA site Embodiment 289 is the gRNA according to any one of the embodiments, wherein at least one YA modification contains a 2'-OMe modification ​​​​It is the gRNA according to any one of 161 to 288. Embodiment 290 is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 YA sites, and is the gR NA according to any one of Embodiments 161 to 289, which contains 2'-OMe modification. Embodiment 291 is the gRNA according to any one of Embodiments 161 to 290, wherein each modified conserved region YA site contains a modification at the pyrimidine position of the YA site. Embodiment 292 is the gRNA according to any one of Embodiments 161 to 291, wherein each modified guide region YA site, or each modified conserved region and guide region YA site, contains a modification at the pyrimidine position of the YA site. Embodiment 293 is the gRNA according to any one of Embodiments 161 to 292, wherein each modified conserved region YA site contains a modification at the adenine position of the YA site. Embodiment 294 is the gRNA according to any one of Embodiments 161 to 293, wherein each modified guide region YA site, or each modified conserved region and guide region YA site, contains a modification at the adenine position of the YA site. Embodiment 295 is the sgRNA containing the modification at LS5, and is the gRNA according to any one of Embodiments 161 to 2 94. Embodiment 296 is the sgRNA containing the modification at LS7, and is the gRNA according to any one of Embodiments 161 to 2 95. Embodiment 297 is the sgRNA containing the modification at LS9, and optionally, when LS9 is modified and LS5, LS7, and LS12 are unmodified, the modification of LS9 is other than 2'-fluoro, and is the gRNA according to any one of Embodiments 161 to 296. Embodiment 298 is an sgRNA comprising a modification in LS12, and optionally, when LS12 is modified and LS9 is unmodified, the modification of LS12 is other than 2'-OMe and is the gRNA according to any one of Embodiments 161 to 297. Embodiment 299 is an sgRNA comprising at least one YA modification that stabilizes the secondary structure, and optionally, the secondary structure is the lower stem, and is the gRNA according to any one of Embodiments 161 to 298. Embodiment 300 is an sgRNA comprising at least one modification of at least one of LS8 and / or LS11, and optionally, the modification of LS8 and / or LS11 stabilizes the secondary structure, and is the gRNA according to any one of Embodiments 161 to 299. Embodiment 301 is a. ENA, b. LNA, or c. an sgRNA comprising a YA modification that stabilizes the secondary structure selected from bicyclic ribose modifications, and is the gRNA according to any one of Embodiments 161 to 300. Embodiment 302 is an sgRNA comprising a modification at N6, and is the gRNA according to any one of Embodiments 161 to 301. Embodiment 303 is an sgRNA comprising a modification at N14, and is the gRNA according to any one of Embodiments 161 to 302. Embodiment 304 is an sgRNA comprising a modification at N17, and optionally, when N17 is modified and N6 and N14 are unmodified, the modification of N17 is other than 2'-fluoro and other than 2'-OMe, and is the gRNA according to any one of Embodiments 161 to 303. Embodiment 305 is at least one of nucleotides 1 to 3 from the 5'-end of the 5'-terminal portion, 2, or 3, contain deoxyribonucleotides, and optionally, nucleotides 1 to 3 from the 5' end of the 5' end contain PS modifications, any one of Embodiments 161 to 304 is the gRNA described in any one of them. Embodiment 306 is such that the gRNA is an sgRNA, and at least 1, 2, or 3 of nucleotides 1 to 3 from the 3' end of the 3' end contain deoxyribonucleotides, and optionally, nucleotides 2 to 3 from the 3' end of the 3' end contain PS modifications, any one of Embodiments 161 to 305 is the gRNA described in any one of them. Embodiment 307 is such that the gRNA is an sgRNA, nucleotide 4 from the 3' end of the 3' end contains a PS modification, and optionally, nucleotide 4 from the 3' end of the 3' end contains a 2'-OMe modification, any one of Embodiments 161 to 306 is the gRNA described in any one of them. Embodiment 308 is such that the gRNA is an sgRNA, hairpin 2 contains deoxyribonucleotides, and optionally, all or almost all of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of hairpin 1 and hairpin 2 are deoxyribonucleotides, any one of Embodiments 161 to 307 is the gRNA described in any one of them. Embodiment 309 is such that the gRNA is an sgRNA, hairpin 1 and hairpin 2 contain deoxyribonucleotides, and optionally, all or almost all of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 nucleotides of hairpin 1 and hairpin 2 are deoxyribonucleotides, any one of Embodiments 161 to 308 is the gRNA described in any one of them. ​​​​​​​​​​Embodiment 310 is such that the gRNA is an sgRNA, and from the start of hairpin 1 to the 3' end of the sgRNA all or almost all of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 nucleotides are deoxyribonucleotides, and optionally, nucleotides 1 to 3 from the 3' end of the 3' end portion are deoxyribonucleotides which is the gRNA according to any one of Embodiments 161 to 309. Embodiment 311 is such that the gRNA is an sgRNA, and the upper stem contains deoxyribonucle otides, which is the gRNA according to any one of Embodiments 161 to 310. Embodiment 312 is such that the gRNA is an sgRNA, and all or almost all of 1, 2, 3, 4, 5, 6, 7, 8 , 9, or 10 nucleotides of the upper stem are deoxyribonucle otides, which is the gRNA according to any one of Embodiments 161 to 311. Embodiment 313 is such that at least 1, 2, or 3 of nucleotides 1 to 3 from the 5' end of the 5' end portion contain ENA, and optionally, nucleotides 1 to 3 from the 5' end of the 5' end portion contain PS modification, which is the gRNA according to any one of Embodiments 161 to 312. NA. Embodiment 314 is such that the gRNA is an sgRNA, and at least 1, 2, or 3 of nucleotides 2 to 4 from the 3' end of the 3' end portion contain ENA, and optionally, nucleotides 2 to 3 from the 3' end of the 3' end portion contain PS modification, which is the gRNA according to any one of Embodiments 161 to 3 13. Embodiment 315 is such that at least 1, 2, or 3 of nucleotides 1 to 3 from the 5' end of the 5' end portion contain UNA, and optionally, nucleotides 1 to 3 from the 5' end of the 5' end portion The oligonucleotides 1-3 are the gR described in any one of Embodiments 161-314, including PS modification. NA. Embodiment 316 is such that the gRNA is an sgRNA, and at least 1, 2, or 3 of nucleotides 2-4 from the 3'-end of the 3'-terminal portion contain UNA, and optionally, nucleotides 2-3 from the 3'-end of the 3'-terminal portion contain PS modification, and it is the gRNA described in any one of Embodiments 161-315. Embodiment 317 is such that the gRNA is an sgRNA, and nucleotide 4 from the 3'-end of the 3'-terminal portion contains PS modification, and optionally, nucleotide 4 from the 3'-end of the 3'-terminal portion contains 2'-OMe modification, and it is the gRNA described in any one of Embodiments 161-316. RNA. Embodiment 318 is such that the gRNA is an sgRNA containing 3'-terminal modification, and it is the gRNA described in any one of Embodiments 161-317. Embodiment 319 is an sgRNA containing 3'-terminal modification, and the 3'-terminal modification is a protected 3'-terminal modification, and it is the gRNA described in any one of Embodiments 161-318. Embodiment 320 is such that the gRNA is an sgRNA containing a 3'-tail, and it is the gRNA described in any one of Embodiments 161-319. Embodiment 321 is such that the 3'-tail contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, and it is the gRNA described in Embodiment 320. Embodiment 322 is such that the 3'-tail contains about 1-2, 1-3, 1-4, 1-5, 1-7, 1-10, at least 1-5, at least 1-3, at least 1-4, at least 1-5, at least 1-5, at least 1-7, or at least 1-10 nucleotides, and it is the gRNA described in Embodiment 320. ​​​​​​​​​​​​ Embodiment 323 is the sgRNA according to Embodiment 16, which includes a modification in the hairpin region and is the gRNA according to any one of 1 to 322. Embodiment 324 is an sgRNA including a 3'-end modification and a modification in the hairpin region and is the gRNA according to any one of Embodiments 161 to 323. Embodiment 325 is the gRNA according to Embodiment 323 or 324, wherein the modification in the hairpin region is a modified nucleotide selected from a 2'-O-methyl (2'-O-Me )-modified nucleotide, a 2'-fluoro (2'-F)-modified nucleotide, or a combination thereof and is the gRNA according to Embodiment 323 or 324, including a modified nucleotide selected from a 2'-O-methyl (2'-O-Me )-modified nucleotide, a 2'-fluoro (2'-F)-modified nucleotide, or a combination thereof. Embodiment 326 is the gRNA according to any one of Embodiments 323 to 325, wherein the modification in the hairpin region includes or further includes a 2'-O-methyl (2'-O-Me )-modified nucleotide and is the gRNA according to any one of Embodiments 323 to 325, including or further including a 2'-O-methyl (2'-O-Me )-modified nucleotide. Embodiment 327 is the gRNA according to any one of Embodiments 323 to 326, wherein the modification in the hairpin region includes or further includes a 2'-fluoro (2'-F)-modified nucleotide and is the gRNA according to any one of Embodiments 323 to 326, including or further including a 2'-fluoro (2'-F)-modified nucleotide. Embodiment 328 is the gRNA according to any one of Embodiments 161 to 327, including a 3'- and / or 5'-protected end modification(s) and is the gRNA according to any one of Embodiments 161 to 327, including a 3'- and / or 5'-protected end modification(s). Embodiment 329 is the gRNA according to Embodiment 328, wherein the 3'- and / or 5'-end modification is a modified nucleotide selected from a 2'-O-methyl (2'-O -Me)-modified nucleotide, a 2'-O-(2-methoxyethyl) (2'-O-moe)-modified nucleotide, a 2'-fluoro (2'-F)-modified nucleotide, an internucleotide phosphorothioate (PS) bond, an inverted deoxyabasic modified nucleotide, or a combination thereof and is the gRNA according to Embodiment 328, including a modified nucleotide selected from a 2'-O-methyl (2'-O -Me)-modified nucleotide, a 2'-O-(2-methoxyethyl) (2'-O-moe)-modified nucleotide, a 2'-fluoro (2'-F)-modified nucleotide, an internucleotide phosphorothioate (PS) bond, an inverted deoxyabasic modified nucleotide, or a combination thereof. and is the gRNA according to Embodiment 328, including a modified nucleotide selected from a 2'-O-methyl (2'-O Embodiment 330 is the gRNA according to Embodiment 328 or 329, wherein the 3' and / or 5' end modification contains or further contains a 2'-O-methyl (2'-O -Me) modified nucleotide. Embodiment 331 is the gRNA according to Embodiment 328 or 329, wherein the 3' and / or 5' end modification contains or further contains a 2'-fluoro (2'-F) modified nucleotide. Embodiment 332 is the gRNA according to Embodiment 328 or 329, wherein the 3' and / or 5' end modification contains or further contains an internucleotide phosphorothioate (PS) bond. Embodiment 333 is the gRNA according to Embodiment 328 or 329, wherein the 3' and / or 5' end modification contains or further contains an inverted abasic modified nucleotide . Embodiment 334 is the gRNA, wherein the gRNA is a sgRNA, and when the sgRNA contains a 3' end modification, the 3' end modification is as follows: i. One or more modifications of any one of the last 7, 6, 5, 4, 3, 2, or 1 nucleotides ii. One modified nucleotide iii. Two modified nucleotides iv. Three modified nucleotides v. Four modified nucleotides vi. Five modified nucleotides, and vii. Six modified nucleotides, and viii. One or more of any one of seven modified nucleotides, which is the gRNA according to any one of Embodiments 16 1 to 333. Embodiment 335 is the gRNA according to Embodiment 334, wherein the 3' end modification contains a modification of 1 to 7, 1 to 5, 1 to 4, or 2 to 4 nucleotides. Embodiment 336 is such that the gRNA is an sgRNA containing a 3'-end modification, and the 3'-end modification is as follows: i. internucleotide phosphorothioate (PS) bond, ii. 2'-O-Me modified nucleotide, iii. 2'-O-moe modified nucleotide, iv. 2'-F modified nucleotide, v. inverted abasic modified nucleotide vi. ENA, UNA, and / or DNA, and vii. or any one or more of combinations thereof, and is the gRNA according to any one of Embodiments 16 to 335. Embodiment 337 is such that the gRNA is an sgRNA containing a 3'-tail, and the 3'-tail is i. internucleotide phosphorothioate (PS) bond, ii. 2'-O-Me modified nucleotide, iii. 2'-O-moe modified nucleotide, iv. 2'-F modified nucleotide, v. inverted abasic modified nucleotide vi. ENA, UNA, and / or DNA, and vii. or any one or more of combinations thereof, and is the gRNA according to any one of Embodiments 16 to 336. Embodiment 338 is such that the 3'-end modification is i. 1, 2, 3, 4, 5, 6, or 7 internucleotide PS bonds, ii. about 1 to 3, 1 to 5, 1 to 6, or 1 to 7 internucleotide PS bonds, or or iii. contains PS bonds between each nucleotide, and is the gRNA according to Embodiment 336 thereof. Embodiment 339 is such that the 3'-end modification is at least one 2'-O-Me, 2'-O-m oe, inverted abasic, or 2'-F modified nucleotides. The gRNA is any one of the gRNAs listed in any one of claims 1 to 328. Embodiment 340 is an embodiment in which the 3' end modification comprises at least one PS bond; i. There is one PS bond, and the bond is between the last nucleotide and the penultimate nucleotide. Between the Ochid and the ii. there are two PS bonds between the last three nucleotides; iii. There is a PS bond between any one or more of the last four nucleotides do, iv. There is a PS bond between any one or more of the last five nucleotides. or v. of the last 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides Any one of embodiments 326 to 329, wherein a PS bond is present between any one or more of The gRNA is described in Embodiment 341 is an embodiment in which the 3' end modification is i. one or more modifications of the last 1 to 7 nucleotides, Bonds, inverted abasic nucleotides, 2'-O-Me, 2'-O-moe, 2'-F, or combinations thereof, ii. 2'-O-Me, 2'-O-moe, 2'-F, or a combination thereof Modification of the last nucleotide by, and the adjacent nucleotide and / or the 3' nucleotide by optional one or two PS bonds to the first nucleotide of the sequence; iii. 2'-O-Me, 2'-O-moe, 2'-F, or a combination thereof Modification of the last and / or penultimate nucleotide by nucleotide substitution, and optionally One or more PS bonds, iv. 2'-O-Me, 2'-O-moe, 2'-F, or a combination thereof modification of the last, second to last, and / or third to last nucleotide by and optionally one or more PS bonds. v. 2'-O-Me, 2'-O-moe, 2'-F, or a combination thereof The last, second to last, third to last, and / or fourth to last modification of the oxidase and, optionally, one or more PS bonds, or vi. 2'-O-Me, 2'-O-moe, 2'-F, or a combination thereof last, second to last, third to last, fourth to last, and / or last by and optionally one or more PS linkages. , a gRNA described in any one of embodiments 336 to 340. Embodiment 342 is an embodiment in which the gRNA is an sgRNA comprising a 3' tail, the 3' tail comprising: 3. The method of claim 1, further comprising the steps of: The gRNA is any one of 61 to 341. Embodiment 343 is the gamma nucleotide sequence of embodiment 342, wherein the 3' tail is fully modified. It is RNA. In embodiment 344, the 3' tail is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 to 2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, or 1-10 nucleotides, and optionally any one or more of these nucleotides The gRNA of embodiment 342, wherein the gRNA is modified. Embodiment 345 is directed to a method for the preparation of a 3' end modification comprising the steps of: i. A 3'-end modification as set forth in any one of SEQ ID NOs: 401-532; ii. (i) At the last nucleotide of the conserved region of the sgRNA or short sgRNA a 2’O-Me modified nucleotide, (ii) three consecutive 2’O-moe modified nucleotides immediately 5’ to the 2’O-Me modified nucleotide, and (iii) three consecutive PS bonds between the last three nucleotides, iii. (i) Five consecutive 2’O-Me modified nucleotides from the 3’ end of the 3’ end region, and (ii) three PS bonds between the last three nucleotides, iv. An inverted abasic modified nucleotide at the last nucleotide of the conserved region of the sgRNA or short sgRNA, v. (i) An inverted abasic modified nucleotide at the last nucleotide of the conserved region of the sgRNA or short sgRNA, and (ii) three consecutive 2’O-Me modified nucleotides at the last three nucleotides of the conserved region of the sgRNA or short sgRNA, vi. (i) Fifteen consecutive 2’O-Me modified nucleotides from the 3’ end of the 3’ end region, (ii) five consecutive 2’-F modified nucleotides immediately 5’ to the 2’O-Me modified nucleotide, and (iii) three PS bonds between the last three nucleotides, vii. (i) Alternating 2’O-Me modified nucleotides and 2’-F modified nucleotides in the last 20 nucleotides of the conserved region of the sgRNA or short sgRNA, and (ii) three PS bonds between the last three nucleotides, viii. (i) Two or three consecutive 2’O-Me modified nucleotides, and (ii) three PS bonds between the last three nucleotides, ix. One PS bond between the last nucleotide and the nucleotide adjacent to the last one, and ​​​​​x.15 or 20 consecutive 2’O-Me modified nucleotides, and (ii) any one or more of the 3 PS linkages among the last 3 nucleotides An embodiment comprising The gRNA according to any one of 336 to 344 Embodiment 346 is as follows: i. Modification of any one or more of nucleotides 1 to 7 in the guide region ii. One modified nucleotide iii. Two modified nucleotides iv. Three modified nucleotides v. Four modified nucleotides vi. Five modified nucleotides vii. Six modified nucleotides, and viii. A 5'-end modification comprising any one or more of seven modified nucleotides The gRNA according to any one of Embodiments 161 to 345 Embodiment 347 comprises a 5'-end modification, and the 5'-end modification is a protecting 5'-end modification The gRNA according to any one of Embodiments 161 to 346 Embodiment 348 comprises a 5'-end modification, and the 5'-end modification comprises modifications of 1 to 7, 1 to 5, 1 to 4 1 to 3, or 1 to 2 nucleotides The gRNA according to any one of Embodiments 161 to 347 Embodiment 349 comprises a 5'-end modification, and the 5'-end modification is as follows: i. Modifications of 1, 2, 3, 4, 5, 6, or 7 of the first 7 nucleotides , ii. Approximately 1 to 3, 1 to 4, 1 to 5, 1 to 6 modifications of 1 to 7 of the first 7 nucleotides, and iii. Modifications at the first, second, third, fourth, fifth, sixth, and / or seventh nucleotides at the 5'-end, optionally with the modifications being consecutive is any one of Embodiments 161 to 348, including one or more of the modifications the gRNA described. Embodiment 350 includes a 5'-end modification, and the 5'-end modification is i. internucleotide phosphorothioate (PS) bond, ii. 2'-O-Me modified nucleotide, iii. 2'-O-moe modified nucleotide, iv. 2'-F modified nucleotide, v. inverted abasic modified nucleotide vi. ENA, UNA, and / or DNA, and vii. is the gRNA described in any one of Embodiments 161 to 3 49, including one or more of these combinations. Embodiment 351 includes a 5'-end modification, and the 5'-end modification is i. 1, 2, 3, 4, 5, 6, and / or 7 internucleotide PS bonds, or or ii. about 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, or 1 to 7 nucleotide is the gRNA described in any one of Embodiments 161 to 350, including PS bonds between them. Embodiment 352 is such that the sgRNA includes a 5'-end modification, and the 5'-end modification is at least one 2'-O-Me, 2'-O-moe, inverted abasic, 2'-H, inosine, or also includes a 2'-F modified nucleotide, and is the g RNA described in any one of Embodiments 161 to 351. Embodiment 353 is such that the 5'-end modification includes at least one PS bond, i. one PS bond is present, and the bond is at nucleotide 1 of the guide region, ii. two PS bonds are present, and the bonds are at nucleotides 1 and 2 of the guide region are iii. to any one or more of nucleotides 1, 2, and 3 of the guide region P wherein there is an S bond iv. in any one or more of nucleotides 1, 2, 3, and 4 of the guide region wherein there is a PS bond v. any one or more of nucleotides 1, 2, 3, 4, and 5 of the guide region wherein there is a PS bond vi. in any one or more of nucleotides 1, 2, 3, 4, 5, and 6 of the guide region, or vii. in any one or more of nucleotides 1, 2, 3, 4, 5, 6, and 7 of the guide region, the gRNA according to Embodiment 352, wherein there is a PS bond Embodiment 354 is such that the 5'-end modification is i. a modification of one or more of nucleotides 1 to 7 in the variable region, the modification being a PS bond, a reversed abasic nucleotide, 2'-O-Me, 2'-O-moe, 2'-F, 2 '-H, inosine, and / or a combination thereof ii. an optional PS bond to the first nucleotide of the guide region and the adjacent nucleotide by 2'-O-Me, 2'-O-moe, 2'-F, 2'-H, inosine, or a combination thereof iii. a modification of the first and / or second nucleotide of the variable region by 2'-O-Me, 2'-O-moe, 2'-F, 2'-H, inosine, or a combination thereof, and optionally, one or more PS bonds iv. a modification of the first, second, and / or third nucleotide of the variable region by 2'-O-Me, 2'-O-moe, 2'-F, 2'-H, inosine, or a combination thereof, and optionally, one or more PS bonds v. 2'-O-Me, 2'-O-moe, 2'-F, 2'-H, inosine, or a combination thereof modification of the first, second, third, and / or fourth nucleotide of the variable region by a combination thereof, and optionally, one or more PS bonds vi. 2'-O-Me, 2'-O-moe, 2'-F, 2'-H, inosine, or a combination thereof modification of the first, second, third, fourth, and / or fifth nucleotide of the variable region by a combination thereof, and optionally, one or more PS bonds v. 2'-O-Me, 2'-O-moe, 2'-F, 2'-H, inosine, or modification of the first, second, third, and / or fourth nucleotides of the variable region by combinations thereof, and optionally, one or more PS linkages, or vi. 2'-O-Me, 2'-O-moe, 2'-F, 2'-H, inosine, or modification of the first, second, third, fourth, and / or fifth nucleotides of the variable region by combinations thereof, and optionally, one or more PS linkages, and is the gRNA described in Embodiment 352 or 353. is the gRNA described in Embodiment 352 or 353. Embodiment 355 includes a 5' end modification, and the 5' end modification is as follows: i. a 5' end modification shown in any one of SEQ ID NOs: 1-54, 401-532, 1001, 1007-1132, 1205 ~1212, 1322~1406, 1417~1501, 1511~1596, 3018 ~3059, 3063~3104, 3108~3149, 3153~3194, 3198 ~3239, 3243~3284, 3295~3341, 3343~3385, 3388 ~3430, or 3549~3552; ii. 2'-OMe modified nucleotides at nucleotides 1, 2, and 3 of the guide region; iii. 2'-OMe modified nucleotides at nucleotides 1, 2, and 3 of the guide region, and PS linkages between nucleotides 1 and 2, 2 and 3, and 3 and 4 of the guide region; iv. 2'-OMe modified nucleotides at 1, 2, 3, 4, and 5 of the guide region; v. 2'-OMe modified nucleotides at 1, 2, 3, 4, and nucleotide 5 of the guide region, and PS linkages between nucleotides 1 and 2, 2 and 3, and 3 and 4 of the guide region. iii. 2'-OMe modified nucleotides at nucleotides 1, 2, and 3 of the guide region, and PS linkages between nucleotides 1 and 2, 2 and 3, and 3 and 4 of the guide region; iii. 2'-OMe modified nucleotides at nucleotides 1, 2, and 3 of the guide region, and PS linkages between nucleotides 1 and 2, 2 and 3, and 3 and 4 of the guide region; iii. 2'-OMe modified nucleotides at nucleotides 1, 2, and 3 of the guide region, and PS linkages between nucleotides 1 and 2, 2 and 3, and 3 and 4 of the guide region; iv. 2'-OMe modified nucleotides at 1, 2, 3, 4, and 5 of the guide region; v. 2'-OMe modified nucleotides at 1, 2, 3, 4, and nucleotide 5 of the guide region, and PS linkages between nucleotides 1 and 2, 2 and 3, and 3 and 4 of the guide region. v. 2'-OMe modified nucleotides at 1, 2, 3, 4, and nucleotide 5 of the guide region, and PS linkages between nucleotides 1 and 2, 2 and 3, and 3 and 4 of the guide region. v. 2'-OMe modified nucleotides at 1, 2, 3, 4, and nucleotide 5 of the guide region, and PS linkages between nucleotides 1 and 2, 2 and 3, and 3 and 4 of the guide region. PS linkages between it and 4 and 5, and between 5 and 6, vi. 2’O-moe modified nucleotides at nucleotides 1, 2, and 3 of the guide region nucleotides, vii. 2’O-moe modified nucleotides at nucleotides 1, 2, and 3 of the guide region nucleotides, and PS linkages between nucleotides 1 and 2, 2 and 3, and 3 and 4 of the guide region, viii. Inverted abasic modified nucleotide at nucleotide 1 of the guide region, ix. Inverted abasic modified nucleotide at nucleotide 1 of the guide region, and also 2’-OMe modified nucleotides at nucleotides 1, 2, and 3 of the guide region , x. Inverted abasic modified nucleotide at nucleotide 1 of the guide region, guide region 2’-OMe modified nucleotides at nucleotides 1, 2, and 3, and PS linkages between nucleotides 1 and 2, 2 and 3, 3 and 4, 4 and 5, and 5 and 6 of the variable region, including any one or more of them, Embodiments 161 - 354 The gRNA according to any one of. Embodiment 356 is the gRNA according to any one of Embodiments 161 - 355, wherein the gRNA is a sgRNA and the upper stem region contains at least 1 modification. Embodiment 357 is that the upper stem modification is as follows: i. Modification of any one or more of US1 - US12 in the upper stem region, ii. Modification of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 0, 11, or all 12 nucleotides in the upper stem region, and iii. About 1 - 2, 1 - 3, 1 - 4, 1 - 5, 1 - 6, 1 - 7, any one or more of the modifications of 1 to 8, 1 to 9, 1 to 10, or 1 to 12 nucleotides including the gRNA described in Embodiment 346. Embodiment 358, where the upper stem modification is i. 2'-O-Me modified nucleotide, ii. 2'-H modified nucleotide, iii. 2'-F modified nucleotide, and iv. any one or more of their combinations, is the gRNA described in Embodiment 356 or 357. Embodiment 359, where the gRNA is an sgRNA containing one or more modifications in the hairpin 1 region is the gRNA described in any one of Embodiments 161 to 358. Embodiment 360, where the sgRNA contains a modification in H1-1, is the gRNA described in Embodiment 359. Embodiment 361, where the gRNA is an sgRNA containing one or more modifications in the hairpin 2 region is the gRNA described in any one of Embodiments 161 to 360. Embodiment 362, where the sgRNA contains a modification in H2-1, is the gRNA described in Embodiment 361. Embodiment 363, where the gRNA is an sgRNA containing modifications in H1-1 to H1-12 is the gRNA described in any one of Embodiments 161 to 362. Embodiment 364, where the gRNA is an sgRNA containing modifications in H2-1 to H2-15 is the gRNA described in any one of Embodiments 161 to 363. Embodiment 365, where the gRNA is an sgRNA containing one or more modifications in each of the upper stem region, hairpin 1 region, and hairpin 2 region is the gRNA described in any one of Embodiments 161 to 364. is the gRNA described in any one of Embodiments 161 to 364. Embodiment 366 is the gRNA according to any one of Embodiments 161 to 365, wherein the gRNA contains a modified nucleotide between the hairpin 1 region and the hairpin 2 region. Embodiment 367 is the gRNA according to any one of Embodiments 161 to 366, wherein the sgRNA further contains a lower stem region containing a modification. A. Embodiment 368 is the gRNA according to any one of Embodiments 161 to 367, further comprising a 3'-end modification. Embodiment 369 is the gRNA according to Embodiment 368, wherein at least two of the last four nucleotides at the 3'-end of the 3'-terminal portion are modified. Embodiment 370 is the gRNA according to Embodiment 369, wherein at least two of the last four nucleotides at the 3'-end of the 3'-terminal portion are modified by 2'-O-Me, 2'-F, or 2'-O-moe. Embodiment 371 is the gRNA according to any one of Embodiments 368 to 370, further comprising a phosphorothioate (PS) bond between one or more of the last four nucleotides at the 3'-end of the 3'-terminal portion. Embodiment 372 is the gRNA according to any one of Embodiments 161 to 371, wherein the sgRNA further contains a bulge region containing a modification. Embodiment 373 is the gRNA according to any one of Embodiments 161 to 372, wherein the sgRNA further contains a nexus region containing a modification. Embodiment 374 is the sgRNA containing any one of SEQ ID NOs: 401 to 535, 601, 607 to 732, 801, 807 to 932, 1001, or 1007 to 1132, including the modifications in Table 1. Embodiment 375 is the sgRNA according to any one of Embodiments 161 to 374, further comprising a 5'-end modification. Embodiment 376 is the gRNA according to Embodiment 375, wherein at least two of the first four nucleotides at the 5'-end of the 5'-terminal portion are modified. Embodiment 377 is the gRNA according to Embodiment 376, wherein at least two of the first four nucleotides at the 5'-end of the 5'-terminal portion are modified by 2'-O-Me, 2'-F, or 2'-O-moe. Embodiment 378 is the gRNA according to any one of Embodiments 375 to 377, further comprising a phosphorothioate (PS) bond between one or more of the first four nucleotides at the 5'-end of the 5'-terminal portion. Embodiment 379 is the gRNA according to any one of Embodiments 161 to 378, further comprising a modified nucleotide at the 5'-end of the 5'-terminal portion. Embodiment 380 is the gRNA according to any one of Embodiments 161 to 379, further comprising a modified nucleotide at the 3'-end of the 3'-terminal portion. Embodiment 381 is the gRNA according to any one of Embodiments 161 to 380, further comprising a modified nucleotide at the 5'-end of the 5'-terminal portion and a modified nucleotide at the 3'-end of the 3'-terminal portion. Embodiment 382 is the gRNA according to any one of Embodiments 161 to 381, further comprising a modified nucleotide at the 5'-end of the 5'-terminal portion, a modified nucleotide at the 3'-end of the 3'-terminal portion, and a modified nucleotide between the hairpin 1 region and the hairpin 2 region. Embodiment 383 is the gRNA according to any one of Embodiments 161 to 382, further comprising a modified nucleotide at the 5'-end of the 5'-terminal portion, a modified nucleotide at the 3'-end of the 3'-terminal portion, a modified nucleotide between the hairpin 1 region and the hairpin 2 region, and a lower stem region containing a modification. Embodiment 384 is the sgRNA according to any one of Embodiments 161 to 383, further comprising a 5'-end modification and a 3'-end modification. Embodiment 385 is the gRNA according to Embodiment 384, wherein at least two of the first four nucleotides at the 5'-end of the 5'-terminal portion and at least two of the last four nucleotides at the 3'-end of the 3'-terminal portion are modified. Embodiment 386 is the gRNA according to Embodiment 385, wherein at least two of the first four nucleotides at the 5'-end of the 5'-terminal portion and at least two of the last four nucleotides at the 3'-end of the 3'-terminal portion are modified by 2'-O-Me, 2'-F, or 2'-O-moe. Embodiment 375 is an sgRNA comprising any one nucleic acid of SEQ ID NOs: 401 to 535, 601, 607 to 732, 801, 80 7 to 932, 1001, or 1007 to 1132, and at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75 , or 70% identity nucleic acid, and the modification at each nucleotide of the sgRNA corresponding to the nucleotide of the reference sequence identifier in Table 1 is the same or equivalent to the modification shown in the reference sequence identifier in Table 1, which is an sgRNA. Embodiment 376 is the gRNA according to any one of Embodiments 161 to 375, wherein the modification reduces the degradation of the gRNA without significantly changing the ability of the guide to cleave the target nucleic acid. A. Embodiment 377 is the gRNA according to any one of Embodiments 161 to 376, which comprises a YA modification, and the modification comprises 2'-fluoro, 2'-H, 2'-O- Me, ENA, UNA, or PS. Embodiment 378 is the gRNA according to any one of Embodiments 161 to 377, which comprises a YA modification, and the modification changes the structure of the dinucleotide motif to reduce the RNA endonuclease activity. Embodiment 379 is the gRNA according to any one of Embodiments 161 to 378, which comprises a YA modification, and the modification prevents the recognition or cleavage of the YA site by RNase and / or stabilizes the RNA structure. Embodiment 380 is the gRNA which comprises a YA modification, and the modification is a. a ribose modification selected from 2'-O-alkyl, 2'-F, 2'-moe, 2'-F arabinose, and 2'-H (deoxyribose), b. bicyclic ribose analogs such as LNA, BNA, and ENA, ​​​​​​​c. Locked nucleic acid modification, d. Base modifications such as inosine, pseudouridine, and 5'-methylcytosine, and and e. Include any one or more of internucleoside bond modifications such as phosphorothioate , The gRNA according to any one of Embodiments 161 to 379. Embodiment 381 is a gRNA in which the guide region contains a modification at nucleotide 5, and optionally, the guide region contains 2'-OMe modifications at nucleotides 1 to 4, phosphorothioate modifications at nucleotides 1 to 3 and 6 to 10, and / or 2'-F modifications at nucleotides 8 to 11, 13, 14, 17, and 18, according to any one of Embodiments 1 to 380. and optionally, the guide region contains 2'-OMe modifications at nucleotides 1 to 4, phosphorothioate modifications at nucleotides 1 to 3 and 6 to 10, and / or 2'-F modifications at nucleotides 8 to 11, 13, 14, 17, and 18, according to any one of Embodiments 1 to 380. and / or 2'-F modifications at nucleotides 8 to 11, 13, 14, 17, and 18, according to any one of Embodiments 1 to 380. The gRNA according to any one of Embodiments 1 to 380. Embodiment 382 is a gRNA in which the guide region contains a modification at nucleotide 12, and optionally, the guide region contains 2'-OMe modifications at nucleotides 1 to 4, phosphorothioate modifications at nucleotides 1 to 3 and 6 to 10, and / or 2'-F modifications at nucleotides 8 to 11, 13, 14, 17, and 18, according to any one of Embodiments 1 to 381. and optionally, the guide region contains 2'-OMe modifications at nucleotides 1 to 4, phosphorothioate modifications at nucleotides 1 to 3 and 6 to 10, and / or 2'-F modifications at nucleotides 8 to 11, 13, 14, 17, and 18, according to any one of Embodiments 1 to 381. and / or 2'-F modifications at nucleotides 8 to 11, 13, 14, 17, and 18, according to any one of Embodiments 1 to 381. The gRNA according to any one of Embodiments 1 to 381. Embodiment 383 is a gRNA that contains a guide region with 2'-OMe modifications at nucleotide 5 and / or nucleotide 12, and optionally, the guide region contains 2'-OMe modifications at nucleotides 1 to 4, phosphorothioate modifications at nucleotides 1 to 3 and 6 to 10, and / or 2'-F modifications at nucleotides 8 to 11, 13, 14, 17, and 18, according to any one of Embodiments 1 to 382. The gRNA according to any one of Embodiments 1 to 382. Embodiment 383 is a gRNA that contains a guide region with 2'-OMe modifications at nucleotide 5 and / or nucleotide 12, and optionally, the guide region contains 2'-OMe modifications at nucleotides 1 to 4, phosphorothioate modifications at nucleotides 1 to 3 and 6 to 10, and / or 2'-F modifications at nucleotides 8 to 11, 13, 14, 17, and 18, according to any one of Embodiments 1 to 382. and optionally, the guide region contains 2'-OMe modifications at nucleotides 1 to 4, phosphorothioate modifications at nucleotides 1 to 3 and 6 to 10, and / or 2'-F modifications at nucleotides 8 to 11, 13, 14, 17, and 18, according to any one of Embodiments 1 to 382. and / or 2'-F modifications at nucleotides 8 to 11, 13, 14, 17, and 18, according to any one of Embodiments 1 to 382. The gRNA according to any one of Embodiments 1 to 382. The gRNA according to any one of Embodiments 1 to 382. NA. Embodiment 384 is such that the gRNA contains a guide region including a 2'-F modification at nucleotide 5 and / or nucleotide 12, and optionally, the guide region contains a 2'-OMe modification at nucleotides 1 to 4, a phosphorothioate modification at nucleotides 1 to 3 and 6 to 10, and / or a 2'-F modification at nucleotides 8 to 11, 13, 14, 17, and 18, and is the gRNA according to any one of Embodiments 1 to 383. Embodiment 385 is such that the gRNA contains a guide region including a 2'-H modification at nucleotide 5 and / or nucleotide 12, and optionally, the guide region contains a 2'-OMe modification at nucleotides 1 to 4, a phosphorothioate modification at nucleotides 1 to 3 and 6 to 10, and / or a 2'-F modification at nucleotides 8 to 11, 13, 14, 17, and 18, and is the gRNA according to any one of Embodiments 1 to 384. Embodiment 386 is such that the gRNA contains a guide region including a phosphorothioate modification at the nucleotide at position 5 and / or 12, and optionally, the guide region contains a 2'-OMe modification at nucleotides 1 to 4, a phosphorothioate modification at nucleotides 1 to 3 and 6 to 10, and / or a 2'-F modification at the nucleotides at positions 8 to 11, 13, 14, 17, and 18, and is the gRNA according to any one of Embodiments 1 to 385. Embodiment 387 is such that the gRNA is i. nucleotides 8 to 10, ii. nucleotides 8 and 9, iii. nucleotides 8 and 10, or Embodiment 384 is such that the gRNA contains a guide region including a 2'-F modification at nucleotide 5 and / or nucleotide 12, and optionally, the guide region contains a 2'-OMe modification at nucleotides 1 to 4, a phosphorothioate modification at nucleotides 1 to 3 and 6 to 10, and / or a 2'-F modification at nucleotides 8 to 11, 13, 14, 17, and 18, and is the gRNA according to any one of Embodiments 1 to 383. Embodiment 385 is such that the gRNA contains a guide region including a 2'-H modification at nucleotide 5 and / or nucleotide 12, and optionally, the guide region contains a 2'-OMe modification at nucleotides 1 to 4, a phosphorothioate modification at nucleotides 1 to 3 and 6 to 10, and / or a 2'-F modification at nucleotides 8 to 11, 13, 14, 17, and 18, and is the gRNA according to any one of Embodiments 1 to 384. Embodiment 386 is such that the gRNA contains a guide region including a phosphorothioate modification at the nucleotide at position 5 and / or 12, and optionally, the guide region contains a 2'-OMe modification at nucleotides 1 to 4, a phosphorothioate modification at nucleotides 1 to 3 and 6 to 10, and / or a 2'-F modification at the nucleotides at positions 8 to 11, 13, 14, 17, and 18, and is the gRNA according to any one of Embodiments 1 to 385. Embodiment 387 is such that the gRNA is i. nucleotides 8 to 10, ii. nucleotides 8 and 9, iii. nucleotides 8 and 10, or Embodiment 384 is such that the gRNA contains a guide region including a 2'-F modification at nucleotide 5 and / or nucleotide 12, and optionally, the guide region contains a 2'-OMe modification at nucleotides 1 to 4, a phosphorothioate modification at nucleotides 1 to 3 and 6 to 10, and / or a 2'-F modification at nucleotides 8 to 11, 13, 14, 17, and 18, and is the gRNA according to any one of Embodiments 1 to 383. Embodiment 385 is such that the gRNA contains a guide region including a 2'-H modification at nucleotide 5 and / or nucleotide 12, and optionally, the guide region contains a 2'-OMe modification at nucleotides 1 to 4, a phosphorothioate modification at nucleotides 1 to 3 and 6 to 10, and / or a 2'-F modification at nucleotides 8 to 11, 13, 14, 17, and 18, and is the gRNA according to any one of Embodiments 1 to 384. Embodiment 386 is such that the gRNA contains a guide region including a phosphorothioate modification at the nucleotide at position 5 and / or 12, and optionally, the guide region contains a 2'-OMe modification at nucleotides 1 to 4, a phosphorothioate modification at nucleotides 1 to 3 and 6 to 10, and / or a 2'-F modification at the nucleotides at positions 8 to 11, 13, 14, 17, and 18, and is the gRNA according to any one of Embodiments 1 to 385. Embodiment 387 is such that the gRNA is Embodiment 387 is such that the gRNA is i. nucleotides 8 to 10, ii. nucleotides 8 and 9, iii. nucleotides 8 and 10, or iv. Comprising a guide region containing modifications at nucleotides 9 and 10, Optionally, the guide region comprises 2'-OMe modifications at nucleotides 1-4, the guide region comprises phosphorothioate modifications at nucleotides 1-3 and 6-7, and / or the guide region comprises 2'-F modifications at nucleotides 11, 13, 14, 17, and 18, and is a gRNA according to any one of Embodiments 1-3 86. Embodiment 388 is such that the gRNA i. nucleotides 8-10, ii. nucleotides 8 and 9, iii. nucleotides 8 and 10, iv. nucleotides 9 and 10, or v. comprises a guide region containing a 2'-F modification at nucleotide 8, Optionally, the guide region comprises 2'-OMe modifications at nucleotides 1-4, the guide region comprises phosphorothioate modifications at nucleotides 1-3 and 6-7, and / or the guide region comprises 2'-F modifications at nucleotides 11, 13, 14, 17, and 18, and is a gRNA according to any one of Embodiments 1-3 87. Embodiment 389 is such that the gRNA i. nucleotides 8-10, ii. nucleotides 8 and 9, iii. nucleotides 8 and 10, iv. nucleotides 9 and 10, or v. comprises a guide region containing a 2'-F modification at nucleotide 8, wherein nucleotides 8-10 do not comprise phosphorothioate modifications, and optionally, the guide region comprises 2'-OMe modifications at nucleotides 1-4, phosphorothioate modifications at nucleotides 1-3 and 6-7 and / or 2'-F modifications at nucleotides 11, 13, 14, 1 7. and the 2'-F modification at 18, and is the gRNA according to any one of Embodiments 1 to 388. . Embodiment 390 is such that the gRNA has a 2'-F modification at nucleotides 8 to 10, and also i. a phosphorothioate modification at 1, 2, or 3 of nucleotides 8 to 10, ii. a phosphorothioate modification at nucleotide 8, iii. a phosphorothioate modification at nucleotide 9, iv. a phosphorothioate modification at nucleotide 10, v. phosphorothioate modifications at nucleotides 8 and 9, vi. phosphorothioate modifications at nucleotides 8 and 10, vii. phosphorothioate modifications at nucleotides 9 and 10, or vii. includes phosphorothioate modifications at nucleotides 8 to 10, optionally, the guide region includes a 2'-OMe modification at nucleotides 1 to 4, a phosphorothioate modification at nucleotides 1 to 3 and 6 to 7, and / or a 2'-F modification at nucleotides 11, 13, 14, 17, and 18, and is the gRNA according to any one of Embodiments 1 to 389. . Embodiment 391 is such that the gRNA i. has a 2'-F or phosphorothioate modification at nucleotides 5 and 6, ii. has a 2'-F modification at nucleotides 5 and 6, iii. has a phosphorothioate modification at nucleotides 5 and 6, iv. has a 2'-F modification at nucleotide 5 and a phosphorothioate modification at nucleotide 6, or v. has a 2'-F modification at nucleotide 6 and a phosphorothioate modification at nucleotide 5, and includes a guide region, Optionally, the guide region has 2'-OMe modifications at nucleotides 1-4, 1-3 and phosphorothioate modifications at nucleotides 7 - 10, and / or the guide region contains 2'-F modifications at nucleotides 8-11, 13, 14, 17, and 18, and is the gRNA according to any one of 1-390. Embodiment 392 is a gRNA wherein the guide region contains at least 1, 2, 3, 4, 5, or 6 2'-F modifications at nucleotides 6-11, and optionally, the guide region contains 2'-OMe modifications at nucleotides 1-4, phosphorothioate modifications at nucleotides 1-3, and / or 2'-F modifications at nucleotides 13, 14, 17, and 18, and is the gRNA according to any one of Embodiments 1-391. Embodiment 393 is a gRNA wherein the guide region contains 2'-F modifications at at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of nucleotides 1-4 and 6-11, and optionally, the guide region contains phosphorothioate modifications at nucleotides 1-3, and / or 2'-F modifications at nucleotides 13, 14, 17, and 18, and is the gRNA according to any one of Embodiments 1-392. Embodiment 394 is a gRNA wherein the guide region contains 2'-F modifications at nucleotides 6-11, and optionally, the guide region contains 2'-OMe modifications at nucleotides 1-4, phosphorothioate modifications at nucleotides 1-3, and / or 2'-F modifications at nucleotides 13, 14, 17, and 18, and is the gRNA according to any one of Embodiments 1-393. Embodiment 395 is a gRNA wherein the guide region contains 2'-F modifications at nucleotides 1-4 and optionally, the guide region contains phosphorothioate modifications at nucleotides 1-3, and / or 2'-F modifications at nucleotides 13, 14, 17, and 18, and is the gRNA according to any one of Embodiments 1-394. Embodiment 396 is a gRNA wherein the guide region contains 2'-F modifications at nucleotides 13, 14, 17, and 18, and optionally, the guide region contains 2'-OMe modifications at nucleotides 1-4, phosphorothioate modifications at nucleotides 1-3, and / or 2'-F modifications at nucleotides 6-11, and is the gRNA according to any one of Embodiments 1-395. Embodiment 397 is a gRNA wherein the guide region contains 2'-F modifications at nucleotides 13, 14, 17, and 18, and optionally, the guide region contains 2'-OMe modifications at nucleotides 1-4, phosphorothioate modifications at nucleotides 1-3, and / or 2'-F modifications at nucleotides 1-4 and 6-11, and is the gRNA according to any one of Embodiments 1-396. Embodiment 398 is a gRNA wherein the guide region contains 2'-F modifications at nucleotides 13, 14, 17, and 18, and optionally, the guide region contains 2'-OMe modifications at nucleotides 1-4, phosphorothioate modifications at nucleotides 1-3, and / or 2'-F modifications at nucleotides 1-11, and is the gRNA according to any one of Embodiments 1-397. Embodiment 399 is a gRNA wherein the guide region contains 2'-F modifications at nucleotides 13, 14, 17, and 18, and optionally, the guide region comprising a domain d, and optionally, the guide region has phosphorothioate modifications at nucleotides 1 to 3 and 6 to 10, and / or 2'-F modifications at nucleotides 6 to 11, 13, 14, 17, and 18, the gRNA according to any one of embodiments 1 to 394. Embodiment 396 is a gRNA in which the guide region comprises a 2'-F modification at nucleotide 9 and does not comprise a phosphorothioate modification at nucleotide 9, and optionally, the guide region comprises a 2'-OMe modification at nucleotides 1 to 4, phosphorothioate modifications at nucleotides 1 to 3 and 6 to nucleotide 8 and 10, and / or 2'-F modifications at nucleotides 8, 10, 11, 13, 14, 17, and 18, the gRNA according to any one of embodiments 1 to 395. Embodiment 397 is a gRNA in which the guide region does not comprise at least 1, 2, 3, 4, 5, 6, 7, or 8 of the 2'-F modifications at nucleotides 8 to 11, 13, 14, 17, and 18, and optionally, the guide region comprises a 2'-OMe modification at nucleotides 1 to 4, and / or phosphorothioate modifications at nucleotides 1 to 3 and 6 to 10, the gRNA according to any one of embodiments 1 to 396. Embodiment 398 is a gRNA in which the guide region does not comprise 2'-F modifications at nucleotides 8 to 11, 13, 14, 17, and 18, and optionally, the guide region comprises a 2'-OMe modification at nucleotides 1 to 4, and / or phosphorothioate modifications at nucleotides 1 to 3 and 6 to 10, the gRNA according to any one of embodiments 1 to 397. Embodiment 399 is such that the gRNA contains a guide region comprising at least one, two, three, or four 2'-OMe modifications at nucleotides 9, 11, 13, and 14, and optionally , the guide region contains 2'-OMe modifications at nucleotides 1 to 4, and / or phosphorothioate modifications at nucleotides 1 to 3 and 6 to 10, and is the gRNA according to any one of Embodiments 1 to 398. Embodiment 400 is such that the gRNA contains a guide region comprising 2 '-OMe modifications at nucleotides 9, 11, 13, and 14, and optionally, the guide region contains 2'-OMe modifications at nucleotides 1 to 4, and / or phosphorothioate modifications at nucleotides 1 to 3 and 6 to 10, and is the g RNA according to any one of Embodiments 1 to 399. Embodiment 401 is such that the gRNA contains a guide region comprising phosphorothioate modifications at one or both of 8 and nucleotide 10, and optionally, the guide region contains 2'-OMe modifications at nucleotides 1 to 4, phosphorothioate modifications at nucleotides 1 to 3 and 6 to 7, and / or 2'-F modifications at nucleotides 8 to 11, 13, 14, 17, and 18, and is the gRNA according to any one of Embodiments 1 to 400. Embodiment 402 is such that it contains a guide region comprising modifications at at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen, or all of the following nucleotides 1, 2, 3, 4, 6, 7, 8, 9, 10, 11 , 13, 14, 17, and 18, and optionally, the modification is a 2'-OMe, 2'-fluoro, or phosphorothioate modification, and is the gRNA according to any one of Embodiments 1 to 401. ​ Embodiment 403 is a gRNA comprising a guide region in which the gRNA contains modifications at the 1st, 2nd, 3rd, 4th, 6th, 7th, 8th, 9th, 10th, 11th, 13th , 14th, 17th, and 18th nucleotides, and optionally, the modification is a 2'-OMe, 2'-fluoro, or phosphorothioate modification, which is the gRNA according to any one of Embodiments 1 to 402. Embodiment 404 is the gRNA according to any one of Embodiments 1 to 403, in which the 2'-OMe modification is absent from the guide region at nucleotides 6 to 11 and the 13th end. . Embodiment 405 is the gRNA according to any one of Embodiments 1 to 404, in which the 2'-fluoro modification is absent from the guide region at nucleotides 1 to 7, 15, 16, and the 19th end. Embodiment 406 is the gRNA according to any one of Embodiments 1 to 405, in which the phosphorothioate modification is absent from the guide region at nucleotides 4, 5, 11 to 14, 1 7, and 18. Embodiment 407 is the gRNA according to any one of Embodiments 1 to 40 6, in which the guide region contains unmodified nucleotide 20. Embodiment 408 is the gRNA according to any one of Embodiments 1 to 40 7, in which the guide region consists of 20 nucleotides. Embodiment 409 is the gRNA according to any one of Embodiments 1 to 408, in which the guide region contains a YA site at nucleotides 5 to 6 and a modification at nucleotide 5. Embodiment 410 is the gRNA according to any one of Embodiments 1 to 409, in which the guide region contains a YA site at nucleotides 12 to 13 and a modification at nucleotide 12. Embodiment 411 is the gRNA according to any one of Embodiments 1 to 410, wherein the guide region contains the YA site at nucleotides 15-16 and the modification of nucleotide 15. Embodiment 412 is the gRNA according to any one of Embodiments 1 to 411, wherein the guide region contains the YA site at nucleotides 16-17 and the modification of nucleotide 16. Embodiment 413 is the gRNA according to any one of Embodiments 1 to 412, wherein the guide region contains the YA site at nucleotides 19-20 and the modification of nucleotide 19. Embodiment 414 is the gRNA according to any one of Embodiments 1 to 413, wherein the guide region does not contain the YA site at nucleotides 5-6, and nucleotide 5 is unmodified. Embodiment 415 is the gRNA according to any one of Embodiments 1 to 414, wherein the guide region does not contain the YA site at nucleotides 12-13, and nucleotide 12 is unmodified. Embodiment 416 is the gRNA according to any one of Embodiments 1 to 415, wherein the guide region does not contain the YA site at nucleotides 15-16, and nucleotide 15 is unmodified. Embodiment 417 is the gRNA according to any one of Embodiments 1 to 416, wherein the guide region does not contain the YA site at nucleotides 16-17, and nucleotide 16 is unmodified. Embodiment 418 is the gRNA according to any one of Embodiments 1 to 417, wherein the guide region does not contain the YA site at nucleotides 19-20, and nucleotide 19 is unmodified. Embodiment 419 is that the gRNA is as follows: ​​​​​​​​​​​​​​​a. 2'-OMe and phosphorothioate modifications at nucleotide 1, b. 2'-OMe and phosphorothioate modifications at nucleotide 2, c. 2'-OMe and phosphorothioate modifications at nucleotide 3, d. 2'-OMe modification at nucleotide 4, e. phosphorothioate modification at nucleotide 6, f. phosphorothioate modification at nucleotide 7, g. 2'-fluoro and phosphorothioate modifications at nucleotide 8, h. 2'-fluoro and phosphorothioate modifications at nucleotide 9, i. 2'-fluoro and phosphorothioate modifications at nucleotide 10, j. 2'-fluoro modification at nucleotide 11, k. 2'-fluoro modification at nucleotide 13, l. 2'-fluoro modification at nucleotide 14, m. 2'-fluoro modification at nucleotide 17, and n. at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or all of the 2'-fluoro modifications at nucleotide 18, , and is the gRNA according to any one of Embodiments 1 to 418. Embodiment 420 is the gRNA according to any one of Embodiments 1 to 419, wherein the guide region contains each of the modifications described in the previous embodiments. Embodiment 421 is the gRNA according to any one of Embodiments 1 to 419, wherein the guide region contains each of the modifications described in the previous embodiments. Embodiment 421 is as follows: i. When nucleotides 5 and 6 form a YA site, 2'-OMe modification at nucleotide 5 ', ii. When nucleotides 12 and 13 form a YA site, 2'-OMe modification at nucleotide 12 ', iii. When nucleotides 15 and 16 form a YA site, the phosphorothioate modification at nucleotide 15 and iv. When nucleotides 16 and 17 form a YA site, the phosphorothioate modification at nucleotide 16, and and v. When nucleotides 19 and 20 form a YA site, at least 1, 2, 3, or 4 of the phosphorothioate modification or 2'-fluoro modification at nucleotide 19, wherein the gRNA is any one of Embodiments 1 to 420. Embodiment 422 is the gRNA according to any one of Embodiments 1 to 421, wherein the guide region comprises a YA site at nucleotides 5 to 6 and a 2'-OMe modification at nucleotide 5. Embodiment 423 is the gRNA according to any one of Embodiments 1 to 422, wherein the guide region comprises a YA site at nucleotides 12 to 13 and a 2'-OMe modification at nucleotide 12. Embodiment 424 is the gRNA according to any one of Embodiments 1 to 423, wherein the guide region comprises a YA site at nucleotides 15 to 16 and a phosphorothioate modification at nucleotide 15. Embodiment 425 is the gRNA according to any one of Embodiments 1 to 424, wherein the guide region comprises a YA site at nucleotides 16 to 17 and a phosphorothioate modification at nucleotide 16. Embodiment 426 is the gRNA according to any one of Embodiments 1 to 425, wherein the guide region comprises a YA site at nucleotides 19 to 20 and a phosphorothioate modification at nucleotide 19. ​​​​​​​​​​Embodiment 427 is the gRNA according to any one of Embodiments 1 to 426, wherein the guide region contains a 2'-fluoro modification at nucleotide 19. Embodiment 428 is the gRNA according to any one of Embodiments 1 to 427, wherein the guide region contains unmodified nucleotide 15 or contains only a phosphorothioate modification at nucleotide 15. Embodiment 429 is the gRNA according to any one of Embodiments 1 to 428, wherein the guide region contains unmodified nucleotide 16 or contains only a phosphorothioate modification at nucleotide 16. Embodiment 430 is the gRNA according to any one of Embodiments 1 to 429, wherein the gRNA is an sgRNA containing a conserved portion of an sgRNA containing a hairpin region, and the hairpin region lacks at least 5 to 10 nucleotides. Embodiment 431 is the gRNA according to Embodiment 430, wherein at least 5 to 10 nucleotides are consecutive. Embodiment 432 is such that at least 5 to 10 missing nucleotides are i. within hairpin 1, ii. within hairpin 1 and within "N" between hairpin 1 and hairpin 2, iii. within hairpin 1 and within 2 nucleotides immediately 3' to hairpin 1, iv. including at least a portion of hairpin 1, v. within hairpin 2, vi. including at least a portion of hairpin 2, vii. within hairpin 1 and hairpin 2, viii. including at least a portion of hairpin 1 and including " N" between hairpin 1 and hairpin 2, ix. including at least a portion of hairpin 2 and including "N" between hairpin 1 and hairpin 2 ​ x. comprising at least a part of Hairpin 1 and including the "N" between Hairpin 1 and Hairpin 2 and comprising at least a part of Hairpin 2 xi. being within Hairpin 1 or Hairpin 2 and optionally including the "N" between Hairpin 1 and Hairpin 2 xii. being continuous xiii. being continuous and including the "N" between Hairpin 1 and Hairpin 2 xiv. being continuous and extending to at least a part of Hairpin 1 and a part of Hairpin 2 xv. being continuous and extending to at least a part of Hairpin 1 and the "N" between Hairpin 1 and Hairpin 2, or xvi. being continuous and extending to at least a part of Hairpin 1 and the 2 nucleotides immediately 3' to Hairpin 1, which is the gRNA according to Embodiment 430 or 431 Embodiment 433 is the gRNA according to any one of Embodiments 430 - 432, wherein at least 5 - 10 nucleotides comprise Nucleotides 54 - 61 of SEQ ID NO: 400, Nucleotides 53 - 60 of SEQ ID NO: 400, or Nucleotides 54 - 58 of SEQ ID NO: 400, and optionally the sgRNA comprises modifications at least at H1 - 1~ H1 - 5 and H2 - 1~H2 - 12 Embodiment 434 is such that at least 5 - 10 nucleotides i. consist of 5 - 10 nucleotides ii. consist of 6 - 10 nucleotides iii. consist of 5 nucleotides iv. consist of 6 nucleotides v. consist of 7 nucleotides vi. consist of 8 nucleotides vii. consist of 9 nucleotides viii. consist of 10 nucleotides ​​​​​​ix. Comprising 5 to 10 consecutive nucleotides, x. Comprising 6 to 10 consecutive nucleotides, xi. Comprising 5 consecutive nucleotides, xii. Comprising 6 consecutive nucleotides, xiii. Comprising 7 consecutive nucleotides, xiv. Comprising 8 consecutive nucleotides, xv. Comprising 9 consecutive nucleotides, or xvi. Comprising 10 consecutive nucleotides, any one of Embodiments 430 to 433 is the gRNA described in any one of them. Embodiment 435 is such that at least 5 to 10 nucleotides are the nucleotides 54 to 61 of SEQ ID NO: 400, the nucleotides 53 to 60 of SEQ ID NO: 400, or the nucleotides 54 to 58 of SEQ ID NO: 400, and optionally, the sgRNA comprises at least the modifications at H1-1 to H1-5 and H2-1 to H2-12, and is the gRNA described in Embodiment 434. NA. Embodiment 436 is such that at least 5 to 10 nucleotides i. comprise the nucleotides 54 to 61 of SEQ ID NO: 400, ii. comprise the nucleotides 53 to 60 of SEQ ID NO: 400, iii. comprise the nucleotides 54 to 58 of SEQ ID NO: 400, iv. consist of the nucleotides 54 to 61 of SEQ ID NO: 400, v. consist of the nucleotides 53 to 60 of SEQ ID NO: 400, or vi. consist of the nucleotides 54 to 58 of SEQ ID NO: 400, and is the gRNA described in any one of Embodiments 430 to 435 Embodiment 437 is such that the gRNA has the sequences SEQ ID NOs: 1-54, 201 to 254, 301 to 354 , 401 to 535, 601, 607 to 732, 801, 807 to 932, 1001, 10 07~1132, 1205~1212, 1322~1406, 1417~1501, 15 11~1596, 3018~3059, 3063~3104, 3108~3149, 31 53~3194, 3198~3239, 3243~3284, 3295~3341, 33 Nucleotide from either gRNA 43 to 3385 or 3388 to 3430 The nucleotide sequence from the 5' end of the 5'-terminus corresponds to the modification pattern in sequences 1 to 20. Modified and / or unmodified nucleotides in at least 15 of positions 1 to 20 The gRNA of any one of embodiments 1 to 436 includes a gRNA. In embodiment 438, the gRNA is selected from the group consisting of SEQ ID NOs: 1 to 54, 201 to 254, 301 to 354. , 401~535, 601, 607~732, 801, 807~932, 1001, 10 07~1132, 1205~1212, 1322~1406, 1417~1501, 15 11~1596, 3018~3059, 3063~3104, 3108~3149, 31 53~3194, 3198~3239, 3243~3284, 3295~3341, 33 Nucleotide from either gRNA 43 to 3385 or 3388 to 3430 The nucleotide sequence from the 5' end of the 5'-terminus corresponds to the modification pattern in sequences 1 to 20. Modified and / or unmodified nucleotides in at least 16 of positions 1 to 20 The gRNA of any one of embodiments 1 to 437 includes a gRNA. Embodiment 439 is a method for the preparation of a gRNA comprising the steps of: At least 17 of nucleotides 1 to 20 from the 5' end of the 5' end that match the modified and / or unmodified nucleotides, and the gRNA is 01 to 254, 301 to 354, 401 to 535, 601, 607 to 732, 801, 8 07 to 932, 1001, 1007 to 1132, 1205 to 1212, 1322 to 140 6, 1417 to 1501, 1511 to 1596, 3018 to 3059, 3063 to 310 4, 3108 to 3149, 3153 to 3194, 3198 to 3239, 3243 to 328 4, 3295 to 3341, 3343 to 3385, or any one of 3388 to 3430 It is the gRNA according to any one of Embodiments 1 to 438. In Embodiment 440, the gRNA has SEQ ID NOs: 1 to 54, 201 to 254, 301 to 354 , 401 to 535, 601, 607 to 732, 801, 807 to 932, 1001, 10 07 to 1132, 1205 to 1212, 1322 to 1406, 1417 to 1501, 15 11 to 1596, 3018 to 3059, 3063 to 3104, 3108 to 3149, 31 53 to 3194, 3198 to 3239, 3243 to 3284, 3295 to 3341, 33 43 to 3385, or any one of the gRNAs of 3388 to 3430, and is consistent with the modification pattern in nucleotides 1 to 20 of at least 18 out of nucleotides 1 to 20 from the 5'-end of the 5'-terminal part, and contains modified and / or unmodified nucleotides It is the gRNA according to any one of Embodiments 1 to 439. In Embodiment 441, the gRNA has SEQ ID NOs: 1 to 54, 201 to 254, 301 to 354 , 401 to 535, 601, 607 to 732, 801, 807 to 932, 1001, 10 , 07 to 1132, 1205 to 1212, 1322 to 1406, 1417 to 1501, 15 , 11 to 1596, 3018 to 3059, 3063 to 3104, 3108 to 3149, 31 , 11 to 1596, 3018 to 3059, 3063 to 3104, 3108 to 3149, 31 53~3194, 3198~3239, 3243~3284, 3295~3341, 33 Nucleotide from either gRNA 43 to 3385 or 3388 to 3430 The nucleotide sequence from the 5' end of the 5'-terminus corresponds to the modification pattern in sequences 1 to 20. Modified and / or unmodified nucleotides in at least 19 of positions 1 to 20 The gRNA of any one of embodiments 1 to 440 includes a gRNA. In embodiment 442, the gRNA is selected from the group consisting of SEQ ID NOs: 1 to 54, 201 to 254, 301 to 354. , 401~535, 601, 607~732, 801, 807~932, 1001, 10 07~1132, 1205~1212, 1322~1406, 1417~1501, 15 11~1596, 3018~3059, 3063~3104, 3108~3149, 31 53~3194, 3198~3239, 3243~3284, 3295~3341, 33 Nucleotide from either gRNA 43 to 3385 or 3388 to 3430 The nucleotide sequence from the 5' end of the 5'-terminus corresponds to the modification pattern in sequences 1 to 20. 441, comprising modified and / or unmodified nucleotides in sequences 1 to 20 of any one of embodiments 1 to 441. The gRNA is any one of the gRNAs described above. In embodiment 443, the gRNA is selected from the group consisting of SEQ ID NOs: 1 to 54, 201 to 254, 301 to 354. , 401~535, 601, 607~732, 801, 807~932, 1001, 10 07~1132, 1205~1212, 1322~1406, 1417~1501, 15 11~1596, 3018~3059, 3063~3104, 3108~3149, 31 53~3194, 3198~3239, 3243~3284, 3295~3341, 33 One of the modification patterns of any one of the gRNAs of 43 to 3385 or 3388 to 3430 Including a modification pattern that matches at least 75% of the modification pattern, any one of Embodiments 1 to 442 The gRNA described in any one of them. Embodiment 444: The gRNA includes a modification pattern of any one of the gRNAs in Table 1, The modification pattern is SEQ ID NOs: 1 to 54, 201 to 254, 301 to 354, 401 ~535, 601, 607~732, 801, 807~932, 1001, 1007~1 132, 1205~1212, 1322~1406, 1417~1501, 1511~1 596, 3018~3059, 3063~3104, 3108~3149, 3153~3 194, 3198~3239, 3243~3284, 3295~3341, 3343~3 385, or the same as any one of the gRNAs of 3388 to 3430, the gRNA described in any one of Embodiments 1 to 443 One. Embodiment 445: The gRNA further includes a sequence having at least 75% identity with the sequence at the 21st nucleotide end of the gRNA, any one of Embodiments 437 to 444 The gRNA described in one. One. Embodiment 446: The gRNA further includes a sequence having at least 80% identity with the sequence at the 21st nucleotide end of the gRNA, any one of Embodiments 437 to 444 The gRNA described in one. One. Embodiment 447: The gRNA further includes a sequence having at least 85% identity with the sequence at the 21st nucleotide end of the gRNA, any one of Embodiments 437 to 444 The gRNA described in one. One. Embodiment 448: The gRNA further includes a sequence having at least 90% identity with the sequence at the 21st nucleotide end of the gRNA, any one of Embodiments 437 to 444 The gRNA described in one. It is. Embodiment 449 is the gRNA according to any one of Embodiments 437 to 444, further comprising a sequence having at least 95% identity with the sequence at the 21-nucleotide end of the gRNA It is. Embodiment 450 is the gRNA according to any one of Embodiments 437 to 444, further comprising a sequence having at least 98% identity with the sequence at the 21-nucleotide end of the gRNA It is. Embodiment 451 is the gRNA according to any one of Embodiments 437 to 444, further comprising a sequence having 100% identity with the sequence at the 21-nucleotide end of the gRNA. Embodiment 452 is an LNP composition comprising the gRNA according to any one of the preceding embodiments It is. Embodiment 453 is a composition comprising the gRNA according to any one of Embodiments 1 to 451 associated with a lipid nanoparticle (LNP) Embodiment 454 is a composition further comprising a nuclease or an mRNA encoding a nuclease, the gRNA according to any one of Embodiments 1 to 451 or the composition according to any one of Embodiments 452 to 45 3 Embodiment 455 is the composition according to Embodiment 454, wherein the nuclease is a Cas protein Embodiment 456 is the composition according to Embodiment 455, wherein the Cas protein is Cas9 Embodiment 457 is the composition according to Embodiment 456, wherein Cas9 is S. pyogenes Cas9 or S. aure us Cas9 Embodiment 458 is the composition according to any one of Embodiments 453 to 457, wherein the nuclease is a nickase or dCas ​​​​​​​​Embodiment 459 is the composition according to any one of Embodiments 453 to 458, wherein the nuclease is modified. One of them. Embodiment 460 is the composition according to Embodiment 459, wherein the modified nuclease contains a nuclear localization signal (NLS). One of them. Embodiment 461 is the composition according to any one of Embodiments 452 to 460, which contains mRNA encoding a nuclease. One of them. Embodiment 462 is the composition according to Embodiment 461, wherein the mRNA contains any one of the sequences of SEQ ID NOs: 3499 to 3527 or 3529 to 3546. One of them. Embodiment 463 is a pharmaceutical preparation, which contains the gRNA according to any one of Embodiments 1 to 451 or the composition according to any one of Embodiments 452 to 462, and a pharmaceutically acceptable carrier. One of them. One of them. Embodiment 464 is a method for modifying a target DNA, which includes a Cas protein or a nucleic acid encoding a Cas protein, and the following: One of them. i. The gRNA according to any one of Embodiments 1 to 451, ii. The composition according to any one of Embodiments 452 to 462, and iii. Delivering any one or more of the pharmaceutical preparations of Embodiment 463 to a cell. One of them. Embodiment 465 is the method according to Embodiment 464, wherein the method results in gene insertion or deletion. One of them. Embodiment 466 is the method according to Embodiment 464 or Embodiment 465, which further includes delivering a template to a cell, and at least a part of the template is incorporated into the target DNA at or near the double-strand break site induced by the Cas protein. One of them. One of them. Embodiment 467 is for use in the preparation of a medicament for treating a disease or disorder. The gRNA according to any one of Forms 1 to 451, the composition according to Forms 452 to 462, or the pharmaceutical preparation according to Form 463. Form 468 is for the production of a medicament for treating a disease or disorder, any one of Forms 1 to 451 of the gRNA described, the composition according to Forms 452 to 462, or the use of the pharmaceutical preparation according to Form 463.

Brief Description of Drawings

[0009]

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Mode for Carrying Out the Invention

[0010] Modified guide RNAs (gRNAs) for use in gene editing methods are provided herein and the sequences of the gRNAs that were engineered and tested are shown in Table 1.

[0011] Certain ones of the gRNAs provided herein are modified dual guide RNAs (dgRNAs) for use in gene editing methods and the sequences of the dgRNAs that were engineered and tested are shown in Table 1. Certain ones of the dgRNAs have specific modifications at the YA site in the dgRNA, including modifications in the crRNA and / or trRNA.

[0012] Certain ones of the gRNAs provided herein are modified dual guide RNAs (dgRNAs) for use in gene editing methods It is a modified single guide RNA (sgRNA). The sequences of the sgRNAs that were manipulated and tested are shown in Table 1. Certain ones of the sgRNAs have specific modifications at the YA site in the sgRNA, including modifications in the crRNA portion and / or the trRNA portion of the sgRNA. The sequences of certain ones of the sgRNAs include specific modifications at the YA site in the sgRNA, including modifications in the crRNA portion and / or the trRNA portion of the sgRNA. / or have specific modifications at the YA site in the sgRNA, including modifications in the crRNA portion and / or the trRNA portion of the sgRNA. have specific modifications at the YA site in the sgRNA.

[0013] Optionally modified short single guide RNAs (short sgRNAs) for use in gene editing methods are also provided herein. The sequences of the short sgRNAs that were manipulated and tested are shown in Table 1. Certain ones of the short sgRNAs have specific modifications at the YA site in the short sgRNA, including modifications in the crRNA portion and / or the trRNA portion of the short sgRNA. The sequences of the short sgRNAs that were manipulated and tested are shown in Table 1. Certain ones of the short sgRNAs have specific modifications at the YA site in the short sgRNA, including modifications in the crRNA portion and / or the trRNA portion of the short sgRNA. Certain ones of the short sgRNAs have specific modifications at the YA site in the short sgRNA, including modifications in the crRNA portion and / or the trRNA portion of the short sgRNA. NA portion and / or have specific modifications at the YA site in the short sgRNA, including modifications in the crRNA portion and / or the trRNA portion of the short sgRNA. have specific modifications at the YA site in the short sgRNA.

[0014] The present disclosure further provides the use of these gRNAs (e.g., sgRNAs, short sgRNAs, dgRNAs, or crRNAs) for changing the genome of a target nucleic acid in vitro (e.g., in cells cultured in vitro for use in ex vivo therapy or other uses of gene-edited cells) or in cells in a subject such as a human (e.g., for use in in vivo therapy). The present disclosure also provides methods for preventing or treating a disease in a subject by modifying a target gene associated with the disease. The disclosed gRNAs can be used in any cell type and at any locus suitable for nuclease-mediated genome editing technology. The present disclosure further provides the use of these gRNAs (e.g., sgRNAs, short sgRNAs, dgRNAs, or crRNAs) for changing the genome of a target nucleic acid in vitro (e.g., in cells cultured in vitro for use in ex vivo therapy or other uses of gene-edited cells) or in cells in a subject such as a human (e.g., for use in in vivo therapy). The present disclosure also provides methods for preventing or treating a disease in a subject by modifying a target gene associated with the disease. The disclosed gRNAs can be used in any cell type and at any locus suitable for nuclease-mediated genome editing technology. of these gRNAs (e.g., sgRNAs, short sgRNAs, dgRNAs, or crRNAs) for changing the genome of a target nucleic acid in vitro (e.g., in cells cultured in vitro for use in ex vivo therapy or other uses of gene-edited cells) or in cells in a subject such as a human (e.g., for use in in vivo therapy). The present disclosure also provides methods for preventing or treating a disease in a subject by modifying a target gene associated with the disease. The disclosed gRNAs can be used in any cell type and at any locus suitable for nuclease-mediated genome editing technology. The present disclosure also provides methods for preventing or treating a disease in a subject by modifying a target gene associated with the disease. The disclosed gRNAs can be used in any cell type and at any locus suitable for nuclease-mediated genome editing technology. The disclosed gRNAs can be used in any cell type and at any locus suitable for nuclease-mediated genome editing technology. can be used. Table 1 (Sequence Listing):

Table 1-1

Table 1-2

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Table 1-369

[0015] In Table 1, (N) x represents x consecutive nucleotides, where x is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25.

[0016] Nucleotide modifications are shown in Table 1 as follows: m: 2'-OMe; *: PS linkage. ;f: 2'-fluoro;(invd): inverted abasic;moe: 2'-moe;e: E NA; d: deoxyribonucleotide (T is always a deoxyribonucleotide) Note also that x is UNA. Thus, for example, mA is 2'-O-methyl amine. represents adenosine, xA represents a UNA nucleotide having an adenine nucleobase, and eA represents represents an ENA nucleotide having an adenine nucleobase, and dA represents an adenosine deoxyribonucleic acid. Represents a polynucleotide.

[0017] The sgRNA designation may be provided with one or more leading zeros immediately following the G. This does not affect the designated meaning. Thus, for example, G000282, G0 282, G00282, and G282 refer to the same sgRNA. Similarly, the designation of crRNA and / or trRNA may each be provided with one or more leading zeros immediately following CR or TR, which does not affect the designated meaning. Thus, for example, CR000100, CR00100, CR0100, and CR100 refer to the same crRNA, and TR000200, TR00200, TR0200, and T R200 refer to the same trRNA.

[0018] For SEQ ID NOs: 401 to 535, 1001, and 1007 to 1032, the positions correspond to the following sgRNA regions: 1 to 20, guide region; 21 to 26 and 45 to 50, lower stem; 27 to 28 and 41 to 44, bulge; 29 to 40, upper stem (of which 33 to 36 is the tetraloop); 51 to 68, nexus; 69 to 80, hairpin 1; 82 to 96, hairpin 2 (position 81 is the nucleotide between hairpin 1 and hairpin 2); 97 to 100, 3'-terminal region.

[0019] For SEQ ID NOs: 601 and 607 to 732, the guide region is not shown, and the corresponding positions in the remaining regions are each 20 less compared to the positions given for SEQ ID NOs: 401 to 532. For SEQ ID NOs: 801 and 807 to 932, the spacer is of length x, and the corresponding positions in the remaining regions are each 20 less and x more compared

[0020] Definitions As used herein, "editing efficiency" or "editing percentage" or "editing percent" refers to the total number of sequence reads having nucleotide insertions or deletions in the target region relative to the total number of sequence reads after cleavage by Cas RNP. The "region" as used herein describes conserved groups of nucleic acids. A region may also be referred to as a "module" or "domain". The region of the sgRNA can perform specific functions such as inducing the endonuclease activity of the RNP, as described in, for example, Briner AE et al., Molecular Cell 56:333-339 (2014). Exemplary regions of the sgRNA are described in Table 3.

[0021] As used herein, "hairpin" describes a double-stranded nucleic acid generated when a nucleic acid strand is folded and forms base pairs with another segment of the same strand. A hairpin can form a structure that includes a loop or a U-shape. In some embodiments, the hairpin can be composed of an RNA loop. A hairpin can be formed by two complementary sequences during the binding of a single nucleic acid molecule with folding or wrinkling of the molecule. In some embodiments, the hairpin includes a stem or a stem-loop structure. As used herein, "hairpin region" refers to hairpin 1 and hairpin 2, and the "n" between hairpin 1 and hairpin 2 in the conserved portion of the sgRNA.

[0022] As used herein, "ribonucleoprotein" (RNP) or "RNP complex" describes an sgRNA in association with a nuclease such as, for example, a Cas protein.

[0023] ​​​​​​​​​​​​​​In embodiments, the RNP comprises Cas9 and a gRNA (e.g., a sgRNA, a short sgRNA, , dgRNA, or crRNA).

[0024] As used herein, a "stem loop" refers to a nucleic acid sequence that terminates in a loop of unpaired nucleic acid. The secondary structure of nucleotides that form the base-paired "stem" Explain the structure. Two regions of the same nucleic acid strand have a small difference in sequence when read in opposite directions. When the two sequences are at least partially complementary to each other, a stem can be formed. A "base pair" is a nucleic acid that does not contain a base pair that can cap the stem (i.e., is not complementary). Describes a region of a nucleotide. "Tetraloop" describes a loop of four nucleotides. As used herein, the upper stem of the sgRNA can contain a tetraloop.

[0025] "Guide RNA", "gRNA", and "guide" refer to the CRISPR RNA), or a combination of crRNA and trRNA (traRNA These terms are used interchangeably herein to refer to either the crRNA and trRNA are single-stranded RNA molecules (single guide RNA (sgRNA) NA) or as two separate RNA molecules (dual guide RNA ( The term "guide RNA" or "gRNA" refers to each type of RNA. The trRNA may be a naturally occurring sequence or may be modified compared to the naturally occurring sequence. The guide RNA may be a trRNA sequence having a mutated or unmutated trRNA sequence as described herein. The modified RNA may include such modified RNA.

[0026] In some embodiments, the gRNA (e.g., sgRNA) includes, for example, a "guide region" that is referred to as a "spacer" or "spacer region" (however, as used herein, this is applicable to all guide RNAs) as in Brine r AE et al., Molecular Cell 56:333-339 (20 14). The guide region or spacer region may also be referred to as a "variable region", "guide domain", or "targeting domain". In some embodiments the "guide region" is present immediately prior to the "conserved portion of the sgRNA" at its 5' end, and in some embodiments, the sgRNA is a short sgRNA. An exemplary "conserved portion of the sgRNA" is shown in Table 2. In some embodiments, the "guide region" includes a series of nucleotides at the 5' end of the crR NA. In some embodiments, the guide region includes one or more YA sites ("guide region YA sites"). In some embodiments the guide region includes one or more YA sites positioned at a given nucleotide position relative to the end of the guide region from the 5' end. The range of such positions is referred to, for example, as the "5' end, 6th end, 7th end, 8th end, 9th end, or 10th end from the 5' end of the 5' end portion", and the "end" at the "5' end" etc. refers to most of the 3' nucleotides in the guide region. ( Similarly, expressions such as "nucleotide 21 end of the gRNA" refer to the range from the 21st nucleotide from the 5' end of the 5' end portion of the gRNA to the last nucleotide at the 3' end of the gRNA. ) Further, for example, the 6 nucleotides from the 5' end of a particular sgRNA segment are the 6th nucleotide of that segment, or 5 ' end. In some embodiments, the guide region includes a series of nucleotides at the 5' end of the crRNA. In some embodiments, the guide region includes one or more YA sites ("guide region YA sites"). In some embodiments the guide region includes one or more YA sites positioned at a given nucleotide position relative to the end of the guide region from the 5' end. The range of such positions is referred to, for example, as the "5' end, 6th end, 7th end, 8th end, 9th end, or 10th end from the 5' end of the 5' end portion", and the "end" at the "5' end" etc. refers to most of the 3' nucleotides in the guide region. ( Similarly, expressions such as "nucleotide 21 end of the gRNA" refer to the range from the 21st nucleotide from the 5' end of the 5' end portion of the gRNA to the last nucleotide at the 3' end of the gRNA. ) Further, for example, the 6 nucleotides from the 5' end of a particular sgRNA segment are the 6th nucleotide of that segment, or 5 ' end. ' end. ' end. ' end. ' end. ' end. ' end. “Nucleotide 6” from the ’end, e.g., is XXXXXN, where N is the 6th nucleotide from the 5’ end. The range of nucleotides located “before or after” the 6 nucleotides from the 5’ end starts with the 6th nucleotide and proceeds down the strand towards the 3’ end. Similarly, for example, a nucleotide that is 5 nucleotides from the 3’ end of the strand is the 5th nucleotide when counted from the 3’ end of the strand, e.g., NXXXX. The position or range by number in the guide region refers to the position determined from the 5’ end, unless another reference point is specified. For example, “nucleotide 5” in the guide

[0027] region is the 5th nucleotide from the 5’ end. In some embodiments, the gRNA comprises nucleotides that “match the modification pattern” at the corresponding or designated nucleotides of the gRNA described herein. This means that nucleotides that match the modification pattern have the same modification (e.g., phosphorothioate, 2’-fluoro, 2’-OMe, etc.) as the nucleotides at the corresponding positions of the gRNA described herein, regardless of whether the nucleobases at those positions are the same. For example, in a first gRNA, if nucleotides 5 and 6 have 2’-OMe and phosphorothioate modifications, respectively, this gRNA has the same modification pattern at nucleotides 5 and 6 as a second gRNA that also has 2’-OMe and phosphorothioate modifications It has. However, at nucleotide 6 but not at nucleotide 7, the 2'- OMe modification is at nucleotide 7 but not at nucleotide 6, unlike the 2'-OMe modification So, it is not the same modification pattern at nucleotides 6 and 7. Similarly, herein A modification pattern that matches at least 75% of the modification patterns of the gRNAs described herein means that at least 75% of the nucleotides have the same modification as the corresponding positions of the gRNAs described herein. The corresponding positions can be determined by pairwise or structural alignment.

[0028] The "conserved region" of S.pyogenes Cas9 (also referred to as "spyCas9" or "spCas9") is shown in Table 2. The first row shows the nucleotide numbering, the second row shows the sequence (e.g., SEQ ID NO: 400), and the third row shows the region.

[0029] As used herein, "short single-guide RNA" ("short sgRNA") is a sgRNA that includes the conserved portion of the sgRNA that contains the hairpin region, and the hairpin region lacks at least 5 to 10 nucleotides or 6 to 10 nucleotides. In some embodiments, the short sgRNA lacks at least nucleotides 54 ~58 (AAAAA) of the conserved portion of the S.pyogenes Cas9 ("spyCas9") sgRNA as shown in Table 2. In some embodiments, the short sgRNA lacks the nucleotides corresponding to nucleotides 54 - 58 (AAAAA) of the conserved portion of spyCas9 determined, for example, by pairwise or structural alignment, for non-spy ​​​​​​​​​It is a Cas9 sgRNA. In some embodiments, the short sgRNA lacks the nucleotides 54-61 (AAAAAGUG) of the conserved portion of spyCas9 sgRNA. In some embodiments, the short sgRNA lacks the nucleotides 53-60 (GAAAAAGU) of the conserved portion of spyCas9 sgRNA. In some embodiments, the short sgRNA lacks 4, 5, 6, 7, or 8 nucleotides of nucleotides 53-60 (GAAAAAGU) or nucleotides 54-61 (AAAAAGUG) of the conserved portion of spyCas9 sgRNA, or the corresponding nucleotides of the conserved portion of non-spyCas9 sgRNA determined by, for example, pairwaies or structural alignments. The "YA site" as used herein refers to the 5'-pyrimidine-adenine-3' dinucleotide. For the sake of clarity, the "YA site" in the original sequence modified by modifying the base is still regarded as the (modified) YA site in the resulting sequence. The "conserved region YA site" is present in the conserved region of the sgRNA. The "guide region YA site" is present in the guide region of the sgRNA. The unmodified YA site in the sgRNA may be susceptible to cleavage by an RNase-A-like endonuclease, such as RNase A. In some embodiments, the short sgRNA contains about 10 YA sites in its conserved region. In some embodiments, the sgRNA contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 YA sites in its conserved region. Exemplary conserved region YA sites are shown in FIG. 10B. The guide region can have any number of at least nucleotides 54-61 (AAAAAGUG) of the conserved portion of s9 sgRNA is missing. In some embodiments, the short sgRNA lacks the spyCas9 sgRN at least nucleotides 53-60 (GAAAAAGU) of the conserved portion of A. In some embodiments, the short sgRNA is the conserved portion of the spyCas9 sgRNA nucleotides 53-60 (GAAAAAGU) or nucleotides 54-61 (AAA AAGUG), 4, 5, 6, 7, or 8 nucleotides, or, for example, pairwa is or the corresponding nucleotides of the conserved portion of non-spyCas9 sgRNA determined by structural alignment is missing.

[0030] The "YA site" as used herein refers to the 5'-pyrimidine-adenine-3' dinucleotide. For the sake of clarity, the "YA site" in the original sequence modified by modifying the base is still regarded as the (modified) YA site in the resulting sequence. The "conserved region YA site" is present in the conserved region of the sgRNA. The "guide region YA site" is present in the guide region of the sgRNA. For the sake of clarity, the "YA site" in the original sequence modified by modifying the base is still regarded as the (modified) YA site in the resulting sequence even in the absence of the literal YA dinucleotide. The "conserved region YA site" is present in the conserved region of the sgRNA. The "guide region YA site" is present in the guide region of the sgRNA. The "conserved region YA site" is present in the conserved region of the sgRNA. The "guide region YA site" is present in the guide region of the sgRNA. The "guide region YA site" is present in the guide region of the sgRNA. The unmodified YA site in the sgRNA may be susceptible to cleavage by an RNase-A-like endonuclease, such as RNase A. In some embodiments, the short sgRNA contains about 10 YA sites in its conserved region. In some embodiments, s gRNA contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 Y A sites in its conserved region. Exemplary conserved region YA sites are shown in FIG. 10B. The guide region can have any number of Since it can be any array containing the YA region, the exemplary guide region YA region is not shown in FIG. 10B. In some embodiments, the sgRNA contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the YA regions shown in FIG. 10B. In some embodiments, the sgRNA contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 YA regions at the following positions or subsets thereof: LS5-LS6, US3-US4, US9-U S10, US12-B3, LS7-LS8, LS12-N1, N6-N7, N14-N1 5, N17-N18, and H2-2 to H2-3. In some embodiments, the YA region contains a modification, which means that at least one nucleotide of the YA region is modified. In some embodiments, the pyrimidines of the YA region (also referred to as pyrimidine positions) contain a modification (including a modification that changes the internucleoside bond immediately 3' to the sugar of the pyrimidine). In some embodiments, the adenines of the YA region (also referred to as adenine positions) contain a modification (including a modification that changes the internucleoside bond immediately 3' to the sugar of the adenine). In some embodiments, the pyrimidine positions and adenine positions of the YA region contain a modification. In some embodiments, the short sgRNA guide region or short sgRNA conserved region described herein contains one or more YA regions ("guide region YA regions" or "conserved region YA regions"). In some embodiments, the crRNA or trRNA described herein contains one or more YA regions. As discussed herein, the nucleotides described for the spyCas9 gRNA or short sgRNA conserved region contains one or more YA regions ("guide region YA regions" or "conserved region YA regions"). In some embodiments, the crRNA or trRNA described herein contains one or more YA regions.

[0031] As described herein with respect to the spyCas9 gRNA, the nucleotides The position of the nucleotide corresponding to d can be identified in another gRNA having sequence and / or structural similarity by pairwise or structural alignment. Structural alignment is useful when molecules share a similar structure despite large sequence variations. For example, spyCas9 and Staphylococcus aureus Cas9 ("SaCas9") have different sequences but have significant structural alignment. For example, see Figure 2(F) of Nishimasu et al., Cell 162(5):1113-11 26(2015). Structural alignment corresponds to specific positions such as nucleotides 54-58 (AAAAA) in the conserved portion of the spyCas9 s gRNA and can be used to identify nucleotides in saCas9 or other sgRNAs. can be.

[0032] Structural alignment involves (i) modeling the structure of the first sequence using the known structure of the second sequence, or (ii) comparing the structures of the first and second sequences, both of which are known, to identify corresponding residues across two (or more) sequences, and to identify the residue in the first sequence that is most similar to the residue of interest in the second sequence. The corresponding residues are based on the minimization of the distance at a given position in the superimposed structure (e.g., for polynucleotides, the nucleobase at position 1 or the 1' carbon of the pentose ring, or for polypeptides, the alpha carbon), and are identified in some algorithms (e.g., which pair of opposing positions provides the minimum mean square deviation for alignment). The position in the non-spyCas9 gRNA corresponding to the position described for the spyCa s9 gRNA When identifying, the spyCas9 gRNA can be the "second" sequence. The non-s pyCas9 gRNA has no known structure available, but another with a known structure is more closely related to the non-spyCas9 gRNA. If so, use the known structure of the closely related non-spyCas9 gRNA to model the non-spyCas9 gRNA of interest, and then compare that model to the spyCas9 gRNA structure to identify the desired corresponding residues in the non-spyCas9 gRNA of interest. This may be most effective. Extensive literature exists on protein structure modeling and alignment, and representative disclosures include US6859736, US8738343, and Aslam et al., Electronic Journal of Biotechnology 20(2 016)9-13. For considerations of structure modeling based on known related structures, see, for example, Bordoli et al., Nature Pr otocols 4(2009)1-13, and the references cited therein. For nucleic acid alignment, also see Figure 2(F) of Nishimasu et al., Cell 162(5):1113-1126(2015).

[0033] As used herein, "target sequence" refers to the sequence of a nucleic acid that the guide region induces a nuclease to cleave. In some embodiments, the spyCas9 protein can be induced to the target sequence by nucleotides present in the guide region. In some embodiments, the sgRNA does not include a spacer region.

[0034] As used herein, "5' end" refers to the first nucleotide of a gRNA (dgR NA (typically, the 5' end of the crRNA of the dgRNA), sgRNA, or short sg RNA), and the 5' position is not attached to another nucleotide.

[0035] As used herein, "5' end modification" refers to a gRNA comprising a guide region having a modification at one or more of about seven nucleotides at its 5' end, optionally where the first nucleotide (from the 5' end) of the gRNA is modified.

[0036] As used herein, "3' end" refers to the end or terminal nucleotide of a gRNA where the 3' position is not attached to another nucleotide. In some embodiments, the 3 ' end is in the 3' tail. In some embodiments, the 3' end is in the conserved portion of the gRNA.

[0037] As used herein, "3' end modification" refers to a gRNA having a modification at one or more of about seven nucleotides at the 3' end, optionally where the last nucleotide (i.e., the most 3' nucleotide) of the gRNA is modified. If a 3' tail is present, 1 to about seven nucleotides can be within the 3' tail. If no 3' tail is present, 1 to about seven nucleotides can be within the conserved portion of the sgRNA.

[0038] "Last", "second to last", "third to last", etc. refer to the most 3' nucleotide, the second most 3' nucleotide, the third most 3' nucleotide, etc., respectively, in a given sequence. ​​​​​​​​​​​. For example, in the sequence 5’-AAACTG-3’, the last, the second last, and the third last nucleotides are G, T, and C, respectively. The phrase “the last three nucleotides” refers to the last, the second last, and the third last nucleotides, and more generally, “the last N nucleotides” refers to the nucleotides from the last to the Nth last (inclusive). The “third nucleotide from the 3’ end of the 3’ end region” is equal to “the third last nucleotide”. Similarly, the “third nucleotide from the 5’ end of the 5’ end region” is equal to “the third nucleotide at the 5’ end”. The last three nucleotides are G, T, and C, respectively. The phrase “the last three nucleotides” refers to the last, the second last, and the third last nucleotides, and more generally, “the last N nucleotides” refers to the nucleotides from the last to the Nth last (inclusive). The “third nucleotide from the 3’ end of the 3’ end region” is equal to “the third last nucleotide”. Similarly, the “third nucleotide from the 5’ end of the 5’ end region” is equal to “the third nucleotide at the 5’ end”. The phrase “the last three nucleotides” refers to the last, the second last, and the third last nucleotides, and more generally, “the last N nucleotides” refers to the nucleotides from the last to the Nth last (inclusive). and more generally, “the last N nucleotides” refers to the nucleotides from the last to the Nth last (inclusive). The “third nucleotide from the 3’ end of the 3’ end region” is equal to “the third last nucleotide”. Similarly, the “third nucleotide from the 5’ end of the 5’ end region” is equal to “the third nucleotide at the 5’ end”. The “third nucleotide from the 3’ end of the 3’ end region” is equal to “the third last nucleotide”. Similarly, the “third nucleotide from the 5’ end of the 5’ end region” is equal to “the third nucleotide at the 5’ end”. The “third nucleotide from the 5’ end of the 5’ end region” is equal to “the third nucleotide at the 5’ end”.

[0039] As used herein, “protected end modification” (such as protected 5’ end modification or protected 3’ end modification) refers to the modification of one or more nucleotides within the 7 nucleotides at the end of the sgRNA that reduces the degradation of the sgRNA, such as exonuclease degradation. In some embodiments, the protected end modification includes the modification of at least two or at least three nucleotides within the 7 nucleotides at the end of the sgRNA. In some embodiments, the modification includes phosphorothioate bond, 2’ modification, such as 2’-OMe or 2’-fluoro, 2’-H (DNA), ENA, UNA, or a combination thereof. In some embodiments, the modification includes phosphorothioate bond and 2’-OMe modification. In some embodiments, at least three terminal nucleotides are modified, for example, by phosphorothioate bond or a combination of phosphorothioate bond and 2’-OME modification. Modifications that reduce exonuclease degradation known to those skilled in the art are included. As used herein, “protected end modification” (such as protected 5’ end modification or protected 3’ end modification) refers to the modification of one or more nucleotides within the 7 nucleotides at the end of the sgRNA that reduces the degradation of the sgRNA, such as exonuclease degradation. In some embodiments, the protected end modification includes the modification of at least two or at least three nucleotides within the 7 nucleotides at the end of the sgRNA. In some embodiments, the protected end modification includes the modification of at least two or at least three nucleotides within the 7 nucleotides at the end of the sgRNA. In some embodiments, the modification includes phosphorothioate bond, 2’ modification, such as 2’-OMe or 2’-fluoro, 2’-H (DNA), ENA, UNA, or a combination thereof. In some embodiments, the modification includes phosphorothioate bond, 2’ modification, such as 2’-OMe or 2’-fluoro, 2’-H (DNA), ENA, UNA, or a combination thereof. In some embodiments, the modification includes phosphorothioate bond, 2’ modification, such as 2’-OMe or 2’-fluoro, 2’-H (DNA), ENA, UNA, or a combination thereof. In some embodiments, the modification includes phosphorothioate bond and 2’-OMe modification. In some embodiments, at least three terminal nucleotides are modified, for example, by phosphorothioate bond or a combination of phosphorothioate bond and 2’-OME modification. In some embodiments, at least three terminal nucleotides are modified, for example, by phosphorothioate bond or a combination of phosphorothioate bond and 2’-OME modification. Modifications that reduce exonuclease degradation known to those skilled in the art are included.

[0040] In some embodiments, a "3' tail" comprising 1 to about 20 nucleotides follows a conserved portion of the sgRNA at its 3' end.

[0041] As used herein, an "RNA-Guided DNA binding agent" means a polypeptide or complex of polypeptides having RNA and DNA binding activities, or a DNA binding subunit of such a complex, wherein the DNA binding activity is sequence specific and depends on the sequence of the RNA. Exemplary RNA-guided DNA binding agents include Cas endonucleases / nickases and their inactivated forms ("dCas DNA binding agents"). As used herein, a "Cas nuclease," also referred to as a "Cas protein," includes Cas endonucleases, Cas nickases, and dCas DNA binding agents. Cas endonucleases / nickases and dCas DNA binding agents include a Csm or Cmr complex of a type III CRISPR system, the Cas10, Csm1, or Cmr2 subunit thereof, a Cascade complex of a type I CRISPR system, the Cas3 subunit thereof, and Class 2 Cas nucleases. As used herein, a "Class 2 Cas nuclease" is a single-stranded polypeptide having RNA-guided DNA binding activity, such as a Cas9 nuclease or a Cpf1 nuclease. Class 2 Cas nucleases further include Class 2 Cas endonucleases and Class 2 Cas nickases having RNA-guided DNA cleavage or nickase activity (e.g., H840A, D10A nucleases, etc.). Class 2 Cas nucleases further include Class 2 Cas endonucleases and Class 2 Cas nickases having RNA-guided DNA cleavage or nickase activity (e.g., H840A, D10A ​​​​ or the N863A variant), and a class where the cleavage / nicking activity is inactivated 2 dCas DNA binders are included. Class 2 Cas nucleases include, for example, C as9, Cpf1, C2c1, C2c2, C2c3, HF Cas9 (e.g., N497 A, R661A, Q695A, Q926A variants), HypaCas9 (e.g., N 692A, M694A, Q695A, H698A variants), eSPCas9(1.0 )(e.g., K810A, K1003A, R1060A variants), and eSPCa s9(1.1)(e.g., K848A, K1003A, R1060A variants) proteins, as well as modifications thereof. The Cpf1 protein, Zetsche et al., Cell, 163:1-13(2015) is homologous to Cas9 and contains an RuvC -like nuclease domain. The Cpf1 sequence of Zetsche is incorporated in its entirety by reference. See, for example, Tables S1 and S3 of Zetsche. "Ca s9" includes Spy Cas9, the variants of Cas9 listed herein, and equivalents thereof. See, for example, Makarova et al., Nat Rev M icrobiol, 13(11):722-36(2015); Shmakov et al., Molecular Cell, 60:385-397(2015) for reference. When used herein, if it is shown that by alignment of the first sequence to the second sequence, X% or more of the entire positions of the second sequence are matched by the first sequence, the first sequence is considered to "include a sequence having at least X% identity" with the second sequence. See also.

[0042] As used herein, when it is shown that by alignment of the first sequence to the second sequence, X% or more of the entire positions of the second sequence are matched by the first sequence, the first sequence is considered to "include a sequence having at least X% identity" with the second sequence. See also. . For example, the sequence AAGA includes sequences that have 100% identity with the sequence AAG, because the alignment gives 100% identity in that there is a match at all three positions of the second sequence. The differences between RNA and DNA (generally, exchanging thymidine for uridine, or vice versa), and the presence of nucleoside analogs such as modified uridine, do not contribute to differences in identity or complementarity between polynucleotides as long as the relevant nucleotides (such as thymidine, uridine, or modified uridine) have the same complement (for example, adenosine for all of thymidine, uridine, or modified uridine; another example is cytosine and 5-methylcytosine, both of which have guanosine or modified guanosine as the complement). Thus, for example, the sequence 5'-AXG, where X is any modified uridine, such as pseudouridine, N1-methylpseudouridine, or 5-methoxyuridine, is considered to be 100% identical to AUG in that both are completely complementary to the same sequence (5'-CAU). Exemplary alignment algorithms are the Smith-Waterman and Needleman-Wunsch algorithms, which are well known in the art. One of ordinary skill in the art will understand which choice of algorithm and parameter settings is appropriate for a given pair of sequences to be aligned. Generally, for sequences with lengths and expected identities that are generally similar, for amino acids, sequences greater than 50%, or for nucleotides, sequences greater than 75%, the default settings of the Needleman-Wunsch algorithm interface provided by EBI at the www.ebi.ac.uk web server are used. because there is a match at all three positions of the second sequence, giving 100% identity. The difference between RNA and DNA (generally, exchanging thymidine for uridine, or vice versa), and the presence of nucleoside analogs such as modified uridine, do not contribute to differences in identity or complementarity between polynucleotides as long as the relevant nucleotides (such as thymidine, uridine, or modified uridine) have the same complement (for example, adenosine for all of thymidine, uridine, or modified uridine; another example is cytosine and 5-methylcytosine, both of which have guanosine or modified guanosine as the complement). Thus, for example, the sequence 5'-AXG, where X is any modified uridine, such as pseudouridine, N1-methylpseudouridine, or 5-methoxyuridine, is considered to be 100% identical to AUG in that both are completely complementary to the same sequence (5'-CAU). Exemplary alignment algorithms are the Smith-Waterman and Needleman-Wunsch algorithms, which are well known in the art. One of ordinary skill in the art will understand which choice of algorithm and parameter settings is appropriate for a given pair of sequences to be aligned. Generally, for sequences with lengths and expected identities that are generally similar, for amino acids, sequences greater than 50%, or for nucleotides, sequences greater than 75%, the default settings of the Needleman-Wunsch algorithm interface provided by EBI at the www.ebi.ac.uk web server are used. Exemplary alignment algorithms are the Smith-Waterman and Needleman-Wunsch algorithms, which are well known in the art. One of ordinary skill in the art will understand which choice of algorithm and parameter settings is appropriate for a given pair of sequences to be aligned. Generally, for sequences with lengths and expected identities that are generally similar, for amino acids, sequences greater than 50%, or for nucleotides, sequences greater than 75%, the default settings of the Needleman-Wunsch algorithm interface provided by EBI at the www.ebi.ac.uk web server are used. Generally, for sequences with lengths and expected identities that are generally similar, for amino acids, sequences greater than 50%, or for nucleotides, sequences greater than 75%, the default settings of the Needleman-Wunsch algorithm interface provided by EBI at the www.ebi.ac.uk web server are used. .ac.uk web server are used. h algorithm interface are used. The sch algorithm is generally appropriate.

[0043] As used herein, "mRNA" refers to a polynucleotide that is RNA or modified RNA and that can be translated into a polypeptide (i.e., can function as a substrate for translation by ribosomes and aminoacylated tRNA). The mRNA can include a ribose residue or an analog thereof, such as a 2'-methoxyribose residue, and can include a phosphosugar backbone. In some embodiments, the sugar of the nucleic acid phosphosugar backbone consists essentially of ribose residues, 2'-methoxyribose residues, or combinations thereof. Generally, mRNA does not contain a substantial amount of thymidine residues (e.g., 0 residues or less than 30, 20, 10, 5, 4, 3, or 2 thymidine residues; or a thymidine content of less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1%). The mRNA can include modified uridine at some or all of its uridine positions. and can function as) an open reading frame that includes a ribosome and an aminoacylated tRNA for translation. For the purpose of being referred to herein. The mRNA can include a ribose residue or an analog thereof, such as a 2'- methoxyribose residue, and can include a phosphosugar backbone. In some embodiments the sugar of the nucleic acid phosphosugar backbone consists essentially of ribose residues, 2'-methoxyribose residues, or combinations thereof. Generally, mRNA does not contain a substantial amount of thymidine residues (e.g., 0 residues or less than 30, 20, 10, 5, 4, 3, or 2 thymidine residues; or a thymidine content of less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1%). The mRNA can include modified uridine at some or all of its uridine positions. For the purpose of being referred to herein. For example, 0 residues or less than 30, 20, 10, 5, 4, 3, or 2 thymidine residues; or a thymidine content of less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1%. The mRNA can include modified uridine at some or all of its uridine positions. For the purpose of being referred to herein. .5%, 0.2%, or less than 0.1% thymidine content) and does not contain. The mRNA can include modified uridine at some or all of its uridine positions. The mRNA can include modified uridine at some or all of its uridine positions.

[0044] As used herein, the "minimum uridine content" of a given ORF is the uridine content of an ORF that (a) uses the minimum uridine codon at all positions and (b) encodes the same amino acid sequence as the given ORF. The minimum uridine codon(s) for a given amino acid is (are) the codon(s) having the fewest uridines (usually 0 or 1, except for the codons for phenylalanine, which have 2 uridines). Modified uridine residues are considered equivalent to uridine for the purpose of evaluating the minimum uridine content. For the purpose of being referred to herein. For the purpose of being referred to herein. For the purpose of being referred to herein. For the purpose of being referred to herein. For the purpose of being referred to herein. For the purpose of being referred to herein.

[0045] As used herein, the "minimum uridine nucleotide content" of a given ORF is (a) using the minimum uridine codon at all positions (as described above), and (b) the possible minimum uridine dinucleotide (U U) content of an ORF that encodes the same amino acid sequence as the given O U). The uridine dinucleotide (UU) content can be expressed in absolute terms, either as an enumeration of the UU dinucleotides in the ORF or as the rate of the positions occupied by the uridine of the uridine dinucleotide (e.g., AUUAU has a uridine dinucleotide content of 40% because 2 out of 5 positions are occupied by the uridine of the uridine dinucleotide). Modified uridine residues are considered equivalent to uridine for the purpose of assessing the minimum uridine dinucleotide content. As used herein, the "minimum adenine content" of a given open reading frame (ORF) is (a) using the minimum adenine codon at all positions, and (b) the adenine content of an ORF that encodes the same amino acid sequence as the given OR F. The minimum adenine codon(s) for a given amino acid is / are the codon(s) having the fewest adenines (usually 0 or 1, except for the codons for lysine and asparagine, and the minimum adenine codon has 2 adenines). Modified adenine residues are considered equivalent to adenine for the purpose of assessing the minimum adenine content.

[0046] As used herein, the "minimum a denine content" of a given open reading frame (ORF) is (a) using the minimum adenine codon at all positions, and (b) the adenine content of an ORF that encodes the same amino acid sequence as the given OR F. The minimum adenine codon(s) for a given amino acid is / are the codon(s) having the fewest adenines (usually 0 or 1, except for the codons for lysine and asparagine, and the minimum adenine codon has 2 adenines). Modified adenine residues are considered equivalent to adenine for the purpose of assessing the minimum adenine content. (usually 0 or 1, except for the codons for lysine and asparagine, and the minimum adenine codon has 2 adenines). Modified adenine residues are considered equivalent to adenine for the purpose of assessing the minimum adenine content. (usually 0 or 1, except for the codons for lysine and asparagine, and the minimum adenine codon has 2 adenines). Modified adenine residues are considered equivalent to adenine for the purpose of assessing the minimum adenine content. (usually 0 or 1, except for the codons for lysine and asparagine, and the minimum adenine codon has 2 adenines). Modified adenine residues are considered equivalent to adenine for the purpose of assessing the minimum adenine content.

[0047] As used herein, the "minimum a The "adenine dinucleotide content" is the minimum adenine codon (as described above) at all positions, and (b) the minimum adenine dinucleotide (AA) content of an ORF that encodes the same amino acid sequence as the given ORF. The adenine dinucleotide (AA) content can be expressed in absolute terms, either as an enumeration of AA dinucleotides in the ORF or as a rate-based percentage of the positions occupied by adenine of the adenine dinucleotide (e.g., UAAUA has an adenine dinucleotide content of 40% because 2 out of 5 positions are occupied by adenine of the adenine dinucleotide). Modified adenine residues are considered equivalent to adenine for the purpose of evaluating the minimum adenine dinucleotide content. Using the minimum adenine codon (as described above) at all positions, and (b) the minimum adenine dinucleotide (AA) content of an ORF that encodes the same amino acid sequence as the given ORF. The adenine dinucleotide (AA) content can be expressed in absolute terms, either as an enumeration of AA dinucleotides in the ORF or as a rate-based percentage of the positions occupied by adenine of the adenine dinucleotide (e.g., UAAUA has an adenine dinucleotide content of 40% because 2 out of 5 positions are occupied by adenine of the adenine dinucleotide). Modified adenine residues are considered equivalent to adenine for the purpose of evaluating the minimum adenine dinucleotide content. For the purposes of evaluating the minimum adenine dinucleotide content, modified adenine residues are considered equivalent to adenine. For example, UAAUA has an adenine dinucleotide content of 40% because 2 out of 5 positions are occupied by adenine of the adenine dinucleotide. Modified adenine residues are considered equivalent to adenine for the purpose of evaluating the minimum adenine dinucleotide content. Modified adenine residues are considered equivalent to adenine for the purpose of evaluating the minimum adenine dinucleotide content. Modified adenine residues are considered equivalent to adenine for the purpose of evaluating the minimum adenine dinucleotide content.

[0048] As used herein, "subject" refers to any member of the animal kingdom. In some embodiments, "subject" refers to a human. In some embodiments, "subject" refers to a non-human animal. In some embodiments, "subject" refers to a primate. In some embodiments, the subject includes, but is not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In certain embodiments, the non-human subject is a mammal (e.g., rodents, mice, rats, rabbits, monkeys, dogs, cats, sheep, cows, primates, and / or pigs). In some embodiments, the subject may be a transgenic animal, a genetically engineered animal, and / or a clone. In certain embodiments of the invention, the subject is an adult, adolescent, or infant. In some embodiments, the terms "individual" or "patient" are used and are intended to be interchangeable with "subject". As used herein, "subject" refers to any member of the animal kingdom. In some embodiments, "subject" refers to a human. In some embodiments, "subject" refers to a non-human animal. In some embodiments, "subject" refers to a primate. In some embodiments, the subject includes, but is not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In certain embodiments, the non-human subject is a mammal (e.g., rodents, mice, rats, rabbits, monkeys, dogs, cats, sheep, cows, primates, and / or pigs). In some embodiments, the subject may be a transgenic animal, a genetically engineered animal, and / or a clone. In certain embodiments of the invention, the subject is an adult, adolescent, or infant. In some embodiments, the terms "individual" or "patient" are used and are intended to be interchangeable with "subject". In certain embodiments of the invention, the subject is an adult, adolescent, or infant. In some embodiments, the terms "individual" or "patient" are used and are intended to be interchangeable with "subject". In some embodiments, the terms "individual" or "patient" are used and are intended to be interchangeable with "subject".

[0049] The types of modifications described in this specification Guide RNAs (e.g., sgRNAs, short sgRNAs, d gRNAs, and crRNAs) containing modifications at various positions are disclosed herein. In some embodiments, the positions of the gRNAs containing modifications are modified by any one or more of the following types of modifications to be. Yes.

[0050] 2'-O-methyl modification Modified sugars are the packing of the nucleotide sugar ring, a physical property that affects oligonucleotide binding affinity for complementary strands, duplex formation, and interaction with nucleases (puckering) is thought to be controlled. Thus, substitutions on the sugar ring can alter the conformation and packing of these sugars. For example, 2'-O-methyl (2' -OMe) modification can increase the binding affinity and nuclease stability of oligonucleotides, but as shown in the examples, it is necessary to empirically determine the effect of any modification at a given position in the oligonucleotide be. -OMe) modification can increase the binding affinity and nuclease stability of oligonucleotides, but as shown in the examples, it is necessary to empirically determine the effect of any modification at a given position in the oligonucleotide be. be.

[0051] The terms "mA", "mC", "mU", or "mG" can be used to represent nucleotides modified by 2'-OMe be.

[0052] Ribonucleotides and modified 2'-O-methyl ribonucleotides can be described as follows be. [Chemical formula]

[0053] 2'-O-(2-methoxyethyl) modification In some embodiments, the modification is 2'-O-(2-methoxyethyl) (2'-O-m may be (oe). Modified 2'-O-moe ribonucleotides can be described as follows .

Chemical formula

[0054] The terms "moeA", "moeC", "moeU", or "moeG" can be used to represent nucleotides modified with 2'-O-moe.

[0055] 2'-Fluoro modification Another chemical modification shown to affect the nucleotide sugar ring is halogen substitution. For example, 2'-fluoro (2'-F) substitution on the nucleotide sugar ring can increase oligonucleotide bond affinity and nuclease stability.

[0056] In this application, the terms "fA", "fC", "fU", or "fG" can be used to represent nucleotides substituted with 2'-F.

[0057] Ribonucleotides without 2'-F substitution and ribonucleotides with 2'-F substitution can be described as follows.

Chemical formula

[0058] Phosphorothioate modification A phosphorothioate (PS) linkage or bond refers to a bond in which sulfur replaces one of the non-bridging phosphorus oxygen atoms in a phosphodiester bond between nucleotides, for example. When oligonucleotides are generated using phosphorothioates, the modified oligonucleotides can also be referred to as S-oligos. ​​​​​​

[0059] "*" may be used to indicate a PS modification. In this application, the terms A*, C*, U*, or or G* to the adjacent (e.g., 3') nucleotide via a PS bond. Throughout this application, the PS modification refers to a nucleotide sequence in which the 3' carbon is phosphorylated. Grouped with nucleotides that are bound to thioates, and therefore PS modification at position 1 In the figure, phosphorothioates are attached to the 3' carbon of nucleotide 1 and the 2' carbon of nucleotide 2. This means that the 5' carbon of the oxythion is bonded to the YA moiety. When a position is indicated as being "PS-modified", the PS bond is between Y and A or adjacent to A. It is located between the nucleotides.

[0060] In this application, the terms "mA*", "mC*", "mU*", or "mG*" refer to 2'- A PS bond that may be substituted with OMe and may also be referred to as a "PS bond" (linkage) indicates the nucleotide linked to the adjacent (e.g. 3') nucleotide In this application, the terms "fA*", "fC*", "fU*", or " fG*”, which is replaced by 2'-F and is linked to the adjacent (e.g. 3') nucleotide by a PS bond It can be used to indicate linked nucleotides. PS linkage or bond Equivalents of bonds are encompassed by the embodiments described herein.

[0061] The diagram below shows the substitution of S- for the non-bridging phosphate oxygen, replacing the phosphodiester bond. This produces a PS bond: [ka]

[0062] Inverted abasic modification An abasic nucleotide refers to one lacking a nitrogenous base. The following figure depicts an oligonucleotide having a site lacking a base as abasic (in this case, shown as depurinated. The abasic site can also be a depyrimidinic site, and the description of the abasic site typically refers to a Watson-Crick base pair. A depurinated site refers to a site lacking a nitrogenous base and typically forms a base pair with a pyrimidine site), and the base can be substituted by another site (e.g., a hydroxyl group shown below to form a ribose or deoxyribose site, or hydrogen) at the 1'-position of the furan ring. site being abasic (in this case, shown as depurinated. The abasic site can also be a depyrimidinic site, and the description of the abasic site typically refers to a Watson-Crick base pair. A depurinated site refers to a site lacking a nitrogenous base and typically forms a base pair with a pyrimidine site), and the base can be substituted by another site (e.g., a hydroxyl group shown below to form a ribose or deoxyribose site, or hydrogen) at the 1'-position of the furan ring. site being abasic (in this case, shown as depurinated. The abasic site can also be a depyrimidinic site, and the description of the abasic site typically refers to a Watson-Crick base pair. A depurinated site refers to a site lacking a nitrogenous base and typically forms a base pair with a pyrimidine site), and the base can be substituted by another site (e.g., a hydroxyl group shown below to form a ribose or deoxyribose site, or hydrogen) at the 1'-position of the furan ring. site being abasic (in this case, shown as depurinated. The abasic site can also be a depyrimidinic site, and the description of the abasic site typically refers to a Watson-Crick base pair. A depurinated site refers to a site lacking a nitrogenous base and typically forms a base pair with a pyrimidine site), and the base can be substituted by another site (e.g., a hydroxyl group shown below to form a ribose or deoxyribose site, or hydrogen) at the 1'-position of the furan ring. site being abasic (in this case, shown as depurinated. The abasic site can also be a depyrimidinic site, and the description of the abasic site typically refers to a Watson-Crick base pair. A depurinated site refers to a site lacking a nitrogenous base and typically forms a base pair with a pyrimidine site), and the base can be substituted by another site (e.g., a hydroxyl group shown below to form a ribose or deoxyribose site, or hydrogen) at the 1'-position of the furan ring. site being abasic (in this case, shown as depurinated. The abasic site can also be a depyrimidinic site, and the description of the abasic site typically refers to a Watson-Crick base pair. A depurinated site refers to a site lacking a nitrogenous base and typically forms a base pair with a pyrimidine site), and the base can be substituted by another site (e.g., a hydroxyl group shown below to form a ribose or deoxyribose site, or hydrogen) at the 1'-position of the furan ring. site being abasic (in this case, shown as depurinated. The abasic site can also be a depyrimidinic site, and the description of the abasic site typically refers to a Watson-Crick base pair. A depurinated site refers to a site lacking a nitrogenous base and typically forms a base pair with a pyrimidine site), and the base can be substituted by another site (e.g., a hydroxyl group shown below to form a ribose or deoxyribose site, or hydrogen) at the 1'-position of the furan ring. [Chemical formula]

[0063] An inverted base refers to one having a bond inverted from the normal 5'-to-3' bond (i.e., either a 5'-to-5' bond or a 3'-to-3' bond). For example, An inverted base refers to one having a bond inverted from the normal 5'-to-3' bond (i.e., either a 5'-to-5' bond or a 3'-to-3' bond). For example, [Chemical formula]

[0064] An abasic nucleotide can bond with an inverted bond. For example, an abasic nucleotide can bond to the terminal 5'-nucleotide via a 5'-to-5' bond, or an abasic nucleotide can bond to the terminal 3'-nucleotide via a 3'-to-3' bond. An inverted abasic nucleotide at either the terminal 5' or 3'-nucleotide can also be referred to as an inverted abasic terminal cap. In this application, the term "invd" indicates an inverted abasic nucleotide bond. An abasic nucleotide can bond with an inverted bond. For example, an abasic nucleotide can bond to the terminal 5'-nucleotide via a 5'-to-5' bond, or an abasic nucleotide can bond to the terminal 3'-nucleotide via a 3'-to-3' bond. An inverted abasic nucleotide at either the terminal 5' or 3'-nucleotide can also be referred to as an inverted abasic terminal cap. In this application, the term "invd" indicates an inverted abasic nucleotide bond. An abasic nucleotide can bond with an inverted bond. For example, an abasic nucleotide can bond to the terminal 5'-nucleotide via a 5'-to-5' bond, or an abasic nucleotide can bond to the terminal 3'-nucleotide via a 3'-to-3' bond. An inverted abasic nucleotide at either the terminal 5' or 3'-nucleotide can also be referred to as an inverted abasic terminal cap. In this application, the term "invd" indicates an inverted abasic nucleotide bond. An abasic nucleotide can bond with an inverted bond. For example, an abasic nucleotide can bond to the terminal 5'-nucleotide via a 5'-to-5' bond, or an abasic nucleotide can bond to the terminal 3'-nucleotide via a 3'-to-3' bond. An inverted abasic nucleotide at either the terminal 5' or 3'-nucleotide can also be referred to as an inverted abasic terminal cap. In this application, the term "invd" indicates an inverted abasic nucleotide bond. An abasic nucleotide can bond with an inverted bond. For example, an abasic nucleotide can bond to the terminal 5'-nucleotide via a 5'-to-5' bond, or an abasic nucleotide can bond to the terminal 3'-nucleotide via a 3'-to-3' bond. An inverted abasic nucleotide at either the terminal 5' or 3'-nucleotide can also be referred to as an inverted abasic terminal cap. In this application, the term "invd" indicates an inverted abasic nucleotide bond. An abasic nucleotide can bond with an inverted bond. For example, an abasic nucleotide can bond to the terminal 5'-nucleotide via a 5'-to-5' bond, or an abasic nucleotide can bond to the terminal 3'-nucleotide via a 3'-to-3' bond. An inverted abasic nucleotide at either the terminal 5' or 3'-nucleotide can also be referred to as an inverted abasic terminal cap. In this application, the term "invd" indicates an inverted abasic nucleotide bond.

[0065] Deoxyribonucleotide Deoxyribonucleotides (where the sugar contains the 2'-deoxy position) are considered modified relative to standard RNA by replacement of the proton on the 2'-hydroxyl of the nucleotide, and are considered modifications in the context of gRNA. Unless otherwise indicated, deoxyribonucleotide modifications at positions that are U in unmodified RNA can also include replacement of the U nucleobase with T. in that respect, and are considered modifications in the context of gRNA. Unless otherwise indicated, deoxyribonucleotide modifications at positions that are U in unmodified RNA can also include replacement of the U nucleobase with T. A. Deoxyribonucleotide modifications at positions that are U in unmodified RNA can also include replacement of the U nucleobase with T. A can also include replacing the U nucleobase with T. can include replacing the U nucleobase with T.

[0066] Bicyclic ribose analogs Exemplary bicyclic ribose analogs include locked nucleic acid (LNA), ENA, bridged nucleic acid (BNA), or another LNA-like modification. In some examples, the bicyclic ribose analog has 2' and 4' positions connected via a linker. The linker can be of the formula -X-(CH2)n-, where n is 1 or 2 and X is O, NR, or S, and R is H or C1-C6 alkyl, such as methyl. Examples of bicyclic ribose analogs include LNA including 2'-O-CH2-4' (oxy-LNA) (see WO 98 / 39352 and WO 99 / 14226), 2'-NH-CH2-4', or 2'-N(CH3)-CH2-4' (amino-LNA) (Singh et al., J. Org. Chem. 63:10035-10039 (1998); Singh et al., J. Org. Chem. 63:6078-6079 (1998)); and 2'-S-CH2-4' (thio-LNA) (Singh et al., J. Org. Chem. 63:6078-6079 (1998); Kumar et al., Bi A), or another LNA-like modification. In some examples, the bicyclic ribose analog has 2' and 4' positions connected via a linker. The linker can be of the formula -X-(CH2)n-, where n is 1 or 2 and X is O, NR, or S, and R is H or C1-C6 alkyl, such as methyl. Examples of bicyclic ribose analogs include LNA including 2'-O-CH2-4' (oxy-LNA) (see WO 98 / 39352 and WO 99 / 14226), 2'-NH-CH2-4', or 2'-N(CH3)-CH2-4' (amino-LNA) (Singh et al., J. Org. Chem. 63:10035-10039 (1998); Singh et al., J. Org. Chem. 63:6078-6079 (1998)); and 2'-S-CH2-4' (thio-LNA) (Singh et al., J. Org. Chem. 63:6078-6079 (1998); Kumar et al., Bi A), or another LNA-like modification. In some examples, the bicyclic ribose analog has 2' and 4' positions connected via a linker. The linker can be of the formula -X-(CH2)n-, where n is 1 or 2 and X is O, NR, or S, and R is H or C1-C6 alkyl, such as methyl. Examples of bicyclic ribose analogs include LNA including 2'-O-CH2-4' (oxy-LNA) (see WO 98 / 39352 and WO 99 / 14226), 2'-NH-CH2-4', or 2'-N(CH3)-CH2-4' (amino-LNA) (Singh et al., J. Org. Chem. 63:10035-10039 (1998); Singh et al., J. Org. Chem. 63:6078-6079 (1998)); and 2'-S-CH2-4' (thio-LNA) (Singh et al., J. Org. Chem. 63:6078-6079 (1998); Kumar et al., Bi n- n -n- and X is O, NR, or S, and R is H or C1-C6 alkyl, such as methyl. n- and X is O, NR, or S, and R is H or C1-C6 alkyl, such as methyl. 1-3 alkyl, such as methyl. Examples of bicyclic ribose analogs include LNA including 2'-O-CH2-4' (oxy-LNA) (see WO 98 / 39352 and WO 99 / 14226), 2'-NH-CH2-4', or 2'-N(CH3)-CH2-4' (amino-LNA) (Singh et al., J. Org. Chem. 63:10035-10039 (1998); Singh et al., J. Org. Chem. 63:6078-6079 (1998)); and 2'-S-CH2-4' (thio-LNA) (Singh et al., J. Org. Chem. 63:6078-6079 (1998); Kumar et al., Bi alkyl, such as methyl. Examples of bicyclic ribose analogs include LNA including 2'-O-CH2-4' (oxy-LNA) (see WO 98 / 39352 and WO 99 / 14226), 2'-NH-CH2-4', or 2'-N(CH3)-CH2-4' (amino-LNA) (Singh et al., J. Org. Chem. 63:10035-10039 (1998); Singh et al., J. Org. Chem. 63:6078-6079 (1998)); and 2'-S-CH2-4' (thio-LNA) (Singh et al., J. Org. Chem. 63:6078-6079 (1998); Kumar et al., Bi 98 / 39352 and WO 99 / 14226), 2'-NH-CH2-4', or 2'-N(CH3)-CH2-4' (amino-LNA) (Singh et al., , J. Org. Chem. 63:10035-10039 (1998); Singh et al., J. Org. Chem. 63:6078-6079 (1998)); and 2'-S-CH2-4' (thio-LNA) (Singh et al., J. Org . Chem. 63:6078-6079 (1998); Kumar et al., Bi org.Med.Chem.Lett.8:2219-2222(1998)).

[0067] ENA An ENA modification refers to a nucleotide that contains a 2'-O,4'-C-ethylene modification. Exemplary structures of nucleotides are shown below, with wavy lines indicating connections (or or optionally, if the 3' terminal nucleotide is an ENA nucleotide, indicates a terminal position, with the understanding that it may contain a hydroxyl rather than a phosphate. For further discussion of nucleotides, see, e.g., Koizumi et al., Nu See cleic Acids Res. 31:3267-3273 (2003). . [ka]

[0068] U.N.A. UNA or Non-Locked Nucleic Acid Modifications are nucleotide modifications that contain 2',3'-seco-RNA modifications. UNA refers to a nucleotide in which the 2' and 3' carbons are not directly bonded to each other. Exemplary structures are shown below. Wavy lines indicate connections to adjacent phosphates or replacements of phosphates. For further discussion of UNA nucleotides, see For example, see Snead et al., Molecular Therapy 2: See e103,doi:10.1038 / mtna.2013.36(2013) and. [ka]

[0069] Base Modification Base modifications are any modifications that alter the structure of a nucleobase or its attachment to the backbone, including isomerization (such as pseudouridine). In some embodiments, the base modification includes inosine. In some embodiments, the modification, for example, prevents recognition of the cleavage site by RNase and / or stabilizes an RNA structure (such as a secondary structure) that reduces the accessibility of the cleavage site by RNase, including base modifications that reduce RNase activity. Exemplary base modifications that can stabilize RNA structures are pseudouridine and 5-methylcytosine. See Peacock et al., J Org Chem. 76:7295-7300 (2011). In some embodiments, the base modification, for example, can increase or decrease the melting temperature (Tm) of a nucleic acid by increasing hydrogen bonding in Watson-Crick base pairs, forming non-canonical base pairs, or creating mismatched base pairs.

[0070] The above modifications and their equivalents are included within the scope of the embodiments described herein.

[0071] YA modification Modifications at the YA site (also referred to as YA modifications) can be modifications of the internucleoside bond, modifications of the base (pyrimidine or adenine), for example, by chemical modification, substitution, or other means, and / or modifications of the sugar (such as those at the 2'-position like 2'-O-alkyl, 2'-F, 2'-moe, 2'-F arabinose, 2'-H (deoxyribose), etc.). In some embodiments, "YA modification" by preventing recognition or cleavage of the cleavage site and / or by blocking access of the cleavage site to RNases. By stabilizing RNA structures (e.g., secondary structures) that reduce access to the RNA The structure of the dinucleotide motif may be altered to reduce endonuclease activity. Peacock et al., J Org Chem.76:729 5-7300(2011);Behlke,Oligonucleotides 18: 305-320(2008);Ku et al.,Adv.Drug Deliver y Reviews 104:16-28(2016);Ghidini et al. , Chem. Commun., 2013, 49, 9036. et al., Belhke, Ku, and Ghidini are suitable for YA modification. Exemplary modifications known to those of skill in the art to reduce endonucleolytic degradation are provided below. Exemplary modifications that affect the 2' hydroxyl group involved in RNase cleavage include: The 2' ribose modifications are 2'-H and 2'-O-alkyl, including 2'-O-Me. Bicyclic ribose analogues, UNA, and modified internucleoside linkages of residues at the YA site and the like can be YA modifications. Exemplary bases that can stabilize RNA structures The modifications are pseudouridine and 5-methylcytosine. At least one nucleotide of the YA site is modified. In the YA site, the pyrimidine (also referred to as the "pyrimidine position") is modified (e.g., 2' A modification that changes the internucleoside linkage immediately 3' to the sugar of a pyrimidine at the position In some embodiments, the YA site comprises a nucleotide sequence that is an aryl group, a base modification, and a ribose modification. The adenine (also referred to as the "adenine position") is modified (e.g., a modification that changes the internucleoside linkage immediately 3' to the ribose of the pyrimidine at the 2' position, a modification of the pyrimidine base, and a modification of the ribose). In some embodiments, the pyrimidine and adenine of the YA site include modifications. In some embodiments, the YA modification reduces RNA endonuclease activity.

[0072] The above modifications and their equivalents are included within the scope of the embodiments described herein.

[0073] Domains / regions of the sgRNA Briner AE et al., Molecular Cell 56:333- 339 (2014) describes the functional domains of the sgRNA, herein referred to as "domains", which include a "spacer" domain responsible for targeting, a "lower stem", a "bulge", an "upper stem" (which may include a tetraloop), a "nexus", and "hairpin 1" and "hairpin 2" domains. See Briner et al., page 334, Figure 1A.

[0074] Table 3 provides a schematic diagram of the domains of the sgRNA used herein. In Table 3, "n" between regions represents a variable number of nucleotides, e.g., 0 to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more. In some embodiments, n is equal to 0. In some embodiments, n is equal to 1.

[0075] 5' terminal region ​​​​​In some embodiments, the sgRNA or short sgRNA includes nucleotides at the 5' end as shown in Table 3 , and includes nucleotides at the 5' end. In some embodiments, the 5' end of the sgRNA or short sgRNA includes a spacer or guide region that functions to direct a Cas protein, such as Cas9 protein, to a target nucleotide sequence. In some embodiments, the 5' end does not include a guide region. In some embodiments, the 5' end includes a spacer and additional nucleotides that do not function to direct the Cas protein to the target nucleotide region.

[0076] Lower stem In some embodiments, the sgRNA or short sgRNA includes a lower stem (LS) region separated by a loop region and an upper stem region when viewed linearly. See Table 3.

[0077] In some embodiments, the lower stem region includes 1 to 12 nucleotides. For example, in one embodiment, the lower stem region includes LS1-LS12. In some embodiments, the lower stem region includes fewer nucleotides than shown in Table 3. In some embodiments, the lower stem region includes more nucleotides than shown in Table 3. If the lower stem region includes fewer or more nucleotides than shown in the schematic of Table 3, it is necessary to maintain the modification pattern, as will be apparent to those skilled in the art.

[0078] In some embodiments, the lower stem region has nucleotides that are complementary in the nucleic acid sequence when read in the opposite direction. In some embodiments, the nucleic acid sequence of the lower stem Complementarity in the columns results in the secondary structure of the stem in the sgRNA or short sgRNA (e.g., the regions can form base pairs with each other). In some embodiments, the lower stem region may not be completely complementary to each other when read in opposite directions.

[0079] Bulge In some embodiments, the sgRNA or short sgRNA includes a bulge region containing 6 nucleotides B 1 to B6. When viewed linearly, the bulge region is separated into two regions. See Table 3. In some embodiments, the bulge region contains 6 nucleotides and is followed by the first 2 nucleotides, then the upper stem region, and then the last 4 nucleotides of the bulge. In some embodiments, the bulge region contains fewer nucleotides than shown in Table 3. In some embodiments, the bulge region contains more nucleotides than shown in Table 3. If the bulge region contains fewer or more nucleotides than shown in the schematic of Table 3, it is necessary to maintain the modification pattern as will be apparent to those skilled in the art.

[0080] In some embodiments, the presence of the bulge results in a directional constriction between the upper stem module and the lower stem module in the sgRNA or short sgRNA.

[0081] Upper stem In some embodiments, the sgRNA or short sgRNA includes an upper stem region containing 12 nucleotides . In some embodiments, the upper stem region contains a loop sequence . In some examples, the loop is a tetraloop (a loop consisting of 4 nucleotides ).

[0082] ​​ In some embodiments, the upper stem region contains fewer nucleotides than shown in Table 3. In some embodiments, the upper stem region contains more nucleotides than shown in Table 3. If the upper stem region contains fewer or more nucleotides than shown in the schematic of Table 3, it is necessary to maintain the modification pattern, as will be apparent to those skilled in the art.

[0083] In some embodiments, the upper stem region has nucleotides that are complementary in the nucleic acid sequence when read in the opposite direction. In some embodiments, the complementarity in the nucleic acid sequence of the upper stem results in the secondary structure of the stem in the sgRNA or short sgRNA (e.g., the regions can form base pairs with each other). In some embodiments, the upper stem region may not be completely complementary to each other when read in the opposite direction.

[0084] Nexus In some embodiments, the sgRNA or short sgRNA includes a nexus region located between the lower stem region and the hairpin 1 region. In some embodiments, the target sequence contains 18 nucleotides. In some embodiments, the nexus region contains nucleotides N1 to N18 shown in Table 3.

[0085] In some embodiments, the nexus region contains fewer nucleotides than shown in Table 3. In some embodiments, the nexus region contains more nucleotides than shown in Table 3. If the nexus region contains fewer or more nucleotides than shown in the schematic of Table 3, it is necessary to maintain the modification pattern, as will be apparent to those skilled in the art.

[0086] In some embodiments, the nexus region has nucleotides that are complementary in the nucleic acid sequence when read in the opposite direction. In some embodiments, the complementarity in the nucleic acid sequence results in the secondary structure of a stem and / or stem-loop in the sgRNA or short sgRNA (e.g., specific nucleotides in the nexus region can base pair with each other). In some embodiments, the nexus region may not be completely complementary to each other when read in the opposite direction. Hairpin In some embodiments, the sgRNA or short sgRNA contains one or more hairpin regions. In some embodiments, the hairpin region is downstream (e.g., on the 3' side) of the nexus region.

[0087] Hairpin In some embodiments, the sgRNA or short sgRNA contains one or more hairpin regions. In some embodiments, the hairpin region is downstream (e.g., on the 3' side) of the nexus region. In some embodiments, the region of nucleotides immediately downstream of the nexus region is referred to as "hairpin 1" or "H1". In some embodiments, the region from nucleotide 3' to the hairpin 1 region is referred to as "hairpin 2" or "H2". In some embodiments, the hairpin region includes both hairpin 1 and hairpin 2. In some embodiments, the sgRNA or short sgRNA includes hairpin 1 or hairpin 2. In some embodiments, the hairpin 1 region includes 12 nucleic acids immediately downstream of the nexus region.

[0088] In some embodiments, the hairpin 1 region includes nucleotides H1-1 to H1-12 as shown in Table 3. In some embodiments, the hairpin 1 region includes 12 nucleic acids immediately downstream of the nexus region. In some embodiments, the hairpin 1 region includes nucleotides H1-1 to H1-12 as shown in Table 3.

[0089] In some embodiments, the hairpin 2 region includes 15 nucleic acids downstream of the hairpin 1 region. In some embodiments, the hairpin 2 region includes nucleotides H2 as shown in Table 3. -1 to H2-15 are included.

[0090] In some embodiments, one or more nucleotides are present between the hairpin 1 region and the hairpin 2 region. One or more nucleotides between the hairpin 1 region and the hairpin 2 region can be modified or unmodified. In some embodiments, hairpin 1 and hairpin 2 are separated by one nucleotide. In some embodiments, the hairpin region contains fewer nucleotides than shown in Table 3. In some embodiments, the hairpin region contains more nucleotides than shown in Table 3. When the hairpin region contains fewer or more nucleotides than shown in the schematic diagram of Table 3, it will be apparent to those skilled in the art that the modification pattern needs to be maintained.

[0091] In some embodiments, it has nucleotides that are complementary in the nucleic acid sequence when read in the opposite direction. In some embodiments, the hairpin regions may not be completely complementary to each other when read in the opposite direction (e.g., the top or loop of the hairpin contains nucleotides that do not pair up).

[0092] In some embodiments, the sgRNA or short sgRNA includes replacing hairpin 1 with nucleotide "n", where "n" is an integer from 1 to 50, 40, 30, 20, 15, 1 0, 5, 4, 3, and 2. In some embodiments, the hairpin 1 region of the sgRNA is replaced with two nucleotides.

[0093] 3' end The sgRNA or short sgRNA is the 3' end, which is the last nucleotide of the sgRNA. ​​​​​​​It has. The 3'-terminal region contains the last 1 to 7 nucleotides from the 3'-end. In some embodiments, the 3'-end is the end of hairpin 2. In some embodiments , the sgRNA contains nucleotides after the hairpin region(s). In some embodiments the sgRNA contains a 3'-terminal region, in which case the last nucleotide of the 3'-end is the 3'-end. In some embodiments, the 3'-tail contains, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 or more nucleotides that do not associate with the secondary structure of the hairpin. In some embodiments the 3'-tail region contains 1, 2, 3, or 4 nucleotides that do not associate with the secondary structure of the hairpin. In some embodiments the 3'-tail region contains 4 nucleotides that do not associate with the secondary structure of the hairpin. In some embodiments the 3'-tail region contains 1, 2, or 3 nucleotides that do not associate with the secondary structure of the hairpin. In some embodiments the 3'-tail region contains 1, 2, or 3 nucleotides that do not associate with the secondary structure of the hairpin.

Table 2

Table 3

[0094] gRNA containing modifications including modification of the YA site In some embodiments, the gRNA (e.g., sgRNA, short sgRNA, dgRNA, or crRNA) described herein has 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 0, 11, 12, 13, 14, 15, 16 or more modifications in the YA site(s) (e.g., in the conserved region and / or the guide region), and / or at the 5'-end of the 5'-terminal portion and / or YA modifications at nucleotide 6 from or one or more nucleotides located downstream thereof include modifications such as those described below. In some embodiments, the pyrimidine of the YA site includes a modification (including a modification that changes the internucleoside bond immediately 3' to the sugar of the pyrimidine ). In some embodiments, the adenine of the YA site includes a modification (including a modification that changes the internucleoside bond immediately 3' to the sugar of the adenine ). In some embodiments, the pyrimidine and adenine of the YA site include modifications such as sugar, base, or internucleoside bond modifications. The YA modification can be any of the types of modifications described herein. In some embodiments, the YA modification includes one or more of phosphorothioate, 2'-OMe, or 2'-fluoro . In some embodiments, the YA modification includes a pyrimidine modification that includes one or more of phosphorothioate, 2'-OMe, or 2'-fluoro . In some embodiments, the YA modification includes a bicyclic ribose analog (e.g., LNA, BNA, or ENA) within an RNA duplex region that includes one or more YA sites . In some embodiments, the YA modification includes a bicyclic ribose analog (e.g., LNA, BNA, or ENA) within an RNA duplex region that includes the YA site, and the YA modification is distal to the YA site . Any of the above embodiments can be combined with the following: (i) at least one of nucleotides 8-11, 13, 14, 17, or 18 from the 5' end of the 5' end portion does not include a 2' -fluoro modification, and / or (ii) at least one of nucleotides 6-10 from the 5' end of the 5' end portion does not include a phosphorothioate bond . . . . .

[0095] . -fluoro modification, and / or (ii) at least one of nucleotides 6-10 from the 5' end of the 5' end portion does not include a phosphorothioate bond, and . . in (i) at least one of nucleotides 7 to 10 from the 5'-end of the 5'-terminal portion does not contain a 2'-OMe modification, (ii) nucleotide 20 from the 5'-end of the 5'-terminal portion does not contain a 2'-OMe modification, and / or (iii) alternatively, the guide RNA contains a 2'- fluoro modification at any one or more of nucleotides 1 to 20 from the 5'-end of the 5'-terminal portion, and at least one of nucleotides 11, 12, 13, 14, 17, or 18 from the 5'-end of the 5'-terminal portion does not contain a 2'-fluoro modification, and optionally further, nucleotide 12 from the 5'-end of the 5'-terminal portion does not contain a 2'-fluoro modification . Such embodiments may further or alternatively be combined, to the extent possible, with any one or more other embodiments described herein. Modifications of the guide region including modifications of the YA site In some embodiments, the guide region contains one or more modifications and optionally contains a YA site modification. In some embodiments, the guide region may contain 1,

[0096] 2, 3, 4, 5 or more YA sites ( "guide region YA sites") that may contain a YA modification. In some embodiments, one or more YA sites located at the 5'-end, 6'-end, 7'-end, 8'-end, 9'-end or 10'-end from the 5'-end of the 5'-terminal portion ( "5'-end", etc. refers to the 5th position relative to the 3'-end of the guide region, i.e., the most 3'-nucleotide in the guide region) contain a YA modification. In some embodiments, two or more YA sites located at the 5'-end, 6'-end, 7'-end, 8'-end or 10'-end from the 5'-end of the 5'-terminal portion contain a YA modification. In some embodiments, two or more YA sites located at the 5'-end, 6'-end, 7'-end, 8'-end or 10'-end from the 5'-end of the 5'-terminal portion contain a YA modification. In some embodiments, two or more YA sites located at the 5'-end, 6'-end, 7'-end, 8'-end or 10'-end from the 5'-end of the 5'-terminal portion contain a YA modification. In some embodiments, two or more YA sites located at the 5'-end, 6'-end, 7'-end, 8'-end or 10'-end from the 5'-end of the 5'-terminal portion contain a YA modification. In some embodiments, two or more YA sites located at the 5'-end, 6'-end, 7'-end, 8'-end or 10'-end from the 5'-end of the 5'-terminal portion contain a YA modification. In some embodiments, two or more YA sites located at the 5'-end, 6'-end, 7'-end, 8'-end , three or more YA sites located at the 9-end or 10-end contain YA modifications. In some embodiments, four or more YA sites located at the 5-end, 6-end, 7-end, 8-end, 9-end, or 10-end from the 5'-end of the 5'-terminal portion contain YA modifications. In some embodiments five or more YA sites located at the 5-end, 6-end, 7-end, 8-end, 9-end, or 10 end from the 5'-end of the 5'-terminal portion contain YA modifications. The modified guide region YA site contains a Y A modification.

[0097] In some embodiments, the modified guide region YA site is within 17, 16, 15, 14, 13, 12, 11, 10, or 9 nucleotides of the 3'-terminal nucleotide of the guide region. For example, if the modified guide region YA site is within 10 nucleotides of the 3'-terminal nucleotide of the guide region and the guide region is 20 nucleotides in length, the modified nucleotide of the modified guide region YA site is located at any of positions 11-20. In some embodiments, the YA modification is located at 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide within the YA site from the 3'-terminal nucleotide of the guide region. In some embodiments the YA modification is located at 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide from the 3'-terminal nucleotide of the guide region. In some embodiments, the modified guide region YA site is nucleotide 4, 5, 6, 7, 8, 9, 10, or 11 from the 5'-end of the 5'-terminal portion. nucleotide from the 3'-terminal nucleotide of the guide region. is located at 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1

[0098] In some embodiments, the modified guide region YA site is nucleotide 4, 5, 6, 7, 8, 9, 10, or 11 from the 5'-end of the 5'-terminal portion. nucleotide 4, 5, 6, 7, 8, 9, 10, or 11 from the 5'-end of the 5'-terminal portion.

[0099] In some embodiments, the modified guide region YA site is other than a 5'-end modification. For example, the gRNA can include the 5'-end modification described herein and further include the modified guide region YA site. Alternatively, the gRNA can include an unmodified 5'-end and the modified guide region YA site. Alternatively, the gRNA can include a modified 5'-end and an unmodified guide region YA site.

[0100] In some embodiments, the modified guide region YA site includes a modification that does not include at least one nucleotide located on the 5'-side of the guide region YA site. For example, nucleotides 1 to 3 include phosphorothioate, nucleotide 4 includes only a 2'-OMe modification, and if nucleotide 5 is a pyrimidine of the YA site and includes phosphorothioate, the modified guide region YA site includes a modification (phosphorothioate) that does not include at least one nucleotide located on the 5'-side of the guide region YA site (nucleotide 4). In another example, nucleotides 1 to 3 include phosphorothioate, nucleotide 4 is a pyrimidine of the YA site and includes 2'-OMe, and the modified guide region YA site includes a modification (2'-OMe) that does not include at least one nucleotide (any of nucleotides 1 to 3) located on the 5'-side of the guide region YA site. This condition is also always satisfied when unmodified nucleotides are located on the 5'-side of the modified guide region YA site.

[0101] In some embodiments, the guide region includes 1 to 14 modifications among nucleotides 1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 13, 14, 17, and 18 of the guide region. ​​​​​​including. Such modifications can be 2'-OMe, 2'-fluoro, 2'-H, inosine, or phosphorothioate modifications, or combinations thereof. For example, 2'-O Me modifications may be included in any or all of nucleotides 1-4 and 12, and pho sphorothioate modifications may be included in any or all of nucleotides 1-3 and 6-10, and / or 2'-fluoro modifications may be included in any or all of nucleotides 8-11, 13, 14, 1 7, and 18. In negative terms, 2'-OMe modifications may be excluded from nucleotides 6-11 and the 13 terminus, 2'-fluoro modifications may be excluded from nucleotides 1-7, 15, 16, and 20 (if present), and / or phosphorothioate modifications may be excluded from nucleotides 4, 5, 11-14, 17, and 18. In some embodiments, the nucleotides are modified in a YA-site-dependent manner. For example, if the YA-site is present in any of nucleotides 5-6, 12-13, 15-16, 16- 17, or 19-20, at least one nucleotide of the YA-site is modified. For example, at least the pyrimidine of the YA-site is modified. Optionally, nucleotides at positions 5, 12, 15, 16, and 19 are unmodified if they are not pyrimidines of the YA-site. In some embodiments, the modification at nucleotide 5 is 2'-OMe if the modification is a pyrimidine of the YA-site, and the modification at nucleotide 12 is 2'-OMe if the modification is a pyrimidine of the YA-site, the modification at nucleotide 15 is phosphorothioate if the modification is a pyrimidine of the YA-site, and the modification at nucleotide 16 is 2'-OMe if the modification is a pyrimidine of the YA-site. The modification at nucleotide 15 is phosphorothioate if the modification is a pyrimidine of the YA-site. pyrimidine of the YA-site. When it is a phosphothioate, and / or when the modification at nucleotide 19 is such that when the modification is a pyrimidine at the YA site, it is a phosphothioate. Thus, it is recognized that the YA site cannot be present at both positions 15 - 16 and 16 - 17, and, conditional on the presence of the YA site, up to 4 modifications can be present. In an alternative embodiment, the modification at nucleotide 19 can instead be 2'-fluoro. This can be present in a YA site-dependent manner or can be present regardless of whether there is a YA site at positions 19 - 20. In some embodiments, nucleotides 15 and 16 are unmodified or modified only by phosphothioate, e.g., modified only by nucleotides that are pyrimidines at the YA site located at nucleotides 15 - 16 or 16 - 17. In some embodiments, nucleotides 15 and 16 contain unmodified ribose and / or unmodified nucleobases. In some embodiments, nucleotide 5 is unmodified or modified only by 2'-OMe when it is a pyrimidine at the YA site. In some embodiments, nucleotide 12 is unmodified or modified only by 2'-OMe when it is a pyrimidine at the YA site. In some embodiments, nucleotide 20 (or the 3'-terminal nucleotide of the guide region) is unmodified. In any of the previous embodiments, the guide region can consist of 20 nucleotides. In some embodiments, the gRNA is 1 of nucleotides 5 and / or 12 modified. In any of the previous embodiments, the guide region can consist of 20 nucleotides. In some embodiments, the gRNA is 1 of nucleotides 5 and / or 12

[0102] modified. It includes a guide region containing modifications in two or more. The modifications at nucleotides 5 and / or 12 can independently be selected from the modifications described herein, for example, 2'-OMe, 2'-F, phosphorothioate, and 2'-H (deoxyribonucleotide). Such modifications can be combined with other modification patterns or nucleotide modifications described herein, as shown, for example, in the gRNAs described herein. Specific examples of such embodiments are described herein, for example, in the specific numbered embodiments described above and in the modification patterns represented by the sequences in the sequence listing. In some embodiments, such modifications are combined with one or more or all of the 2'-OMe modifications at nucleotides 1-4, the phosphorothioate modifications at nucleotides 1-3 and 6-10, and / or the 2'-F modifications at nucleotides 8-11, 13, 14, 17, and 18. The modifications can independently be selected from the modifications described herein, for example, 2'-OMe, 2'-F, phosphorothioate, and 2'-H (deoxyribonucleotide). Such modifications can be combined with other modification patterns or nucleotide modifications described herein, as shown, for example, in the gRNAs described herein. Specific examples of such embodiments are described herein, for example, in the specific numbered embodiments described above and in the modification patterns represented by the sequences in the sequence listing. In some embodiments, such modifications are combined with one or more or all of the 2'-OMe modifications at nucleotides 1-4, the phosphorothioate modifications at nucleotides 1-3 and 6-10, and / or the 2'-F modifications at nucleotides 8-11, 13, 14, 17, and 18. In some embodiments, the gRNA includes a guide region containing modifications at any one, two, or all of nucleotides 8-10. Such modifications can be combined with other modification patterns or nucleotide modifications described herein, as shown, for example, in the gRNAs described herein. The modifications can independently be selected from the modifications described herein, for example, 2'-F modifications and phosphorothioate modifications, or combinations thereof. In some embodiments, any one, two, or all of nucleotides 8-10 include 2'-F modifications. In some embodiments, any one, two, or all of nucleotides 8-10 include 2'-F modifications but phosphorothioate modifications. In some embodiments, the gRNA includes a guide region containing modifications at any one, two, or all of nucleotides 8-10. Such modifications can be combined with other modification patterns or nucleotide modifications described herein, as shown, for example, in the gRNAs described herein. The modifications can independently be selected from the modifications described herein, for example, 2'-F modifications and phosphorothioate modifications, or combinations thereof. In some embodiments, any one, two, or all of nucleotides 8-10 include 2'-F modifications. In some embodiments, any one, two, or all of nucleotides 8-10 include 2'-F modifications but phosphorothioate modifications.

[0103] In some embodiments, the gRNA includes a guide region containing modifications at any one of nucleotides 8-10, two, or all. Such modifications can be combined with other modification patterns or nucleotide modifications described herein, as shown, for example, in the gRNAs described herein. Such modifications can independently be selected from the modifications described herein, for example, 2'-F modifications and phosphorothioate modifications, or combinations thereof. In some embodiments, any one, two, or all of ...

Claims

1. A guide RNA (gRNA) which is a short single guide RNA (short sgRNA) including a guide region and a conserved portion of the sgRNA, The aforementioned storage portion is from 5' to 3'. (i) The lower stem region, which consists of nucleotides LS1 to LS6. (ii) The bulge region, which consists of nucleotides B1 to B2. (iii) The upper stem region, which consists of nucleotides US1 to US12. (iv) The bulge region, which consists of nucleotides B3 to B6. (v) The lower stem region, which consists of nucleotides LS7 to LS12. (vi) Nexus regions consisting of nucleotides N1 to N18, and (vii) A hairpin area, from 5' to 3', a) Hairpin 1 region, which is nucleotide H1-1 to H1-12 b) Nucleotides, and c) Hairpin 2 regions consisting of nucleotides H2-1 to H2-15 The hairpin region including Includes, Here, (1) The hairpin region lacks 5 to 10 nucleotides, (2) The short sgRNA includes a 5' terminal modification, a 3' terminal modification, or both, (3) At least 10 nucleotides in the upper stem region include a 2'-O-methyl (2'-O-Me) modification, The aforementioned guide RNA.

2. The gRNA according to claim 1, wherein the short sgRNA includes a 3' tail.

3. The gRNA according to claim 1, wherein the 5 to 10 missing nucleotides are consecutive.

4. The 5 to 10 missing nucleotides are (i) Within the hairpin 1 region, (ii) Within the hairpin 1 region and the nucleotide between the hairpin 1 region and the hairpin 2 region, (iii) Within the hairpin 1 region and the two nucleotides immediately 3' to the side of the hairpin 1 region, (iv) Including at least a portion of the hairpin 1 region, The gRNA according to claim 1.

5. (i) The modification in the hairpin region comprises a modified nucleotide selected from a 2'-O-methyl (2'-OMe) modified nucleotide, a 2'-fluoro (2'-F) modified nucleotide, an internucleotide phosphorothioate (PS) bond, or a combination thereof. (ii) The 3' or 5' terminal modification comprises or further comprises a 2'-O-methyl (2'-OMe) modified nucleotide, (iii) The 3' or 5' terminal modification comprises or further comprises a 2'-O-(2-methoxyethyl)(2'-O-moe) modified nucleotide, (iv) The 3' or 5' terminal modification comprises or further comprises a 2'-fluoro(2'-F) modified nucleotide, (v) The 3' or 5' terminal modification comprises or further comprises an internucleotide phosphorothioate (PS) bond, (vi) The 3' or 5' terminal modification comprises or further comprises an inverted debasicized nucleotide, (vii) The modification in the hairpin region comprises or further comprises a 2'-O-methyl (2'-OMe) modified nucleotide. (viiii) The modification in the hairpin region comprises or further comprises a 2'-fluoro(2'-F) modified nucleotide, (ix)(i) to (viiii) are combinations of two or more of the following: The gRNA according to claim 1.

6. The gRNA according to claim 1, wherein the 5 to 10 nucleotides include nucleotides H1-6 to H1-12 and the nucleotides between H1-12 and H2-1, nucleotides H1-5 to H1-12, nucleotides H1-6 to H1-10, or nucleotides H1-5 to H1-8.

7. The gRNA according to claim 6, wherein the short sgRNA includes modifications of at least H1-1 to H1-5 and H2-1 to H2-12.

8. From 5' to 3', a guide region, and the following: GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGCACCGAGUCGG*mU*mG*mC (Sequence ID 206), GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGmGmCmAmCmCmGmAmGmUmCmGmG*mU*mG*mC (Sequence ID 207) GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGCACCGAGUCGG*mU*mG*mC (Sequence ID 215) GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGmGmCmAmCmCmGmAmGmUmCmGmG*mU*mG*mC (Sequence ID 216) GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGCACCGAGUCGG*mU*mG*mC (Sequence ID 223), GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGmGmCmAmCmCmGmAmGmUmCmGmG*mU*mG*mC (Sequence ID 224) GUUUUAGAmGmCmUmAmGmAmAmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmGmCmAmCmCmGmAmGmUmCmGmG*mU*mG*mC (Sequence ID 228) GUUUUAGAmGmCmUmAmGmAmAmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmA*mG*mU*mG (Sequence ID 231) GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAfAfCfUfUfGmGmCmAmCmCmGmAmGmUmCmGmG*mU*mG*mC (Sequence ID 241) GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAfAmCfUmUfGmGfCmAfCmCfGmAfGmUfCmGfG*mU*fG*mC (Sequence ID 242) GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGCACCGAGUCGG*mU*mG*mC (Sequence ID 245), GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGmGmCmAmCmCmGmAmGmUmCmGmG*mU*mG*mC (Sequence ID 246) GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGCACCGAGUCGG*mU*mG*mC (Sequence ID 247) GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGmGmCmAmCmCmGmAmGmUmCmGmG*mU*mG*mC (Sequence ID 248) GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGCACCGAGUCGG*mU*mG*mC (Sequence ID 250), GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGmGmCmAmCmCmGmAmGmUmCmGmG*mU*mG*mC (Sequence ID 251) GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGCACCGAGUCGG*mU*mG*mC (Sequence ID 253), or GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGCACCGAGUCGG*mU*mG*mC (Sequence ID 254) A guide RNA (gRNA) is a short sgRNA that contains a conserved portion which includes one of the following: Here, m represents a nucleotide modified with 2'-O-Me, f represents a nucleotide modified with 2'-F, and * represents a nucleotide linked to an adjacent nucleotide by a phosphorothioate (PS) bond. The aforementioned guide RNA.

9. The guide region of the gRNA comprises at least 20 nucleotides, and furthermore: (1) mN*mN*mN*NNNNNNNNNNNNNNNNN, (2) (invd)mNmNmNNNNNNNNNNNNNNNNNN, (3) (invd)mN*mN*mN*NNNNNNNNNNNNNNNNNN, (4) mN*mN*mN*NNN*N*fN*fN*fN*fNNfNfNNNfNfNNN, (5) moeN*moeN*moeN*NNNNNNNNNNNNNNNNN, or (6) (invd)NNNNNNNNNNNNNNNNNNNN The modified and / or unmodified nucleotides comprise at least 15, 16, 17, 18, 19, or 20 of nucleotides 1 to 20, which match the modification pattern selected from: Here, N is a natural or non-natural nucleotide, "mN" indicates a nucleotide modified with a 2'-O-Me modified nucleotide, "fN" indicates a nucleotide modified with a 2'-fluoro(2'-F) modified nucleotide, "moeN" indicates a nucleotide modified with a 2'-O-(2-methoxyethyl)(2'-O-moe) modified nucleotide, "*" indicates a phosphorothioate (PS) bond between two adjacent nucleotides, and "(invd)" indicates an inverted debased modified nucleotide. The gRNA according to claim 1.

10. The guide region of the gRNA comprises at least 20 nucleotides, and furthermore: (1) mN*mN*mN*NNNNNNNNNNNNNNNNN, (2) (invd)mNmNmNNNNNNNNNNNNNNNNNN, (3) (invd)mN*mN*mN*NNNNNNNNNNNNNNNNNN, (4) mN*mN*mN*NNN*N*fN*fN*fN*fNNfNfNNNfNfNNN, (5) moeN*moeN*moeN*NNNNNNNNNNNNNNNNN, or (6) (invd)NNNNNNNNNNNNNNNNNNNN The modified and / or unmodified nucleotides comprise at least 15, 16, 17, 18, 19, or 20 of nucleotides 1 to 20, which match the modification pattern selected from: Here, N is a natural or non-natural nucleotide, "mN" indicates a nucleotide modified with a 2'-O-Me modified nucleotide, "fN" indicates a nucleotide modified with a 2'-fluoro(2'-F) modified nucleotide, "moeN" indicates a nucleotide modified with a 2'-O-(2-methoxyethyl)(2'-O-moe) modified nucleotide, "*" indicates a phosphorothioate (PS) bond between two adjacent nucleotides, and "(invd)" indicates an inverted debased modified nucleotide. The gRNA according to claim 8.

11. The gRNA according to claim 1, wherein the conserved portion of the gRNA includes a modified sequence that is at least 90% identical to any one of the gRNAs of sequence numbers 206, 207, 215, 216, 223, 224, 228, 231, 241, 242, 245-248, 250, 251, 253, or 254.

12. A lipid nanoparticle (LNP) composition comprising the gRNA described in any one of claims 1 to 11.

13. A composition comprising the gRNA according to any one of claims 1 to 11, associated with lipid nanoparticles (LNPs).

14. A composition selected from a composition comprising the gRNA described in any one of claims 1 to 11, a lipid nanoparticle (LNP) composition comprising the gRNA, and a composition comprising the gRNA associated with lipid nanoparticles (LNP), wherein the composition further comprises a nuclease or mRNA encoding a nuclease.

15. The composition according to claim 14, wherein the nuclease is a Cas protein.

16. The composition according to claim 15, wherein the Cas protein is Cas9.

17. The composition according to claim 16, wherein Cas9 is S. pyogenes Cas9 or S. aureus Cas9.

18. The composition according to claim 14, wherein the nuclease is nicasse or dCas.

19. The composition according to claim 14, wherein the mRNA comprises one of the sequences 3499 to 3527 or 3529 to 3546.

20. (i) gRNA according to any one of claims 1 to 11, a lipid nanoparticle (LNP) composition containing the gRNA, or a composition containing the gRNA associated with lipid nanoparticles (LNP), and (ii) Pharmaceutically acceptable carriers Pharmaceutical preparations, including those mentioned above.

21. A method for modifying target DNA, Cas protein or nucleic acid encoding Cas protein, and (i) gRNA according to any one of claims 1 to 11, (ii) Any of the following: a lipid nanoparticle (LNP) composition containing the gRNA, and a composition containing the gRNA associated with lipid nanoparticles (LNP), and (iii) A pharmaceutical formulation comprising one of the gRNA or the composition and a pharmaceutically acceptable carrier. This includes delivering one or more of the following to ex vivo cells: The aforementioned method.

22. The method according to claim 21, wherein the method results in an insertion or deletion in a gene.

23. The method according to claim 21, further comprising delivering a template to the cell, wherein at least a portion of the template is incorporated into target DNA at or near a double-strand break site induced by the Cas protein.

24. A pharmaceutical product for treating a disease or disorder, wherein the pharmaceutical product is gRNA according to any one of claims 1 to 11, Lipid nanoparticle (LNP) composition containing the gRNA, A composition comprising the gRNA associated with lipid nanoparticles (LNPs), or A pharmaceutical formulation comprising either the gRNA or the composition and a pharmaceutically acceptable carrier. The aforementioned pharmaceutical, including the above.

25. A pharmaceutical agent for modifying target DNA inside or within an object, wherein the pharmaceutical agent is gRNA according to any one of claims 1 to 11, Lipid nanoparticle (LNP) composition containing the gRNA, A composition comprising the gRNA associated with lipid nanoparticles (LNPs), or A pharmaceutical formulation comprising either the gRNA or the composition and a pharmaceutically acceptable carrier. The aforementioned pharmaceutical, including the above.