Modified guide RNA for gene editing

Abstract

The present disclosure relates to modified guide RNAs with improved in vitro and in vivo activity in gene editing methods. [Selection diagram] None

Description

Technical Field

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

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

[0003] Oligonucleotides, particularly RNA, may be degraded in cells and in serum by non-enzymatic cleavage, endonuclease cleavage, or exonuclease cleavage. Improved methods and compositions for preventing such degradation, enhancing the stability of gRNA, and enhancing gene editing efficiency are particularly desirable for therapeutic applications.

Summary of the Invention

[0004] In some embodiments, genome editing tools comprising modified guide RNA (gRNA) are provided. The gRNA modifications described herein may improve the stability of the gRNA and the gRNA / Cas9 complex and improve the activity of Cas9 (e.g., SaCas9, spyCas9, and equivalents) that cleave target DNA.

[0005] In some embodiments, modified crRNA (crRNA) and / or modified tracrRNA (trRNA) are provided. In some embodiments, the modified crRNA and / or modified trRNA include a dual guide RNA (dgRNA). In some embodiments, the modified crRNA and / or modified trRNA include a single guide RNA (sgRNA). The modifications of crRNA and / or trRNA described herein may improve the stability of gRNA and gRNA / Cas9 complexes and improve the activity of Cas9 (e.g., SauCas9, spyCas9, and equivalents) that cleave target DNA. In some embodiments, the crRNA portion of the dgRNA or sgRNA is modified in the targeting domain.

[0006] In some embodiments, genome editing tools are provided that include a short single guide RNA (short sgRNA). In some embodiments, the short sgRNA is modified. The short sgRNAs described herein may improve the stability of the short sgRNA and the short sgRNA / Cas9 complex, and improve the activity of Cas9 (e.g., SauCas9, spyCas9, and equivalents) that cleave target DNA.

[0007] In some embodiments, gRNAs (e.g., sgRNAs, short sgRNAs, dgRNAs, or crRNAs) include modifications at one or more YA sites, as described, for example, in the embodiments, Table 1, and the examples and associated figures below. To avoid doubt, sgRNAs include, but are not limited to, short sgRNAs. As discussed in the Examples section, gRNAs have been found to be highly sensitive to RNase A-like degradation patterns, including, for example, cleavage at unmodified YA sites. Furthermore, YA site modifications can reduce or eliminate such cleavage, and many YA site modifications have been found to be tolerable without adversely affecting the ability to induce cleavage by nucleases such as Cas9. Certain gRNA sites, including, but not limited to YA sites, can be modified despite other descriptions that they should not be modified due to concerns that they may be contacted by Cas9 and lose activity (Yin et al., Nature Biotechnol. 35:1179-1187 (2017)). Such modifications can further reduce undesirable gRNA degradation without impairing activity.

[0008] The following embodiments are included. Embodiment 01 is a guide RNA (gRNA) which is a short single guide RNA (short sgRNA) containing a conserved portion of the sgRNA including a hairpin region, 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' terminal modification. Embodiment 03 is a gRNA described in any one of Embodiments 1 to 2, wherein the short sgRNA includes a 3' terminal modification. Embodiment 04 is a gRNA according to any one of Embodiments 1 to 3, wherein the short sgRNA includes a 5' terminal modification and a 3' terminal modification. Embodiment 05 is a gRNA described in any one of Embodiments 1 to 4, wherein the short sgRNA includes a 3' tail. Embodiment 06 is the gRNA according to Embodiment 5, wherein the 3' tail contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. Embodiment 07 is the gRNA according to Embodiment 5, wherein 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. Embodiment 08 is a gRNA described in any one of Embodiments 1 to 7, wherein the short sgRNA does not contain a 3' tail. Embodiment 09 is a gRNA according to any one of Embodiments 1 to 8, which includes modifications in the hairpin region. Embodiment 10 is a gRNA according to any one of Embodiments 1 to 9, which includes 3' terminal modification and modification in the hairpin region. Embodiment 11 is a gRNA according to any one of Embodiments 1 to 10, comprising a 3' terminal modification, a modification in the hairpin region, and a 5' terminal modification. Embodiment 12 is a gRNA according to any one of Embodiments 1 to 11, including 5' terminal modification and modification in the hairpin region. Embodiment 13 is a gRNA according to any one of Embodiments 1 to 12, having at least 5 to 10 consecutive missing nucleotides. Embodiment 14 has at least 5 to 10 missing nucleotides, i. Inside hairpin 1, ii. Within the "N" between hairpin 1 and hairpin 1 and hairpin 2, iii. In the two nucleotides located at hairpin 1 and immediately 3' to the side of hairpin 1, iv. Including at least a portion of hairpin 1, v. Inside hairpin 2, vi. Including at least a portion of hairpin 2, vii. Inside 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. Includes at least a portion of hairpin 1, includes "N" between hairpin 1 and hairpin 2, and includes at least a portion of hairpin 2, xi. Located within hairpin 1 or hairpin 2, optionally including "N" between hairpin 1 and hairpin 2. xii. Continuously, xiii. Consecutive, including "N" between hairpin 1 and hairpin 2, xiv. Continuously extending to at least a portion of hairpin 1 and a portion of hairpin 2, xv. Continuously extending to at least a portion of hairpin 1 and "N" between hairpin 1 and hairpin 2, or xvi. A gRNA according to any one of Embodiments 1 to 13, which is continuous and extends to at least a portion of hairpin 1 and two nucleotides immediately 3' of hairpin 1. Embodiment 15 is a gRNA according to any one of Embodiments 1 to 14, further comprising a guide region. Embodiment 16 is a gRNA according to any one of Embodiments 1 to 15, wherein the 3' and / or 5' terminal modifications include protective terminal modifications, such as 2'-O-methyl (2'-OMe) modified nucleotides, 2'-O-(2-methoxyethyl) (2'-O-moe) modified nucleotides, 2'-fluoro (2'-F) modified nucleotides, internucleotide phosphorothioate (PS) bonds, inverted debasitized modified nucleotides, or modified nucleotides selected from a combination thereof. Embodiment 17 is a gRNA according to any one of Embodiments 1 to 16, wherein the modification in the hairpin region includes a modified nucleotide selected from 2'-O-methyl (2'-OMe) modified nucleotide, 2'-fluoro (2'-F) modified nucleotide, internucleotide phosphorothioate (PS) bond, or a combination thereof. Embodiment 18 is a gRNA according to any one of Embodiments 1 to 17, wherein the 3' and / or 5' terminal modifications include or further include a 2'-O-methyl (2'-OMe) modified nucleotide. Embodiment 19 is a gRNA according to any one of Embodiments 1 to 18, wherein the 3' and / or 5' terminal modifications include or further include a 2'-fluoro(2'-F) modified nucleotide. Embodiment 20 is a gRNA according to any one of Embodiments 1 to 19, wherein the 3' and / or 5' terminal modifications include or further include internucleotide phosphorothioate (PS) bonds. Embodiment 21 is a gRNA according to any one of Embodiments 1 to 20, wherein the 3' and / or 5' terminal modifications include or further include an inverted debasitized nucleotide. Embodiment 22 is a gRNA according to any one of Embodiments 1 to 21, wherein the modification in the hairpin region includes or further includes a 2'-O-methyl (2'-OMe) modified nucleotide. Embodiment 23 is a gRNA according to any one of Embodiments 1 to 22, wherein the modification in the hairpin region includes or further includes a 2'-fluoro(2'-F) modified nucleotide. Embodiment 24 has a 3'-terminal modification, i. One or more modifications of the last 7, 6, 5, 4, 3, 2, or 1 nucleotide, ii. One modified nucleotide, iii. Two modified nucleotides, iv. Three modified nucleotides, v. 4 modified nucleotides, vi. 5 modified nucleotides, vii. Six modified nucleotides, and viii. A gRNA according to any one of Embodiments 1 to 23, comprising any of the seven modified nucleotides. Embodiment 25 is the gRNA according to any one of Embodiments 1 to 24, wherein 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 modifications H1-1 to H1-5 and H2-1 to H2-12. Embodiment 26 is such that at least 5 to 10 nucleotides i. consist of 5 to 10 nucleotides, ii. consist of 6 to 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. consist of 5 to 10 consecutive nucleotides, x. consist of 6 to 10 consecutive nucleotides, xi. consist of 5 consecutive nucleotides, xii. consist of 6 consecutive nucleotides, xiii. consist of 7 consecutive nucleotides, xiv. consist of 8 consecutive nucleotides, xv. consist of 9 consecutive nucleotides, or xvi. consist of 10 consecutive nucleotides, which is the gRNA according to any one of Embodiments 1 to 25. 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 The gRNA described in any one of Embodiments 1 to 26 includes one or more combinations of vi.(i.) to (v.). Embodiment 28 shows that a short sgRNA is i. Internucleotide phosphorothioate (PS) bonding, ii. 2'-OMe modified nucleotides, iii. 2'-O-moe modified nucleotides, iv. 2'-F modified nucleotide, v. Inverted debasic modified nucleotides, and The gRNA described in any one of Embodiments 1 to 27 includes a 3' tail and contains one or more combinations of vi.(i.) to (v.). Embodiment 29 shows that a short sgRNA is i. PS bonds between 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, ii. PS bonds between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or 18 nucleotides, iii. Approximately 1-3, 1-5, 1-6, 1-7, 1-8, 1-9, or 1-10 nucleotide-to-nucleotide PS bonds, iv. Approximately 1-3, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-12, 1-14, 1-16, 1-18, or 1-20 nucleotide-to-nucleotide PS bonds, v. A gRNA according to any one of Embodiments 1 to 28, comprising one or more PS bonds between each nucleotide. Embodiment 30 has a 3'-terminus modification that includes at least one PS bond. i. There is one PS bond, and the bond is between the last nucleotide and the second to last nucleotide. ii. Two PS bonds exist between the last three nucleotides. iii. A PS bond exists between one or more of the last four nucleotides. iv. A PS bond exists between one or more of the last five nucleotides. v. One or more gRNAs according to any one of Embodiments 1 to 29, wherein a PS bond exists between one or more of the last 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. Embodiment 31 is the gRNA according to Embodiment 31, wherein the 3'-terminal modification further comprises at least one 2'-OMe, 2'-O-moe, inverted debase, or 2'-F modified nucleotide. Embodiment 32 has a 3'-terminal modification, i. One or more modifications among the last 1 to 7 nucleotides, wherein the modification is a PS bond, an inverted debasalized nucleotide, 2'-OMe, 2'-O-moe, 2'-F, or a combination thereof. ii. Modification of the last nucleotide by 2'-OMe, 2'-O-moe, 2'-F, or a combination thereof, and one or two PS bonds of any choice to the adjacent nucleotide and / or the first nucleotide of the 3' tail. iii. Modification of the last and / or second-to-last nucleotide by 2'-OMe, 2'-O-moe, 2'-F, or a combination thereof, and optionally one or more PS bonds, iv. Modification of the last, second to last, and / or third to last nucleotide by 2'-OMe, 2'-O-moe, 2'-F, or a combination thereof, and optionally one or more PS bonds, v. Modification of the last, second to last, third to last, and / or fourth to last nucleotide by 2'-OMe, 2'-O-moe, 2'-F, or a combination thereof, and optionally, one or more PS bonds, vi. A gRNA according to any one of Embodiments 1 to 31, comprising modifications to the last, second to last, third to last, fourth to last, and / or fifth to last nucleotide by 2'-OMe, 2'-O-moe, 2'-F, or a combination thereof, and optionally containing one or more PS links. Embodiment 33 is a gRNA according to any one of Embodiments 1 to 32, wherein the sgRNA includes a 3' tail, and the 3' tail includes one or more modifications of the nucleotides present in the 3' tail. Embodiment 34 is the gRNA described in Embodiment 33, wherein the 3' tail is fully modified. Embodiment 35 is the gRNA described in Embodiment 33, wherein at least 5 to 10 nucleotides include 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 includes at least H1-1 to H1-5 and H2-1 to H2-12 modifications. Embodiment 36 is a short sgRNA, i. A 3' terminal modification shown in any one of sequence numbers 1-54, ii. (i) a 2'-OMe modified nucleotide at the last nucleotide of the conserved region of 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 bonds between the last three nucleotides. iii. (i) Five consecutive 2'-OMe modified nucleotides from the 3' end of the 3' terminal, and (ii) three PS bonds between the last three nucleotides, iv. Inverted debase-modified nucleotide at the last nucleotide of the conserved region of sgRNA or short sgRNA, v. (i) an inverted debasic modified nucleotide 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' terminal, (ii) Five consecutive 2'-F modified nucleotides immediately to the 5' side of the 2'-OMe modified nucleotides, and (iii) Three PS bonds 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 bonds between the last three nucleotides, viii. (i) 2 or 3 consecutive 2'-OMe modified nucleotides, and (ii) 3 PS bonds between the last 3 nucleotides, ix. One PS bond between the last nucleotide and the nucleotide adjacent to the last, and The gRNA according to any one of Embodiments 1 to 35, comprising (ii) 15 or 20 consecutive 2'-OMe modified nucleotides, and (ii) one or more of three PS bonds between the last three nucleotides. Embodiment 37 is a 5'-terminus modification, i. Modification of one or more nucleotides 1-7 in the guide region. ii. One modified nucleotide, iii. Two modified nucleotides, iv. Three modified nucleotides, v. 4 modified nucleotides, vi. 5 modified nucleotides, vii. Six modified nucleotides, and viii. A gRNA according to any one of Embodiments 1 to 36, comprising any of the seven modified nucleotides. Embodiment 38 is a gRNA according to any one of Embodiments 1 to 37, wherein the 5' terminal modification includes modifications of 1 to 7, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 nucleotides. Embodiment 39 contains at least 5 to 10 nucleotides, i. Containing nucleotides 54-61 of sequence number 400, ii. Containing nucleotides 53-60 of sequence number 400, iii. Containing nucleotides 54-58 of sequence number 400, iv. Consists of nucleotides 54-61 of sequence number 400, v. Consists of nucleotides 53-60 of sequence number 400, or vi. The gRNA described in Embodiments 1 to 38, consisting of nucleotides 54 to 58 of Sequence ID No. 400. Embodiment 40 has a 5'-terminus modification, i. Internucleotide phosphorothioate (PS) bonding, ii. 2'-OMe modified nucleotides, iii. 2'-O-moe modified nucleotides, iv. 2'-F modified nucleotide, v. Inverted debasic modified nucleotides, vi. Deoxyribonucleotide, vii. Inosine, and The gRNA described in any one of Embodiments 1 to 39 includes one or more combinations of viii.(i.) to (vii.). Embodiment 41 is a 5' terminal modification, i. PS bonds between nucleotides 1, 2, 3, 4, 5, 6, and / or 7, or ii. The gRNA according to any one of Embodiments 1 to 40, which contains approximately 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, or 1 to 7 nucleotide-to-PS bonds. Embodiment 42 has a 5'-terminus modification that includes at least one PS bond. i. One PS bond is present, and the bond is located between nucleotides 1 and 2 of the guide region. ii. Two PS bonds are present, and the bonds are located between nucleotides 1 and 2 in the guide region, and between nucleotides 2 and 3. iii. One or more PS bonds exist between cleotide 1 and 2, between 2 and 3, and between 3 and 4. iv. One or more PS bonds exist between nucleotides 1 and 2, 2 and 3, 3 and 4, and 4 and 5 in the guide region. v. One or more PS bonds exist between nucleotides 1 and 2, 2 and 3, 3 and 4, 4 and 5, and 5 and 6 in the guide region. vi. One or more PS bonds exist between nucleotides 1 and 2, 2 and 3, 3 and 4, 4 and 5, 5 and 6, and 6 and 7 in the guide region, or vii. A gRNA according to any one of Embodiments 1 to 41, wherein one or more PS bonds are present between nucleotides 1 and 2, 2 and 3, 3 and 4, 4 and 5, 5 and 6, 6 and 7, and 7 and 8 in the guide region. Embodiment 43 is the gRNA according to Embodiment 42, wherein the 5' terminal modification further comprises at least one 2'-OMe, 2'-O-moe, inverted debase, or 2'-F modified nucleotide. Embodiment 44 shows that a short sgRNA is i. Modifications of one or more nucleotides 1-7 in the variable region, wherein the modification is a PS bond, an inverted debasalized nucleotide, 2'-OMe, 2'-O-moe, 2'-F, 2'-H (deoxyribonucleotide), inosine, and / or a combination thereof. ii. Modification of the first nucleotide of the guide region with 2'-OMe, 2'-O-moe, 2'-F, 2'-H, inosine, or a combination thereof, and optional PS binding 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 nucleotides of the variable region by 2'-OMe, 2'-O-moe, 2'-F, 2'-H, inosine, or combinations thereof, and optionally, one or more PS bonds. v. Modification of the first, second, third, and / or fourth nucleotides of the variable region by 2'-OMe, 2'-O-moe, 2'-F, 2'-H, inosine, or combinations thereof, and optionally, one or more PS bonds, or vi. A gRNA according to any one of Embodiments 1 to 43, comprising modifications to the first, second, third, fourth, and / or fifth nucleotides of the variable region by 2'-OMe, 2'-O-moe, 2'-F, 2'-H, inosine, or a combination thereof, and optionally containing one or more PS bonds. Embodiment 45 shows that a short sgRNA is i. A 5' terminal modification shown in any one of sequence numbers 1-54, ii. 2'-OMe modified nucleotides in nucleotides 1, 2, and 3 of the guide region, iii. 2'-OMe modified nucleotides in nucleotides 1, 2, and 3 of the guide region, and PS bonds between nucleotides 1 and 2, 2 and 3, and 3 and 4 of the guide region. iv. 2'-OMe modified nucleotides in guide regions 1, 2, 3, 4, and 5, v. 2'-OMe modified nucleotides in guide region 1, 2, 3, 4 and nucleotide 5, and PS bonds between nucleotides 1 and 2, 2 and 3, 3 and 4, 4 and 5, and 5 and 6 in guide region. vi. 2'O-moe modified nucleotides in nucleotides 1, 2, and 3 of the guide region. vii. 2'O-moe modified nucleotides in nucleotides 1, 2, and 3 of the guide region, and PS bonds between nucleotides 1 and 2, 2 and 3, and 3 and 4 of the guide region. viii. Inverted debasic modified nucleotide at nucleotide 1 in the guide region, ix. Inverted debasic 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. The gRNA according to any one of Embodiments 1 to 44, comprising one or more PS bonds between nucleotides 1 and 2, 2 and 3, 3 and 4, 4 and 5, and 5 and 6 in the variable region. Embodiment 46 is a gRNA according to any one of Embodiments 1 to 45, wherein the upper stem region includes at least one modification. Embodiment 47 has an upper stem modification, i. One or more modifications from US1 to US12 in the upper stem region. ii. Modification of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or all 12 nucleotides in the upper stem region, and iii. The gRNA described in any one of Embodiments 1 to 46, comprising one or more modifications of approximately 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, or 1-12 nucleotides in the upper stem region. Embodiment 48 has an upper stem modification, i. 2'-OMe modified nucleotide, ii. 2'-O-moe modified nucleotides, iii. 2'-F modified nucleotides, and iv. The gRNA described in Embodiment 47 includes one or more combinations of (i) to (iii). Embodiment 49 is a guide RNA which is a short sgRNA containing a conserved portion of the sgRNA including a hairpin region, wherein the hairpin region lacks at least 5 to 10 nucleotides, and the short sgRNA has a 5' terminal modification, and i. Upper stem region, ii. Hairpin 1 area, and iii. Includes one or more modifications in one or more of the hairpin 2 regions, The 5' end modification is a 5' protective end modification, such as a guide RNA containing at least two phosphorothioate (PS) bonds within the first seven nucleotides. Embodiment 50 is the gRNA described in Embodiment 49, wherein at least one modification comprises a 2'-O-methyl (2'-OMe) modified nucleotide. Embodiment 51 is a gRNA according to Embodiment 49 or Embodiment 50, wherein at least one modification comprises a 2'-fluoro(2'-F) modified nucleotide. Embodiment 52 is a gRNA according to any one of Embodiments 49 to 51, wherein at least one modification includes an internucleotide phosphorothioate (PS) bond. Embodiment 53 is a gRNA according to any one of Embodiments 49 to 52, wherein the short sgRNA is a gRNA that includes one or more modifications in the upper stem region. Embodiment 54 is the gRNA described in Embodiment 53, which includes a modification in any one of US1 to US12. Embodiment 55 is a gRNA according to any one of Embodiments 49 to 54, wherein the short sgRNA includes one or more modifications in the hairpin 1 region. Embodiment 56 is the gRNA described in Embodiment 55, wherein the short sgRNA includes modifications at H1-1. Embodiment 57 is a gRNA according to any one of Embodiments 49 to 56, wherein the short sgRNA includes one or more modifications in the hairpin 2 region. Embodiment 58 is the gRNA described in Embodiment 57, wherein the short sgRNA includes modifications at H2-1. Embodiment 59 is a gRNA described in any one of Embodiments 49 to 58, wherein the short sgRNA includes modifications at H1-1 to H1-12. Embodiment 60 is a gRNA described in any one of Embodiments 49 to 59, wherein the short sgRNA includes modifications at H2-1 to H2-15. Embodiment 61 is a gRNA according to any one of Embodiments 49 to 60, wherein the short sgRNA includes one or more modifications in each of the upper stem region, hairpin 1 region, and hairpin 2 region. Embodiment 62 is a gRNA according to any one of Embodiments 49 to 61, wherein the short sgRNA includes a modified nucleotide between the hairpin 1 region and the hairpin 2 region. Embodiment 63 is a gRNA according to any one of Embodiments 49 to 62, further comprising a lower stem region including modifications. Embodiment 64 is a gRNA according to any one of Embodiments 49 to 63, further comprising a 3' terminal modification. 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' terminal are modified. 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' terminal are modified with 2'-OMe, 2'-F, or 2'-O-moe. Embodiment 67 is a gRNA according to any one of Embodiments 64 to 66, further comprising a phosphorothioate (PS) bond between one or more of the last four nucleotides at the 3' end of the 3' terminal. Embodiment 68 is a gRNA according to any one of Embodiments 49 to 67, further comprising a bulge region including modifications. Embodiment 69 is a gRNA according to any one of Embodiments 49 to 68, further comprising a modified nexus region. Embodiment 70 is a gRNA according to any one of Embodiments 49 to 69, wherein at least the first three nucleotides at the 5' end of the variable region and the last three nucleotides at the 3' end of the 3' region are modified. Embodiment 71 is a 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' region are linked by phosphorothioate (PS) bonds. Embodiment 72 is a gRNA according to any one of Embodiments 70 to 71, wherein the terminal modification includes 2'-OMe. Embodiment 73 is a gRNA according to any one of Embodiments 70 to 71, wherein the terminal modification includes a 2'-F. Embodiment 74 is a 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' region 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' region include 2'-OMe modifications. Embodiment 75 is a gRNA according to any one of Embodiments 49 to 74, wherein the first four nucleotides at the 5' end and the last four nucleotides at the 3' end are linked by PS bonds, and the first three nucleotides at the 5' end and the last three nucleotides at the 3' end contain 2'-OMe, 2'-F, and / or 2'-O-moe modifications. Embodiment 76 is a gRNA according to any one of Embodiments 49 to 75, wherein LS1, LS6, LS7, LS8, LS11, and / or LS12 are modified with 2'-OMe. Embodiment 77 is a gRNA according to any one of Embodiments 49 to 76, wherein each nucleotide in the bulge region is modified with 2'-OMe. Embodiment 78 is a gRNA according to any one of Embodiments 49 to 77, wherein at least 50% of the nucleotides in the bulge region are modified with 2'-OMe. Embodiment 79 is a gRNA according to any one of Embodiments 49 to 78, wherein each nucleotide in the upper stem region is modified with 2'-OMe. Embodiment 80 is a gRNA according to any one of Embodiments 49 to 79, wherein N16, N17, and / or N18 in the nexus region are modified with 2'-OMe. Embodiment 81 is a gRNA according to any one of Embodiments 49 to 80, wherein N15, N16, N17, and / or N18 in the nexus region are modified. Embodiment 82 is the gRNA according to Embodiment 80 or 81, wherein the modification in the nexus region is selected from 2'-OMe and 2'F. Embodiment 83 is the gRNA described in any one of Embodiments 80 to 82, wherein N16, N17, and N18 are linked by PS bonds. Embodiment 84 is a gRNA according to any one of Embodiments 49 to 83, wherein each of the nucleotides remaining in the hairpin 1 region is modified with 2'-OMe. Embodiment 85 is a gRNA according to any one of Embodiments 49 to 84, wherein each nucleotide in the hairpin 2 region is modified with 2'-OMe. Embodiment 86 is a guide RNA which is a short sgRNA which includes a conserved portion of the sgRNA which includes a hairpin region, the hairpin region which lacks at least 5 to 10 nucleotides, the short sgRNA which includes a 5' terminal modification or a 3' terminal modification, i. At least one modification in the upper stem region, and ii. A guide RNA that further contains one or more of the 3' tails. Embodiment 87 has an upper stem modification, i. Modification of each nucleotide (US1-US12) in the upper stem region. ii. One or more modifications from US1 to 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 in the upper stem region, and iv. The gRNA according to Embodiment 86, comprising one or more of the following modifications in the upper stem region: approximately 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, or 1-12 nucleotides. Embodiment 88 has a 5'-terminus modification, i. Modification of one or more nucleotides 1-7 in the variable region. ii. One modified nucleotide, iii. Two modified nucleotides, iv. Three modified nucleotides, v. 4 modified nucleotides, vi. 5 modified nucleotides, vii. Six modified nucleotides, and viii. The gRNA described in any one of embodiments 86 to 87, comprising one or more of the seven modified nucleotides. Embodiment 89 has a 5'-terminus modification, i. 5'-terminal modifications shown in any one of the following sequence numbers: 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 in nucleotides 1, 2, and 3 of the variable region, iii. 2'-OMe modified nucleotides in nucleotides 1, 2, and 3 of the variable region, and PS bonds between nucleotides 1 and 2, 2 and 3, and 3 and 4 of the variable region. iv. 2'-OMe modified nucleotides in nucleotides 1, 2, 3, 4, and nucleotide 5 of the variable region, v. 2'-OMe modified nucleotides in variable regions 1, 2, 3, 4, and 5, and PS bonds between nucleotides 1 and 2, 2 and 3, 3 and 4, 4 and 5, and 5 and 6 in variable regions. vi. 2'O-moe modified nucleotides in nucleotides 1, 2, and 3 of the variable region. vii. 2'O-moe modified nucleotides in nucleotides 1, 2, and 3 of the variable region, and PS bonds between nucleotides 1 and 2, 2 and 3, and 3 and 4 of the variable region. viii. Inverted debase-modified nucleotide at nucleotide 1 in the variable region, ix. Inverted debasic modified nucleotide at nucleotide 1 in the variable region, and 2'-OMe modified nucleotides at nucleotides 1, 2, and 3 in the variable region, x. The gRNA according to any one of embodiments 86 to 88, comprising one or more of the following: an inverted debasalized nucleotide at nucleotide 1 in the variable region, 2'-OMe modified nucleotides at nucleotides 1, 2, and 3 in the variable region, and PS bonds between nucleotides 1 and 2, 2 and 3, 3 and 4, 4 and 5, and 5 and 6 in the variable region. Embodiment 90 is the gRNA according to Embodiment 89, which includes 2'-OMe modified nucleotides in at least nucleotides 1, 2, and 3 of the variable region, as well as PS bonds between nucleotides 1 and 2, 2 and 3, and 3 and 4 of the variable region. Embodiment 91 is the gRNA according to Embodiment 89, comprising 2'-OMe modified nucleotides in at least nucleotides 1, 2, 3 and nucleotide 4 of the variable region, as well as PS bindings 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 the last 7, 6, 5, 4, 3, 2, or 1 nucleotide, ii. One modified nucleotide, iii. Two modified nucleotides, iv. Three modified nucleotides, v. 4 modified nucleotides, vi. 5 modified nucleotides, vii. Six modified nucleotides, and viii. A gRNA according to any one of embodiments 86 to 91, comprising a 3' terminal modification, which includes one or more of the seven modified nucleotides. Embodiment 93 is a short sgRNA, i. 3'-terminal modifications shown in any one of the following sequence numbers: 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. (i) a 2'-OMe modified nucleotide at the last nucleotide of the conserved region of 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 bonds between the last three nucleotides. iii. (i) Five consecutive 2'-OMe modified nucleotides, and (ii) three PS bonds between the last three nucleotides, iv. Inverted debase-modified nucleotide at the last nucleotide of the conserved region of sgRNA or short sgRNA, v. (i) an inverted debasic modified nucleotide 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) 15 consecutive 2'-OMe modified nucleotides, (ii) 5 consecutive 2'-F modified nucleotides immediately 5' to the 2'-OMe modified nucleotides, and (iii) 3 PS bonds between the last 3 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 bonds between the last three nucleotides, viii. (i) 2 or 3 consecutive 2'-OMe modified nucleotides, and (ii) 3 PS bonds between the last 3 nucleotides, ix. One PS bond between the last nucleotide and the nucleotide adjacent to the last, and The gRNA is one of any one of embodiments 86 to 92, comprising (ii) 15 or 20 consecutive 2'-OMe modified nucleotides, and (ii) one of three PS bonds between the last three nucleotides. Embodiment 94 is a gRNA according to any one of Embodiments 86 to 93, wherein the sgRNA includes a 3' tail, and the 3' tail includes one or more modifications of the nucleotides present in the 3' tail. Embodiment 95 is the gRNA described in Embodiment 94, wherein the 3' tail is fully modified. Embodiment 96 is the gRNA of Embodiment 94, wherein the 3' tail contains 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 one or more of these nucleotides are optionally modified. Embodiment 97 is a guide RNA, which is a short sgRNA containing any of SEQ ID NOs. 1-54, 201-254, and 301-354, including the modifications shown in Table 1. Embodiment 98 is a guide RNA which is a short sgRNA containing a nucleic acid having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, or 70% identity with any one of the nucleic acids among SEQ ID NOs: 1-54, 201-254, and 301-354, wherein the modifications at each nucleotide of the short sgRNA corresponding to the nucleotide of the reference sequence identifier in Table 1 are identical or equivalent to the modifications shown for the reference sequence identifier in Table 1. Embodiment 99 is a gRNA according to any one of Embodiments 1 to 98, comprising YA modification of at least one guide region YA site. Embodiment 100 is a gRNA according to any one of Embodiments 1 to 99, which includes a YA modification in at least one guide region YA site that is not a 5' terminal modification. Embodiment 101 is a gRNA according to any one of Embodiments 1 to 100, comprising YA modifications in one or more guide region YA sites, wherein the guide region YA site is located at nucleotide 8 from the 5' end of the 5' end or after it. Embodiment 102 includes YA modification in one or more guide region YA sites, and a short sgRNA, a. Modification in one or more of H1-1 and H2-1, b. YA modification in guide region YA sites 1, 2, 3, 4, or 5, c.YA modifications in guide region YA sites 1, 2, 3, 4, or 5, wherein the modification of at least one guide region YA site is different from any 5' terminal modification of the sgRNA, d. YA modifications in one or more guide region YA sites, wherein the guide region YA site is located at nucleotide 8 from the 5' end of the 5' end, or thereafter, e. YA modifications in one or more guide region YA sites, wherein the guide region YA site is located within the 5-terminus, 6-terminus, 7-terminus, 8-terminus, 9-terminus, or 10-terminus of the 5' end of the 5' end. f. YA modifications in one or more guide region YA sites, wherein the guide region YA site is located within 17, 16, 15, 14, 13, 12, 11, 10, or 9 nucleotides of the 3' terminal nucleotide of the guide region. g.YA modification in the guide region YA site other than 5' terminal modification, h. YA modifications in two or more guide region YA sites, wherein the guide region YA site is located at nucleotide 8 from the 5' end of the 5' terminal or thereafter, i. YA modifications in two or more guide region YA sites, wherein the two guide region YA sites are located within the 5th, 6th, 7th, 8th, 9th, or 10th nucleotides from the 5' end of the 5' end of the YA site. j. YA modifications in two or more guide region YA sites, wherein the guide region YA site is located within 17, 16, 15, 14, 13, 12, 11, 10, or 9 nucleotides of the 3' terminal nucleotide of the guide region. YA modifications in two or more guide region YA sites other than k.5' terminal modifications, l. A gRNA according to any one of Embodiments 1 to 101, comprising one or more YA modifications in two or more guide region YA sites, 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. Embodiment 103 is a gRNA according to any one of Embodiments 1 to 102, comprising a YA modification, wherein the modification comprises 2'-fluoro, 2'-H, 2'-OMe, ENA, UNA, inosine, or PS. Embodiment 104 is a gRNA according to any one of Embodiments 1 to 103, wherein the modification includes a YA modification, the modification altering the structure of the dinucleotide motif to reduce RNA endonuclease activity. Embodiment 105 is a gRNA according to any one of Embodiments 1 to 104, wherein the modification includes a YA modification, the modification preventing recognition or cleavage of the YA site by RNase and / or stabilizing the RNA structure. Embodiment 106 includes a YA modification, wherein the modification is 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 modification, d. Base modifications such as inosine, pseudouridine, and 5'-methylcytosine, and e. The gRNA described in any one of Embodiments 1 to 105, comprising one or more of the nucleoside binding modifications such as phosphorothioates. Embodiment 107 is a gRNA according to any one of Embodiments 1 to 106, comprising YA modification in one or more conserved YA sites. Embodiment 108 is a gRNA according to any one of Embodiments 1 to 107, comprising YA modification in one or more of the conserved YA sites 2, 3, 4, and 10. Embodiment 109 is a gRNA according to any one of Embodiments 1 to 108, comprising YA modification in one or more of the conserved YA sites 1 and 8. Embodiment 110 is a gRNA according to any one of Embodiments 1 to 109, which includes YA modification of the conserved region YA site 1. Embodiment 111 is a gRNA according to any one of Embodiments 1 to 110, which includes YA modification of the conserved region YA site 2. Embodiment 112 is a gRNA according to any one of Embodiments 1 to 111, which includes YA modification of the conserved region YA site 3. Embodiment 113 is a gRNA according to any one of Embodiments 1 to 112, which includes YA modification of the conserved region YA site 4. Embodiment 114 is a gRNA according to any one of Embodiments 1 to 113, which includes YA modification of the conserved region YA site 5. Embodiment 115 is a gRNA according to any one of Embodiments 1 to 114, which includes YA modification of the conserved region YA site 6. Embodiment 116 is a gRNA according to any one of Embodiments 1 to 115, which includes YA modification of the conserved region YA site 7. Embodiment 117 is a gRNA according to any one of Embodiments 1 to 116, which includes YA modification of the conserved region YA site 8. Embodiment 118 is a gRNA according to any one of Embodiments 1 to 117, which includes YA modification of the conserved region YA site 9. Embodiment 119 is a gRNA according to any one of Embodiments 1 to 118, which includes YA modification of the conserved region YA site 10. Embodiment 120 is, a. YA modifications of YA sites 2, 3, 4, and 10 in the conserved region. b. YA modification of YA sites 2, 3, and 4 in the conserved region. c. YA modifications of YA sites 2, 3, and 10 in the preserved region. d. YA modification of YA sites 2, 4, and 10 in the preserved region. e. YA modification of YA sites 3, 4, and 10 in the preserved region. f. YA modification of YA sites 2 and 10 in the conserved region. g. YA modification of YA sites 2 and 4 in the conserved region. h. YA modification of YA sites 2 and 3 in the conserved region. i. YA modification of YA sites 3 and 4 in the conserved region. j. YA modification of YA sites 3 and 10 in the conserved region. k. YA modification of YA sites 4 and 10 in the conserved region. l. YA modification of YA sites 1 and 5 in the conserved region. m. YA modification of YA sites 1 and 6 in the preserved region. n. YA modification of YA sites 1 and 7 in the conserved region. o. YA modification of YA sites 1 and 8 in the conserved region. p. YA modification of YA sites 1 and 9 in the conserved region. q. YA modification of YA sites 8 and 5 in the conserved region. r. YA modification of YA sites 8 and 6 in the conserved region. s. YA modification of YA sites 8 and 7 in the preserved region, and t. Containing one or more of the YA modifications of YA sites 8 and 9 in the preserved region, Optionally, the sgRNA is the gRNA described in any one of Embodiments 1 to 119, further comprising YA modifications at conserved YA sites 2, 3, 4, and / or 10. Embodiment 121 is a gRNA according to any one of Embodiments 1 to 120, wherein at least one modified YA site optionally includes a 2'-OMe modification in the pyrimidine of the YA site. Embodiment 122 is a gRNA according to any one of Embodiments 1 to 121, wherein at least one modified YA site optionally includes a 2'-fluoro modification in the pyrimidine of the YA site. Embodiment 123 is a gRNA according to any one of Embodiments 1 to 122, wherein at least one modified YA site comprises a PS modification in the pyrimidine of the YA site, by optional choice. Embodiment 124 is a gRNA according to any one of Embodiments 1 to 123, wherein the gRNA includes a guide region comprising modifications in at least one, two, three, four, five, six, seven, eight, nine, ten, one, one, one, two, one Embodiment 125 is a gRNA according to any one of Embodiments 1 to 124, wherein the short sgRNA includes a guide region comprising modifications at nucleotides 1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 13, 14, 17, and 18, and optionally the modifications are 2'-OMe, 2'-fluoro, 2'-H, inosine, or phosphorothioate modifications. Embodiment 126 is a gRNA according to Embodiments 124-125, wherein the 2'-OMe modification is not present in the guide region at the nucleotides 6-11 and 13. Embodiment 127 is a gRNA according to Embodiments 124-126, wherein the 2'-fluoro modification is absent in the guide regions at the nucleotides 1-7, 15, 16, and 19. Embodiment 128 is a gRNA according to Embodiments 124-127, wherein the phosphorothioate modification is absent in the guide regions of nucleotides 4, 5, 11-14, 17, and 18. Embodiment 129 is a gRNA according to Embodiments 124-128, wherein the guide region contains an 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 includes YA sites at nucleotides 5-6 and modifications at nucleotide 5. Embodiment 132 is a gRNA according to Embodiments 124-131, wherein the guide region includes YA sites at nucleotides 12-13 and modifications at nucleotide 12. Embodiment 133 is a gRNA according to Embodiments 124-132, wherein the guide region includes YA sites at nucleotides 15-16 and modifications at nucleotide 15. Embodiment 134 is a gRNA according to Embodiments 124-133, wherein the guide region includes YA sites at nucleotides 16-17 and modifications at nucleotide 16. Embodiment 135 is a gRNA according to Embodiments 124-134, wherein the guide region includes YA sites at nucleotides 19-20 and modifications at nucleotide 19. Embodiment 136 is a gRNA according to Embodiments 124-130 or 132-135, wherein the guide region does not contain a YA site in nucleotides 5-6, and nucleotide 5 is unmodified. Embodiment 137 is a gRNA according to Embodiments 124-131 or 133-136, wherein the guide region does not contain a YA site in nucleotides 12-13, and nucleotide 12 is unmodified. Embodiment 138 is a gRNA according to Embodiments 124-132 or 134-137, wherein the guide region does not contain a YA site in nucleotides 15-16, and nucleotide 15 is unmodified. Embodiment 139 is a gRNA according to Embodiments 124-133 or 135-138, wherein the guide region does not contain a YA site in nucleotides 16-17, and nucleotide 16 is unmodified. Embodiment 140 is a gRNA according to Embodiments 124-134 or 136-139, wherein the guide region does not contain a YA site in nucleotides 19-20, and nucleotide 19 is unmodified. Embodiment 141 is a short sgRNA, 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) Phosphothioate modification at nucleotide 6, (f) Phosphothioate modification at nucleotide 7, (g) 2'-fluoro and phosphorothioate modifications in 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) nucleotide 17 2'-fluoro modification, and The gRNA according to Embodiments 124 to 140, comprising a guide region containing one or more 2'-fluoro modifications at (n)nucleotide 18. Embodiment 142 is a gRNA according to Embodiments 124-141, wherein the guide region includes each of the modifications described in the previous embodiments. Embodiment 143 has the following guide region: (a) When nucleotides 5 and 6 form the YA site, nucleotide 5 has a 2'-OMe modification, (b) When nucleotides 12 and 13 form the YA site, nucleotide 12 has a 2'-OMe modification, (c) If nucleotides 15 and 16 form the YA site, phosphorothioate or 2'-H modification in nucleotide 15, (d) When nucleotides 16 and 17 form the YA site, phosphorothioate modification at nucleotide 16, (e) gRNAs of Embodiments 124-142, wherein nucleotides 19 and 20 form a YA site, and include at least one, two, three, or four of the phosphorothioates or 2'-fluoro modifications in nucleotide 19. Embodiment 144 is a gRNA of Embodiments 124-143, wherein the guide region includes YA sites at nucleotides 5-6 and a 2'-OMe modification at nucleotide 5. Embodiment 145 is a gRNA according to Embodiments 124-144, wherein the guide region includes a YA site at nucleotides 12-13 and a 2'-OMe modification at nucleotide 12. Embodiment 146 is a gRNA according to Embodiments 124-145, wherein the guide region includes YA sites at nucleotides 15-16 and a phosphorothioate modification at nucleotide 15. Embodiment 147 is a gRNA according to Embodiments 124-146, wherein the guide region includes YA sites at nucleotides 16-17 and a phosphorothioate modification at nucleotide 16. Embodiment 148 is a gRNA according to Embodiments 124-147, wherein the guide region includes YA sites at nucleotides 19-20 and a phosphorothioate modification at nucleotide 19. Embodiment 149 is a gRNA according to Embodiments 124-148, wherein the guide region includes a 2'-fluoro modification at nucleotide 19. Embodiment 150 is a gRNA according to Embodiments 124-149, wherein the guide region includes an unmodified nucleotide 15 or only a phosphorothioate modification at nucleotide 15. Embodiment 151 is a gRNA according to Embodiments 124-150, wherein the guide region includes an unmodified nucleotide 16 or only a phosphorothioate modification at the nucleotide 16. Embodiment 152 is, a. YA modification in two or more guide region YA sites, b. YA modification in one or more of the YA sites 2, 3, 4, and 10 of the conserved region, and c. A single guide RNA (sgRNA) that contains YA modifications in one or more of the conserved regions YA sites 1 and 8. Embodiment 153 is, a. YA modification at nucleotide 8 from the 5' end of the 5' terminal or at one or more guide region YA sites thereafter. b. YA modification in one or more of the YA sites 2, 3, 4, and 10 of the preserved region, and optionally, c. A single guide RNA (sgRNA) that contains YA modifications in one or more of the conserved regions YA sites 1 and 8. Embodiment 154 is, a. YA modification at one or more guide region YA sites within the 13 nucleotides of the 3' terminal nucleotide of the guide region. b. YA modification in one or more of the YA sites 2, 3, 4, and 10 of the conserved region, and c. A single guide RNA (sgRNA) that contains YA modifications in one or more of the conserved regions YA sites 1 and 8. Embodiment 155 is, a. 5' terminal modification and 3' terminal modification, b. YA modification in one or more of the YA sites 2, 3, 4, and 10 of the conserved region, and c. A single guide RNA (sgRNA) that contains YA modifications in one or more of the conserved regions YA sites 1 and 8. Embodiment 156 is, a. A YA modification in at least one guide region YA site, 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. b. YA modification in one or more of the YA sites 2, 3, 4, and 10 of the conserved region, and c. A single guide RNA (sgRNA) that contains YA modifications in one or more of the conserved regions YA sites 1 and 8. Embodiment 157 is, a. YA modification in one or more of the YA sites 2, 3, 4, and 10 of the conserved region, and b. A single guide RNA (sgRNA) containing YA modifications in the conserved regions YA sites 1 and 8, which is a guide RNA. Embodiment 158 ​​is, a. YA modification at nucleotide 8 from the 5' end of the 5' terminal or at one or more guide region YA sites thereafter. b. YA modification in one or more of the YA sites 2, 3, 4, and 10 of the conserved region, and c. A single guide RNA (sgRNA) containing modifications in one or more of H1-1 and H2-1 is a guide RNA. Embodiment 159 is, a. YA modification in one or more of the YA sites 2, 3, 4, and 10 of the conserved region. b. YA modifications in one or more of the YA sites 1, 5, 6, 7, 8, and 9 in the conserved region, and c. A single guide RNA (sgRNA) containing modifications in one or more of H1-1 and H2-1 is a guide RNA. Embodiment 160 is, a. Modifications in the 6 nucleotides from the 5' end of the 5' terminal or one or more nucleotides located thereafter, such as YA modifications. b. YA modification at one or more guide sequence YA sites, c. Modifications in one or more of B3, B4, and B5, wherein B6 does not contain a 2'-OMe modification, or contains a modification other than a 2'-OMe modification, d. Modifications in LS10, wherein LS10 includes modifications other than 2'-fluoro, and / or e. Modifications in N2, N3, N4, N5, N6, N7, N10, or N11 A guide RNA that is an 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 does not contain a 2'-fluoro modification. ii. At least one of the nucleotides 6-10 from the 5' end of the 5' terminal 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 the 2'-OMe modification. vi.H1-1 includes modifications, vii.H2-1 includes modifications, or viii. The guide RNA is one in which 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. Embodiment 161 is, i. Modifications in nucleotide 6 from the 5' end of the 5' terminal or one or more nucleotides located thereafter, such as YA modifications, or ii. YA modification at one or more guide sequence YA sites A guide RNA containing 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 does not contain a 2'-fluoro modification, or b. At least one of the 6-10 nucleotides from the 5' end of the 5' terminal does not contain a phosphorothioate bond. At least one of the following applies: c. At least one of nucleotides 7-10 from the 5' end of the 5' terminal does not contain the 2'-OMe modification. d. Nucleotide 20 from the 5' end of the 5' terminal does not contain the 2'-OMe modification, or e. The guide RNA is a guide RNA in which at least one of nucleotides 1-20 from the 5' end of the 5' end contains a 2'-fluoro modification, at least one of nucleotides 11, 12, 13, 14, 17, or nucleotide 18 from the 5' end of the 5' end does not contain a 2'-fluoro modification, and optionally at least one of nucleotides 12 from the 5' end of the 5' end also does not contain a 2'-fluoro modification. Embodiment 162 is guanosine in N14 and / or the following: a. YA modification at nucleotide 8 from the 5' end of the 5' terminal or at one or more guide region YA sites thereafter. b. YA modifications in one or more of the preserved region YA sites 1, 5, and 6, where YA site 6 is modified at LS12 and LS9 is unmodified, and the modification of LS12 is other than 2'-OMe, YA modifications, c. Modifications in LS9, where LS9 is modified and LS5, LS7, and LS12 are unmodified, the modification of LS9 is other than 2'-fluoro, d. Modification in LS12, where LS12 is modified and LS9 is not, the modification of LS12 is other than 2'-OMe, e. Modifications in LS8 or LS11 in which at least one of LS8 and LS11 includes a modification other than 2'-OMe, and / or f. Modifications in N6, N14, or N17, where N17 is modified and N6 and N14 are unmodified, the modification of N17 is other than 2'-fluoro and other than 2'-OMe. A guide RNA that is an sgRNA containing one or more of the following: i. At least one of nucleotides 8-11, 13-14, 17, or nucleotide 18 from the 5' end of the 5' end does not contain a 2'-fluoro modification. ii. At least one of the nucleotides 6-10 from the 5' end of the 5' terminal 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 the 2'-OMe modification. vi.H1-1 includes modifications, vii.H2-1 includes modifications, or viii. The guide RNA is one in which 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. Embodiment 163 is, a. YA modification in guide region YA sites 1, 2, 3, 4, or 5, b.YA modifications in guide region YA sites 1, 2, 3, 4, or 5, wherein the modification of at least one guide region YA site is different from any 5' terminal modification of the sgRNA, c. YA modification at nucleotide 8 from the 5' end of the 5' terminal or one or more guide region YA sites thereafter. d. YA modification in one or more guide region YA sites located within the 5-terminus, 6-terminus, 7-terminus, 8-terminus, 9-terminus, or 10-terminus of the nucleotide originating from the 5' end of the 5' terminal. e. One or more YA modifications at the guide region YA site within the 17th, 16th, 15th, 14th, 13th, 12th, 11th, 10th, or 9th nucleotides of the 3' terminal nucleotide of the guide region. f.YA modification in the guide region YA site other than 5' terminal modification, or g. A gRNA according to Embodiment 161 or 162, comprising a YA modification in the guide region YA site, 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. Embodiment 164 is, a. YA modification at nucleotide 8 from the 5' end of the 5' terminal or at two or more guide region YA sites thereafter. b. YA modifications in two or more guide region YA sites located within the 5-terminus, 6-terminus, 7-terminus, 8-terminus, 9-terminus, or 10-terminus of the nucleotide originating from the 5' end of the 5' end. c. Two or more YA modifications at the guide region YA site within the 17th, 16th, 15th, 14th, 13th, 12th, 11th, 10th, or 9th nucleotides of the 3' terminal nucleotide of the guide region. d.YA modifications in two or more guide region YA sites other than 5' terminal modifications, or e. The gRNA according to Embodiment 163, comprising YA modifications in two or more guide region YA sites, 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. Embodiment 165 is, a. YA modification at nucleotide 8 from the 5' end of the 5' terminal or at three or more guide region YA sites thereafter. b. YA modifications in three or more guide region YA sites located within the nucleotide 5, 6, 7, 8, 9, or 10 terminals of the 5' end of the nucleotide, c. YA modifications at three 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. d.YA modifications in three or more guide region YA sites other than 5' terminal modifications, or e. The gRNA according to Embodiment 163, comprising YA modifications in three or more guide region YA sites, wherein the modifications of the guide region YA site include modifications that do not include at least one nucleotide located on the 5' side of the guide region YA site. Embodiment 166 is a gRNA according to any one of Embodiments 161 to 165, comprising at least one YA modification in nucleotide 6 from the 5' end of the 5' terminal. Embodiment 167 is a gRNA according to any one of Embodiments 161 to 166, comprising at least one YA modification in nucleotide 7 from the 5' end of the 5' terminal. Embodiment 168 is a gRNA according to any one of Embodiments 161 to 167, comprising at least one YA modification in nucleotide 8 from the 5' end of the 5' terminal. Embodiment 169 is a gRNA according to any one of Embodiments 161 to 168, comprising at least one YA modification in nucleotide 9 from the 5' end of the 5' terminal. Embodiment 170 is a gRNA according to any one of Embodiments 161 to 169, comprising at least one YA modification in nucleotide 10 from the 5' end of the 5' terminal. Embodiment 171 is a gRNA according to any one of Embodiments 161 to 170, comprising at least one YA modification in nucleotide 11 from the 5' end of the 5' terminal. Embodiment 172 is a gRNA according to any one of Embodiments 161 to 171, comprising at least one YA modification in nucleotide 12 from the 5' end of the 5' terminal. Embodiment 173 is a gRNA according to any one of Embodiments 161 to 172, comprising at least one YA modification in nucleotide 13 from the 5' end of the 5' terminal. Embodiment 174 is a gRNA according to any one of Embodiments 161 to 173, comprising at least one YA modification in nucleotide 14 from the 5' end of the 5' terminal. Embodiment 175 is a gRNA according to any one of Embodiments 161 to 174, comprising at least one YA modification in nucleotide 15 from the 5' end of the 5' terminal. Embodiment 176 is a gRNA according to any one of Embodiments 161 to 175, comprising at least one YA modification in nucleotide 16 from the 5' end of the 5' terminal. Embodiment 177 is a gRNA according to any one of Embodiments 161 to 176, comprising at least one YA modification in nucleotide 17 from the 5' end of the 5' terminal. Embodiment 178 is a gRNA according to any one of Embodiments 161 to 177, comprising at least one YA modification in nucleotide 18 from the 5' end of the 5' terminal. Embodiment 179 is a gRNA according to any one of Embodiments 161 to 178, comprising at least one YA modification in nucleotide 19 from the 5' end of the 5' terminal. Embodiment 180 is a gRNA according to any one of Embodiments 161 to 179, comprising at least one YA modification in nucleotide 20 from the 5' end of the 5' terminal. Embodiment 181 is a gRNA according to any one of Embodiments 161 to 180, wherein at least one, two, three, four, five, six, seven, or eight nucleotides from nucleotides 8-11, 13-14, and 17-18 from the 5' end of the 5' terminal contain a YA modification, and optionally the modification contains 2'-fluoro, 2'-H, 2'-OMe, or PS. Embodiment 182 is the gRNA described in Embodiment 181, wherein the modification is 2'-fluoro. Embodiment 183 is the gRNA described in Embodiment 181, wherein the modification is 2'-OMe or 2'-H. Embodiment 184 is the gRNA described in Embodiment 181, wherein the modification is PS. Embodiment 185 is a gRNA according to any one of Embodiments 161 to 184, wherein at least one, two, three, four, or five of the 6 to 10 nucleotides 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 described in Embodiment 185, wherein the modification is PS. Embodiment 187 is the gRNA described in 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. Embodiment 189 is as follows: a. YA modifications of 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides from nucleotides 8-11, 13-14, and 17-18 from the 5' end of the 5' terminal, wherein the YA modifications are optionally 2'-fluoro modifications and modifications other than 2'-fluoro on one or more nucleotides 6-10 from the 5' end of the 5' terminal. b. YA modifications other than PS in one or more nucleotides 8-11, 13-14, and 17-18 from the 5' end of the 5' terminal, and YA modifications 1, 2, 3, 4, or 5 in nucleotides 6-10 from the 5' end of the 5' terminal, where the modification is optionally a PS modification. c.YA modifications in nucleotides 8-11, 13-14, and 17-18 from the 5' end of the 5' terminal, wherein the YA modification is optionally a 2'-fluoro modification, and modifications other than 2'-fluoro in nucleotides 6-10 from the 5' end of the 5' terminal. d.YA modifications other than PS in nucleotides 8-11, 13-14, and 17-18 from the 5' end of the 5' terminal, and YA modifications of 1, 2, 3, 4, or 5 in nucleotides 6-10 from the 5' end of the 5' terminal, wherein the modification is optionally a PS 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' terminal, wherein the YA modification is optionally a 2'-fluoro modification, and one or more PS modifications in any one of nucleotides 6-10 from the 5' end of the 5' terminal. f. At least one 2'-fluoro modification on any one of nucleotides 8-11, 13-14, and 17-18 from the 5' end of the 5' terminal, and YA modifications on nucleotides 1, 2, 3, 4, or 5 from nucleotides 6-10 from the 5' end of the 5' terminal, wherein the modification is optionally a PS modification. g.YA modifications of nucleotides 8-11, 13-14, and 17-18 from the 5' end of the 5' terminal, wherein the YA modifications are optionally 2'-fluoro modifications, and PS modifications in each of nucleotides 6-10 from the 5' end of the 5' terminal. The gRNA according to any one of Embodiments 161 to 188, comprising one or more modifications, including 2'-fluoro modifications in each of nucleotides 8-11, 13-14, and 17-18 from the 5' end of the h.5' terminal, and YA modifications in 1, 2, 3, 4, or 5 of nucleotides 6-10 from the 5' end of the 5' terminal, wherein the modifications are optionally PS modifications. Embodiment 190 is, a. Nucleotides 4-20 from the 5' end of the 5' terminal contain at least 2, 3, or 4 modified YA sites, each containing a first modified YA site with a 2'-OMe modification and a second modified YA site with a 2'-fluoro modification or PS modification. b. Nucleotides 4-20 from the 5' end of the 5' terminal contain at least 2, 3, or 4 modified YA sites, each containing a first modified YA site with a 2'-fluoro modification and a second modified YA site with a 2'-OMe modification or PS modification. c. Nucleotides 4-20 from the 5' end of the 5' terminal contain at least 2, 3, or 4 modified YA sites, including a first modified YA site containing a PS modification and a second modified YA site containing a 2'-OMe modification or a 2'-fluoro modification. d. Nucleotides 4-20 from the 5' end of the 5' terminal contain at least 2, 3, or 4 modified YA sites, including YA modifications. e. Nucleotides 4-20 from the 5' end of the 5' terminal include at least three or four modified YA sites, each including a first modified YA site containing a 2'-OMe modification, a second modified YA site containing a 2'-fluoro modification, and a third modified YA site containing a PS modification. Nucleotides 4-20 from the 5' end of the f.5' terminal include at least three or four modified YA sites, including a first modified YA site containing a 2'-OMe modification, a second modified YA site containing a 2'-fluoro modification, a third modified YA site containing a 2'-fluoro modification, and a fourth modified YA site containing a PS modification. g. Nucleotides 4-20 from the 5' end of the 5' terminal contain at least three or four modified YA sites, including YA modifications. Nucleotides 4-20 from the 5' end of the 5' terminal of the h.5' end contain at least four modified YA sites, including a first modified YA site containing a 2'-OMe modification, a second modified YA site containing a 2'-fluoro modification, a third modified YA site containing a PS modification, and a fourth modified YA site containing a PS modification, or i. The gRNA is one of any one of Embodiments 161 to 189, wherein nucleotides 4 to 40 from the 5' end of the 5' terminal contain at least four modified YA sites, including YA modifications. Embodiment 191 is a gRNA according to any one of Embodiments 161 to 190, wherein nucleotides 4 to 20 from the 5' end of the 5' terminal contain at least five modified YA sites. Embodiment 192 is a gRNA according to any one of Embodiments 161 to 191, wherein 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. Embodiment 193 is a gRNA according to any one of Embodiments 161 to 192, wherein the first, second, and (if applicable) third, fourth, and fifth of at least five modified YA sites are arranged in the 5' to 3' direction. Embodiment 194 is a gRNA according to any one of Embodiments 161 to 193, wherein the first, second, and (if applicable) third, fourth, and fifth of at least five modified YA sites are not located in the 5' to 3' direction. Embodiment 195 is a gRNA according to any one of Embodiments 161 to 194, wherein nucleotides 4 to 20 from the 5' end of the 5' terminal contain at least 2, 3, 4, or 5 modified YA sites, each containing a deoxyribonucleotide, and optionally the deoxyribonucleotide is a pyrimidine at the YA site. Embodiment 196 is, a. The YA modification includes at least 1, 2, 3, or 4 of the nucleotides 8-11 from the 5' end of the 5' terminal being optionally 2'-fluoromodified. b. At least one, two, three, four, five, six, seven, or eight nucleotides from nucleotides 8-11, 13, 14, 17, and 18 from the 5' end of the 5' terminal include a YA modification, which is optionally 2'-OMe if present in nucleotides 8-11, and 2'-fluoro if present in nucleotides 13, 14, 17, or 18. c. The YA modification includes at least one or both of nucleotides 17 and 18 from the 5' end of the 5' terminal, which are optionally 2'-fluoromodified. d. The YA modification includes, or, at least one or both of nucleotides 17 and 18 from the 5' end of the 5' terminal are optionally 2'-fluoromodified. The gRNA described in any one of Embodiments 161 to 195 includes a YA modification, wherein at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 nucleotides from 4 to 14, 17, and nucleotide 18 from the 5' end of the 5' end are optionally 2'-fluoro modifications. Embodiment 197 is a gRNA according to any one of Embodiments 161 to 196, comprising a YA modification in which at least one, two, three, four, five, or six nucleotides from the 5' end of the 5' terminal are optionally 2'-OMe modifications. Embodiment 198 is a gRNA according to any one of Embodiments 161 to 197, which includes a YA modification in which nucleotides 4 to 10 from the 5' end of the 5' end are optionally 2'-OMe modifications. Embodiment 199 is, a. A 5' protective end modification is included, wherein at least one of nucleotides 1-3 from the 5' end of the 5' terminal is optionally a 2'-OMe modification. b. The 5' end contains a 5' protective end modification in which at least two of nucleotides 1-3 from the 5' end of the 5' end are optionally 2'-OMe modifications, or c. A gRNA according to any one of Embodiments 161 to 198, comprising a 5' protective end modification in which each of nucleotides 1 to 3 from the 5' end of the 5' terminal is optionally a 2'-OMe modification. Embodiment 200 is a gRNA according to any one of Embodiments 161 to 199, which includes a 5' end modification in which at least one, two, three, four, or five of nucleotides 11, 13, 14, 17 and nucleotide 18 from the 5' end of the 5' end are optionally 2'-fluoro modifications. 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 is either unmodified or modified only by a 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 is either unmodified or modified only by a 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' end is either unmodified or modified only by a phosphorothioate. Embodiment 204 is a gRNA described in any one of Embodiments 161 to 203, which is either a crRNA or a dgRNA. Embodiment 205 is an sgRNA, which is a gRNA described in any one of Embodiments 161 to 203. Embodiment 206 is a short sgRNA, which is a gRNA described in any one of Embodiments 161 to 203. Embodiment 207 is a gRNA according to either Embodiment 205 or 206, which includes YA modification of the conserved region YA site 1. Embodiment 208 is a gRNA according to any one of Embodiments 205 to 207, which includes YA modification of the conserved region YA site 2. Embodiment 209 is a gRNA according to any one of Embodiments 205 to 208, which includes YA modification of the conserved region YA site 3. Embodiment 210 is a gRNA according to any one of Embodiments 205 to 209, which includes YA modification of the conserved region YA site 4. Embodiment 211 is a gRNA according to any one of Embodiments 205 to 210, which includes YA modification of the conserved region YA site 5. Embodiment 212 is a gRNA according to any one of Embodiments 205 to 211, which includes YA modification of the conserved region YA site 6. Embodiment 213 is a gRNA according to any one of Embodiments 205 to 212, which includes YA modification of the conserved region YA site 7. Embodiment 214 is a gRNA according to any one of Embodiments 205 to 213, which includes YA modification of the conserved region YA site 8. Embodiment 215 is a gRNA according to any one of Embodiments 205 to 214, which includes YA modification of the conserved region YA site 9. Embodiment 216 is a gRNA according to any one of Embodiments 205 to 215, which includes YA modification of the conserved region YA site 10. Embodiment 217 is, a. YA modifications of YA sites 2, 3, 4, and 10 in the conserved region. b. YA modification of YA sites 2, 3, and 4 in the conserved region. c. YA modifications of YA sites 2, 3, and 10 in the preserved region. d. YA modification of YA sites 2, 4, and 10 in the preserved region. e. YA modification of YA sites 3, 4, and 10 in the preserved region. f. YA modification of YA sites 2 and 10 in the conserved region. g. YA modification of YA sites 2 and 4 in the conserved region. h. YA modification of YA sites 2 and 3 in the conserved region. i. YA modification of YA sites 3 and 4 in the conserved region. j. YA modification of YA sites 3 and 10 in the preserved region, or k. The gRNA described in any one of embodiments 205 to 216, comprising YA modifications at YA sites 4 and 10 in the conserved region. Embodiment 218 is, a. YA modification of YA sites 1 and 5 in the conserved region. b. YA modification of YA sites 1 and 6 in the preserved region. c. YA modification of YA sites 1 and 7 in the preserved region. d. YA modification of YA sites 1 and 8 in the preserved region. e. YA modification of YA sites 1 and 9 in the conserved region. f. YA modification of YA sites 8 and 5 in the conserved region. g. YA modification of YA sites 8 and 6 in the conserved region. h. YA modification of YA sites 8 and 7 in the preserved region, or i. Containing YA modifications of YA sites 8 and 9 in the preserved region, Optionally, the sgRNA is the gRNA described in any one of Embodiments 205 to 217, further comprising YA modifications at conserved YA sites 2, 3, 4, and 10. Embodiment 219 is a gRNA according to any one of Embodiments 205 to 218, wherein at least one modified YA site optionally includes a 2'-OMe modification in the pyrimidine of the YA site. Embodiment 220 is a gRNA according to any one of Embodiments 205 to 219, wherein at least one modified YA site optionally includes a 2'-fluoro modification in the pyrimidine of the YA site. Embodiment 221 is a gRNA according to any one of Embodiments 205 to 220, wherein at least one modified YA site optionally includes a PS modification in the pyrimidine of the YA site. Embodiment 222 is a gRNA according to any one of Embodiments 205 to 221, wherein at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 modified YA sites optionally include 2'-OMe modifications in the pyrimidine of the YA sites. Embodiment 223 is one of the gRNAs from Embodiments 205 to 222, wherein at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 modified YA sites optionally include 2'-fluoro modifications in the pyrimidine of the YA sites. Embodiment 224 is one of the gRNAs from Embodiments 205 to 223, wherein at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 modified YA sites include PS modifications in the pyrimidine of the YA sites, by optional selection. Embodiment 225 is one of the gRNAs from Embodiments 205 to 224, wherein at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 modified YA sites include ribose modifications at the 2' position, optionally in the pyrimidine of the YA site, and optionally selected from 2'-O alkyl, 2'-H, and 2'-fluoro modifications. Embodiment 226 is, a. The conserved regions YA sites 1 and 8 optionally contain 2'-fluoromodifications in the pyrimidine of the YA sites. b. Preservation 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 contain 2'-OMe modification in the pyrimidine of the YA site. c. Conserved region YA site 1 contains 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 contain a 2'-OMe modification in the pyrimidine of the YA site. d. Conserved region YA site 8 contains 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 contain a 2'-OMe modification in the pyrimidine of the YA site. e. Conservation region YA site 1 contains a 2'-fluoro modification in the pyrimidine of the YA site, 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 a 2'-OMe modification in the pyrimidine of the YA site. f. The conserved region YA site 8 contains a 2'-fluoro modification in the pyrimidine of the YA site, and 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 a 2'-OMe modification in the pyrimidine of the YA site. g. Conserved regions YA sites 1 and 8 contain 2'-fluoro modifications, and 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 modifications in the pyrimidine of the YA site, h. The gRNA is described in any one of Embodiments 205 to 225, wherein the conserved regions YA sites 1 and 8 contain a 2'-fluoro modification at the pyrimidine of the YA site, 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 a 2'-OMe modification at the pyrimidine of the YA site. Embodiment 227 is a gRNA according to any one of Embodiments 205 to 226, which includes a YA modification in which the conserved regions YA sites 7 and 9 are optionally 2'-OMe modifications. Embodiment 228 is a gRNA according to any one of Embodiments 205 to 227, comprising YA modifications, wherein the conserved YA sites 5, 6, 7, and 9 are optionally 2'-OMe modifications. Embodiment 229 is a gRNA according to any one of Embodiments 205 to 228, wherein the conserved region YA site 8 includes a 2'-fluoro modification. Embodiment 230 is a gRNA according to any one of Embodiments 205 to 229, wherein the conserved region YA site 8 includes a deoxyribonucleotide modification. Embodiment 231 is a gRNA according to any one of Embodiments 205 to 230, wherein the conserved region YA site 8 is eliminated by base substitution, optionally the base substitution eliminates uracil from YA site 8, and optionally the base substitution replaces uracil with guanine. Embodiment 232 is a gRNA according to any one of Embodiments 205 to 231, wherein the conserved region YA site 1 includes a 2'-fluoro modification. Embodiment 233 is a gRNA according to any one of Embodiments 205 to 232, wherein the conserved region YA site 1 includes PS modification. Embodiment 234 is a 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 include modifications, and optionally the modifications are 2'-fluoro and / or 2'-OMe modifications. Embodiment 235 is a gRNA according to any one of Embodiments 205 to 234, wherein the modifications in LS5, LS7, LS9, and LS11, if present, include 2'-fluoro modifications, and optionally each of LS5, LS7, LS9, and LS11 includes 2'-fluoro modifications. Embodiment 236 is a gRNA according to any one of Embodiments 205 to 235, wherein the modifications in LS8, LS10, and LS12, if present, include 2'-OMe modifications, and each of LS8, LS10, and LS12 includes 2'-OMe modifications. Embodiment 237 is a gRNA according to any one of Embodiments 205 to 236, which includes modifications in which one, two, three, four, five, six, seven, eight, nine, or ten of N2, N3, N4, N5, N6, N7, N10, N11, N16, and N17 are optionally 2'-OMe modifications. Embodiment 238 is a gRNA according to any one of Embodiments 205 to 237, wherein H2-2 is modified, and optionally H2 is otherwise unmodified. Embodiment 239 is a gRNA according to any one of Embodiments 205 to 238, wherein H2-2 includes a 2'-OMe modification. Embodiment 240 is a gRNA according to any one of Embodiments 205 to 239, wherein US3, US9, and US12 are modified, and optionally US is otherwise unmodified. Embodiment 241 is a gRNA according to any one of Embodiments 205 to 240, wherein US3, US9, and US12 include a 2'-OMe modification. Embodiment 242 is a gRNA according to any one of Embodiments 205 to 241, wherein nucleotides 6 to 10 from the 5' end of the 5' terminal include a PS modification, and nucleotides 8 to 11, 13, 14, 17, and 18 from the 5' end of the 5' terminal include a 2'-fluoro modification. Embodiment 243 is a gRNA according to any one of Embodiments 205 to 242, wherein each guide region YA site includes a 2'-fluoro modification and optionally excludes nucleotides 15 and / or 16 from the 5' end of the 5' terminal. Embodiment 244 is a gRNA according to any one of Embodiments 205 to 243, wherein nucleotides 4, 8, and 11 from the 5' end of the 5' terminal contain YA modifications, and optionally, nucleotide 4 contains a 2'-OMe modification, and nucleotides 8 and 11 contain a 2'-fluoro modification. Embodiment 245 is a gRNA according to any one of Embodiments 205 to 244, 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 pyrimidine position of the YA site. Embodiment 246 is the gRNA according to Embodiment 245, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modified conserved regions (YA sites) include YA modifications at the pyrimidine positions of the YA sites. Embodiment 247 is a gRNA according to any one of Embodiments 205 to 246, wherein one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, 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 regions (YA sites) include YA site modifications at the adenine positions of the YA sites. Embodiment 249 is, a. Modification of H1-1, b. Modification of H2-1, or c. The gRNA described in any one of embodiments 205 to 248, including modifications to H1-1 and H2-1. Embodiment 250 is the gRNA described in Embodiment 249, wherein H1-1 and / or H2-1 include a 2'-OMe modification. Embodiment 251 is the gRNA according to Embodiment 250, wherein H1-1 and / or H2-1 include a 2'-fluoro modification. Embodiment 252 is the gRNA according to Embodiment 251, wherein H1-1 and / or H2-1 include PS modification. Embodiment 253 is a gRNA according to any one of Embodiments 205 to 252, which includes a modification in B3, and optionally B6 either does not include a 2'-OMe modification or includes a modification other than 2'-OMe. Embodiment 254 is a gRNA according to any one of Embodiments 205 to 253, which includes a modification in B4, and optionally B6 either does not include a 2'-OMe modification or includes a modification other than 2'-OMe. Embodiment 255 is a gRNA according to any one of Embodiments 205 to 254, which includes a modification in B5, and optionally, B6 either does not include a 2'-OMe modification or includes a modification other than 2'-OMe. Embodiment 256 is a gRNA according to any one of Embodiments 205 to 255, comprising modifications in LS10, and optionally, LS10 comprising modifications other than 2'-fluoro. Embodiment 257 is a gRNA according to any one of Embodiments 205 to 256, including modification at N2. Embodiment 258 is a gRNA according to any one of Embodiments 205 to 257, including modification at N3. Embodiment 259 is a gRNA according to any one of Embodiments 205 to 258, including modification at N4. Embodiment 260 is a gRNA according to any one of Embodiments 205 to 259, including modification at N5. Embodiment 261 is a gRNA according to any one of Embodiments 205 to 260, including modification at N6. Embodiment 262 is a gRNA according to any one of Embodiments 205 to 261, including modification at N7. Embodiment 263 is a gRNA according to any one of Embodiments 205 to 262, including modification at N10. Embodiment 264 is a gRNA according to any one of Embodiments 205 to 263, including modification at N11. Embodiment 265 is, a. Nucleotide 8 from the 5' end of the 5' terminal does not contain 2'-fluoro modification. b. Nucleotide 9 from the 5' end of the 5' terminal does not contain 2'-fluoro modification. c. Nucleotide 10 from the 5' end of the 5' terminal does not contain 2'-fluoro modification. Nucleotide 11 from the 5' end of the d.5' terminal does not contain 2'-fluoro modification. e. Nucleotide 13 from the 5' end of the 5' terminal does not contain 2'-fluoro modification. Nucleotide 14 from the 5' end of the f.5' terminal does not contain 2'-fluoromodification. g. Nucleotide 17 from the 5' end of the 5' terminal does not contain a 2'-fluoro modification, and / or The nucleotide 18 from the 5' end of the h.5' terminal is a gRNA according to any one of embodiments 205 to 264, and does not contain a 2'-fluoro modification. Embodiment 266 is, a. Nucleotide 6 from the 5' end of the 5' terminal does not contain 2'-fluoro modification. b. Nucleotide 7 from the 5' end of the 5' terminal does not contain 2'-fluoro modification. c. Nucleotide 8 from the 5' end of the 5' terminal does not contain 2'-fluoromodification. d. Nucleotide 9 from the 5' end of the 5' terminal does not contain a 2'-fluoro modification, and / or e. The nucleotide 10 from the 5' end of the 5' terminal is a gRNA according to any one of embodiments 205 to 265, and does not contain a 2'-fluoro modification. Embodiment 267 is, a. Nucleotide 6 from the 5' end of the 5' terminal does not contain a phosphorothioate bond. b. Nucleotide 7 from the 5' end of the 5' terminal does not contain a phosphorothioate bond. c. Nucleotide 8 from the 5' end of the 5' terminal does not contain a phosphorothioate bond. d. Nucleotide 9 from the 5' end of the 5' terminal does not contain a phosphorothioate bond, and / or e. The nucleotide 10 from the 5' end of the 5' terminal is a gRNA according to any one of embodiments 205 to 266, which does not contain a phosphorothioate bond. Embodiment 268 is, a. Nucleotide 7 from the 5' end of the 5' terminal does not contain the 2'-OMe modification. b. Nucleotide 8 from the 5' end of the 5' terminal does not contain the 2'-OMe modification. c. Nucleotide 9 from the 5' end of the 5' terminal does not contain the 2'-OMe modification, and / or The d. The nucleotide 10 from the 5' end of the 5' terminal is a gRNA according to any one of embodiments 205 to 267, and does not contain a 2'-OMe modification. Embodiment 269 is a gRNA according to any one of Embodiments 205 to 268, wherein nucleotide 20 from the 5' end of the 5' terminal does not contain a 2'-OMe modification. Embodiment 270 is a gRNA according to any one of Embodiments 205 to 269, wherein the guide RNA includes a 2'-fluoro modification in one or more nucleotides 1 to 11 and 13 to 20 from the 5' end of the 5' terminal, and nucleotide 12 from the 5' end of the 5' terminal does not include a 2'-fluoro modification. Embodiment 271 includes a guide RNA comprising a 2'-fluoro modification in one or more nucleotides 1 to 20 from the 5' end of the 5' terminal, a. Nucleotide 11 from the 5' end of the 5' terminal does not contain 2'-fluoro modification. b. Nucleotide 12 from the 5' end of the 5' terminal does not contain 2'-fluoro modification. c. Nucleotide 13 from the 5' end of the 5' terminal does not contain 2'-fluoro modification. Nucleotide 14 from the 5' end of the d.5' terminal does not contain 2'-fluoromodification. e. Nucleotide 17 from the 5' end of the 5' terminal does not contain a 2'-fluoro modification, and / or The nucleotide 18 from the 5' end of the f.5' terminal is a gRNA according to any one of embodiments 205 to 270, and does not contain a 2'-fluoro modification. Embodiment 272 is, a. B2 does not contain the 2'-OMe modification. b.B3 does not contain the 2'-OMe modification. c.B4 does not contain the 2'-OMe modification, and / or d.B5 is a gRNA described in any one of Embodiments 205 to 271, which does not contain the 2'-OMe modification. Embodiment 273 is, a. LS1 does not contain the 2'-OMe modification. b.LS8 does not contain the 2'-OMe modification, and / or c.LS10 is a gRNA described in any one of Embodiments 205 to 272, which does not contain the 2'-OMe modification. Embodiment 274 is, a.N2 does not contain the 2'-OMe modification. b.N3 does not include the 2'-OMe modification. c.N4 does not contain the 2'-OMe modification. d.N5 does not contain the 2'-OMe modification. e.N6 does not contain the 2'-OMe modification. f.N7 does not include the 2'-OMe modification. g.N10 does not contain the 2'-OMe modification. h.N11 does not contain the 2'-OMe modification. i.N16 does not contain the 2'-OMe modification, and / or j.N17 is the gRNA described in any one of Embodiments 205 to 273, which does not contain the 2'-OMe modification. 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 phosphorothioate bonds. 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 phosphorothioate bonds. 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 phosphorothioate bonds. q.H2-8 does not contain a phosphorothioate bond. r.H2-9 does not contain a phosphorothioate bond. s.H2-10 does not contain phosphorothioate bonds. t.H2-11 does not contain phosphorothioate bonds. u.H2-12 does not contain a phosphorothioate bond. v.H2-13 does not contain phosphorothioate bonds. w.H2-14 does not contain phosphorothioate bonds, and / or x.H2-15 is a gRNA described in any one of Embodiments 205 to 274, which does not contain a phosphorothioate bond. Embodiment 276 is, a. Selectively, nucleotides 6-10 from the 5' end of the 5' terminal, which are PS modified. b. Optional 2'-fluoromodifications, nucleotides 8-11, 13, 14, 17, and 18 from the 5' end of the 5' terminal, and c. A gRNA that is optionally modified with 2'-OMe modification, or has modifications in the conserved region YA site 1 or 8, and contains modifications in H1-1 and H2-1. Embodiment 277 is, a. Conserved regions YA sites 1, 5, 6, 7, and 9, which are 2'-OMe modified by arbitrary selection, and b. A gRNA that is a 2'-fluoro modified sgRNA, has a YA modification in the conserved region YA site 8, or has a 2'-fluoro modification by optional selection. Embodiment 278 is a gRNA comprising YA modifications in four guide region YA sites, wherein at least one of the YA sites is located at nucleotide 8 from the 5' end of the 5' end or thereafter. a. The first YA site includes a 2'-OMe modification. b. The second YA site includes a 2'-fluoro modification, c. The third YA site includes a 2'-fluoro or PS modification. d. The fourth YA site includes PS modification, This gRNA is one in which the first, second, third, and fourth YA sites are positioned in the 5' to 3' direction by arbitrary selection. Embodiment 279 is the gRNA of Embodiment 278, wherein the third YA site includes PS modification. Embodiment 280 is the gRNA according to Embodiment 278 or 279, wherein the third YA site includes a 2'-fluoro modification. Embodiment 281 is a gRNA according to any one of Embodiments 278 to 280, further comprising, optionally, a fifth YA site containing a PS modification located 3' to the fourth YA site. Embodiment 282 is a gRNA according to any one of Embodiments 205 to 281, comprising modifications in which the conserved YA sites 1, 5, 6, 7, and 9 are optionally 2'-OMe modifications, and the conserved YA site 8 is optionally 2'-fluoro modifications. Embodiment 283 is, a. Nucleotide 4 from the 5' end of the 5' terminal, wherein the YA modification is optionally a 2'-OMe modification, b. Nucleotides 6-10 from the 5' end of the 5' terminal, which are PS modified by choice. c. Optionally, nucleotides 8-11, 13, 14, 17, and 18 from the 5' end of the 5' terminal are 2'-fluoro modified. d. LS5, LS7, LS9, and LS11, which are optionally 2'-fluoro modified. e. LS8, LS10, and LS12, which are optionally modified with 2'-OMe. f. N2, N3, N4, N5, N6, N7, N10, N11, N16, and N17, which are 2'-OMe modified by any choice, and g. A gRNA that is an sgRNA containing a 2'-fluoro modification, a YA modification at N14, or a gRNA that is optionally modified with 2'-fluoro. Embodiment 284 is, a. Nucleotide 4 from the 5' end of the 5' terminal contains a 2'-OMe modification. b. Nucleotides 6-10 from the 5' end of the 5' terminal include PS modification. c. Nucleotides 8-11, 13, 14, 17, and 18 from the 5' end of the 5' terminal contain 2'-fluoro modifications. d.LS5, LS7, LS9, and LS11 contain 2'-fluoro modifications. e.LS8, LS10, and LS12 include the 2'-OMe modification, f.N2, N3, N4, N5, N6, N7, N10, N11, N16, and N17 include the 2'-OMe modification, and g.N14 is a gRNA described in any one of embodiments 161 to 164, wherein one or more of the gRNAs include 2'-fluoro modifications. Embodiment 285 is a gRNA according to any one of Embodiments 161 to 284, wherein at least one YA modification includes a modification at the pyrimidine site of the YA site. Embodiment 286 is a gRNA according to any one of Embodiments 161 to 285, wherein at least one YA modification includes a modification of the adenine position of the YA site. Embodiment 287 is a gRNA according to any one of Embodiments 161 to 286, wherein at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 YA sites include YA modifications at the pyrimidine positions of the YA sites. Embodiment 288 is a gRNA according to any one of Embodiments 161 to 287, wherein at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 YA sites include YA modifications at the adenine positions of the YA sites. Embodiment 289 is a gRNA according to any one of Embodiments 161 to 288, wherein at least one YA modification includes a 2'-OMe modification. Embodiment 290 is a gRNA according to any one of Embodiments 161 to 289, wherein at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 YA sites contain 2'-OMe modifications. Embodiment 291 is a gRNA according to any one of Embodiments 161 to 290, wherein each modified conserved region YA site includes modification at the pyrimidine position of the YA site. Embodiment 292 is a gRNA according to any one of Embodiments 161 to 291, wherein each modification guide region YA site, or each modification conservation region and guide region YA site, includes modification at the pyrimidine position of the YA site. Embodiment 293 is a gRNA according to any one of Embodiments 161 to 292, wherein each modified conserved region YA site includes modification at the adenine position of the YA site. Embodiment 294 is a gRNA according to any one of Embodiments 161 to 293, wherein each modification guide region YA site, or each modification conservation region and guide region YA site, includes modification at the adenine position of the YA site. Embodiment 295 is a gRNA described in any one of Embodiments 161 to 294, which is an sgRNA that includes modifications at LS5. Embodiment 296 is a gRNA according to any one of Embodiments 161 to 295, which is an sgRNA that includes modifications at LS7. Embodiment 297 is an sgRNA containing a modification at LS9, wherein, optionally, LS9 is modified and LS5, LS7, and LS12 are unmodified, and the modification of LS9 is other than 2'-fluoro, as described in any one of Embodiments 161 to 296. Embodiment 298 is an sgRNA containing a modification at LS12, and optionally, if LS12 is modified and LS9 is unmodified, the modification at LS12 is other than 2'-OMe, and is one of the gRNAs described in any one of Embodiments 161 to 297. Embodiment 299 is an sgRNA containing at least one YA modification that stabilizes the secondary structure, and optionally, the secondary structure is a lower stem, as described in any one of Embodiments 161 to 298. Embodiment 300 is an sgRNA comprising at least one modification of LS8 and / or LS11, wherein the modification of LS8 and / or LS11 optionally stabilizes the secondary structure, and is a gRNA according to any one of Embodiments 161 to 299. Embodiment 301 is, a.ENA, b.LNA, or c. A gRNA according to any one of Embodiments 161 to 300, comprising a YA modification that stabilizes a secondary structure selected from bicyclic ribose modifications. Embodiment 302 is a gRNA according to any one of Embodiments 161 to 301, which is an sgRNA including modification at N6. Embodiment 303 is a gRNA according to any one of Embodiments 161 to 302, which is an sgRNA including modification at N14. Embodiment 304 is an sgRNA containing a modification at N17, and optionally, if N17 is modified and N6 and N14 are unmodified, the modification at N17 is other than 2'-fluoro and other than 2'-OMe, and is the gRNA described in any one of Embodiments 161 to 303. Embodiment 305 is a gRNA according to any one of Embodiments 161 to 304, wherein at least one, two, or three of the nucleotides 1 to 3 from the 5' end of the 5' terminal contain a deoxyribonucleotide, and optionally, the nucleotides 1 to 3 from the 5' end of the 5' terminal contain a PS modification. Embodiment 306 is a gRNA according to any one of Embodiments 161 to 305, wherein the gRNA is an sgRNA, and at least one, two, or three of the nucleotides 1 to 3 from the 3' end of the 3' terminal contain a deoxyribonucleotide, and optionally, the nucleotides 2 to 3 from the 3' end of the 3' terminal contain a PS modification. Embodiment 307 is a gRNA according to any one of Embodiments 161 to 306, wherein the gRNA is an sgRNA, and nucleotide 4 from the 3' end of the 3' terminal contains a PS modification, and optionally, nucleotide 4 from the 3' end of the 3' terminal contains a 2'-OMe modification. Embodiment 308 is a gRNA according to any one of Embodiments 161 to 307, wherein the gRNA is an sgRNA, and hairpin 2 contains a deoxyribonucleotide, and optionally all or nearly all of the 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of hairpin 1 and hairpin 2 are deoxyribonucleotides. Embodiment 309 is a gRNA according to any one of Embodiments 161 to 308, wherein the gRNA is an sgRNA, and hairpin 1 and hairpin 2 contain deoxyribonucleotides, and optionally all or nearly all of the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 nucleotides of hairpin 1 and hairpin 2 are deoxyribonucleotides. Embodiment 310 is a gRNA according to any one of Embodiments 161 to 309, wherein the gRNA is an sgRNA, and all or nearly all of the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 nucleotides from the start of hairpin 1 to the 3' end of the sgRNA are deoxyribonucleotides, and optionally, nucleotides 1 to 3 from the 3' end of the 3' end are deoxyribonucleotides. Embodiment 311 is a gRNA according to any one of Embodiments 161 to 310, wherein the gRNA is an sgRNA and the upper stem contains a deoxyribonucleotide. Embodiment 312 is a gRNA according to any one of Embodiments 161 to 311, wherein the gRNA is an sgRNA and all or nearly all of the nucleotides of the 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 upper stems are deoxyribonucleotides. Embodiment 313 is a gRNA according to any one of Embodiments 161 to 312, wherein at least one, two, or three nucleotides 1 to 3 from the 5' end of the 5' terminal contain ENA, and optionally, nucleotides 1 to 3 from the 5' end of the 5' terminal contain PS modification. Embodiment 314 is a gRNA according to any one of Embodiments 161 to 313, wherein the gRNA is an sgRNA, and at least one, two, or three of the nucleotides 2 to 4 from the 3' end of the 3' terminal contain ENA, and optionally, nucleotides 2 to 3 from the 3' end of the 3' terminal contain PS modification. Embodiment 315 is a gRNA according to any one of Embodiments 161 to 314, wherein at least one, two, or three nucleotides 1 to 3 from the 5' end of the 5' terminal contain UNA, and optionally, nucleotides 1 to 3 from the 5' end of the 5' terminal contain PS modification. Embodiment 316 is a gRNA according to any one of Embodiments 161 to 315, wherein the gRNA is an sgRNA, and at least one, two, or three of the nucleotides 2 to 4 from the 3' end of the 3' terminal contain UNA, and optionally, nucleotides 2 to 3 from the 3' end of the 3' terminal contain PS modification. Embodiment 317 is a gRNA according to any one of Embodiments 161 to 316, wherein the gRNA is an sgRNA, and nucleotide 4 from the 3' end of the 3' terminal contains a PS modification, and optionally, nucleotide 4 from the 3' end of the 3' terminal contains a 2'-OMe modification. Embodiment 318 is a gRNA according to any one of Embodiments 161 to 317, wherein the gRNA is an sgRNA that includes a 3' terminal modification. Embodiment 319 is an sgRNA containing a 3' terminal modification, wherein the 3' terminal modification is a protective 3' terminal modification, and is the gRNA described in any one of Embodiments 161 to 318. Embodiment 320 is a gRNA according to any one of Embodiments 161 to 319, wherein the gRNA is an sgRNA including a 3' tail. Embodiment 321 is the gRNA according to Embodiment 320, wherein the 3' tail contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. Embodiment 322 is the gRNA according to Embodiment 320, wherein 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. Embodiment 323 is a gRNA according to any one of Embodiments 161 to 322, which is an sgRNA including modifications in the hairpin region. Embodiment 324 is a gRNA according to any one of Embodiments 161 to 323, which is an sgRNA including a 3' terminal modification and a modification in the hairpin region. Embodiment 325 is a gRNA according to Embodiment 323 or 324, wherein the modification in the hairpin region includes a modified nucleotide selected from 2'-O-methyl (2'-O-Me) modified nucleotides, 2'-fluoro (2'-F) modified nucleotides, or a combination thereof. Embodiment 326 is a 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. Embodiment 327 is a 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. Embodiment 328 is a gRNA according to any one of Embodiments 161 to 327, comprising 3' and / or 5' protective end modification(s). Embodiment 329 is the gRNA according to Embodiment 328, wherein the 3' and / or 5' terminal modifications include 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 debasitized modified nucleotide, or a combination thereof. Embodiment 330 is the gRNA according to Embodiment 328 or 329, wherein the 3' and / or 5' terminal modifications include or further include 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' terminal modifications include or further include a 2'-fluoro(2'-F) modified nucleotide. Embodiment 332 is the gRNA according to Embodiment 328 or 329, wherein the 3' and / or 5' terminal modifications include or further include internucleotide phosphorothioate (PS) bonds. Embodiment 333 is the gRNA according to Embodiment 328 or 329, wherein the 3' and / or 5' terminal modifications include or further include an inverted debased nucleotide. Embodiment 334 is an sgRNA in which the sgRNA includes a 3' terminal modification, and the 3' terminal modification is as follows: i. One or more modifications of the last 7, 6, 5, 4, 3, 2, or 1 nucleotide, ii. One modified nucleotide, iii. Two modified nucleotides, iv. Three modified nucleotides, v. 4 modified nucleotides, vi. 5 modified nucleotides, vii. Six modified nucleotides, and viii. A gRNA according to any one of embodiments 161 to 333, comprising one or more of the seven modified nucleotides. Embodiment 335 is the gRNA according to Embodiment 334, wherein the 3' terminal modification includes modifications of 1 to 7, 1 to 5, 1 to 4, or 2 to 4 nucleotides. Embodiment 336 is an sgRNA in which the gRNA has a 3' terminal modification, and the 3' terminal modification is as follows: i. Internucleotide phosphorothioate (PS) bonding, ii. 2'-O-Me modified nucleotides, iii. 2'-O-moe modified nucleotides, iv. 2'-F modified nucleotide, v. Inverted debasitized nucleotides vi. ENA, UNA, and / or DNA, and vii. The gRNA described in any one of embodiments 161 to 335, comprising one or more of the above or combinations thereof. Embodiment 337 is an sgRNA that includes a 3' tail, i. Internucleotide phosphorothioate (PS) bonding, ii. 2'-O-Me modified nucleotides, iii. 2'-O-moe modified nucleotides, iv. 2'-F modified nucleotide, v. Inverted debasitized nucleotides vi. ENA, UNA, and / or DNA, and vii. The gRNA described in any one of embodiments 161 to 336, comprising one or more of the above or combinations thereof. Embodiment 338 has a 3' terminal modification, i. PS bonds between 1, 2, 3, 4, 5, 6, or 7 nucleotides, ii. Approximately 1-3, 1-5, 1-6, or 1-7 nucleotide-to-PS bonds, iii. The gRNA according to Embodiment 336, which includes PS bonds between each nucleotide. Embodiment 339 is a gRNA according to any one of Embodiments 326 to 328, wherein the 3'-terminal modification further comprises at least one 2'-O-Me, 2'-O-moe, inverted debase, or 2'-F modified nucleotide. Embodiment 340 has a 3'-terminus modification that includes at least one PS bond. i. There is one PS bond, and the bond is between the last nucleotide and the second to last nucleotide. ii. Two PS bonds exist between the last three nucleotides. iii. A PS bond exists between one or more of the last four nucleotides. iv. A PS bond exists between one or more of the last five nucleotides, or v. The gRNA according to any one of embodiments 326 to 329, wherein a PS bond exists between one or more of the last 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. Embodiment 341 has a 3' terminal modification, i. One or more modifications among the last 1 to 7 nucleotides, wherein the modification is a PS bond, an inverted debasalized nucleotide, 2'-O-Me, 2'-O-moe, 2'-F, or a combination thereof. ii. Modification of the last nucleotide by 2'-O-Me, 2'-O-moe, 2'-F, or a combination thereof, and one or two PS bonds of any choice to the adjacent nucleotide and / or the first nucleotide of the 3' tail. iii. Modification of the last and / or second-to-last nucleotide by 2'-O-Me, 2'-O-moe, 2'-F, or a combination thereof, and optionally one or more PS bonds, iv. Modification of the last, second to last, and / or third to last nucleotide by 2'-O-Me, 2'-O-moe, 2'-F, or a combination thereof, and optionally, one or more PS bonds. v. Modification of the last, second to last, third to last, and / or fourth to last nucleotide by 2'-O-Me, 2'-O-moe, 2'-F, or a combination thereof, and optionally, one or more PS bonds, vi. A gRNA according to any one of embodiments 336 to 340, comprising modifications to the last, second to last, third to last, fourth to last, and / or fifth to last nucleotide by 2'-O-Me, 2'-O-moe, 2'-F, or a combination thereof, and optionally containing one or more PS bonds. Embodiment 342 is a gRNA according to any one of Embodiments 161 to 341, wherein the gRNA is an sgRNA including a 3' tail, and the 3' tail includes one or more modifications of the nucleotides present in the 3' tail. Embodiment 343 is the gRNA described in Embodiment 342, wherein the 3' tail is fully modified. Embodiment 344 is the gRNA described in Embodiment 342, wherein the 3' tail contains 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 one or more of these nucleotides are optionally modified. Embodiment 345 has the following 3' terminal modification: i. A 3' terminal modification shown in any one of sequence numbers 401-532, ii. (i) a 2'O-Me modified nucleotide at the last nucleotide of the conserved region of sgRNA or short sgRNA, (ii) three consecutive 2'O-Me 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' terminal, and (ii) three PS bonds between the last three nucleotides, iv. Inverted debase-modified nucleotide at the last nucleotide of the conserved region of sgRNA or short sgRNA, v. (i) an inverted debasic modified nucleotide at the last nucleotide of the conserved region of sgRNA or short sgRNA, and (ii) three consecutive 2'O-Me modified nucleotides at the last three nucleotides of the conserved region of sgRNA or short sgRNA. vi. (i) Fifteen consecutive 2'O-Me modified nucleotides from the 3' end of the 3' terminal, (ii) five consecutive 2'-F modified nucleotides immediately to the 5' side of the 2'O-Me modified nucleotides, 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 sgRNA or short sgRNA, and (ii) three PS bonds between the last three nucleotides, viii. (i) 2 or 3 consecutive 2'O-Me modified nucleotides, and (ii) 3 PS bonds between the last 3 nucleotides, ix. One PS bond between the last nucleotide and the nucleotide adjacent to the last, and The gRNA according to any one of embodiments 336 to 344, comprising (ii) 15 or 20 consecutive 2'O-Me modified nucleotides, and (ii) one or more of three PS bonds between the last three nucleotides. Embodiment 346 is as follows: i. Modification of one or more nucleotides 1-7 in the guide region. ii. One modified nucleotide, iii. Two modified nucleotides, iv. Three modified nucleotides, v. 4 modified nucleotides, vi. 5 modified nucleotides, vii. Six modified nucleotides, and viii. The gRNA according to any one of embodiments 161 to 345, comprising a 5' terminal modification containing one or more of the seven modified nucleotides. Embodiment 347 is the gRNA according to any one of Embodiments 161 to 346, which includes a 5'-end modification, and the 5'-end modification is a protective 5'-end modification. Embodiment 348 is the gRNA according to any one of Embodiments 161 to 347, which includes a 5'-end modification, and the 5'-end modification includes modifications of 1 to 7, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 nucleotides. Embodiment 349 is the gRNA according to any one of Embodiments 161 to 348, which includes 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. Modifications of about 1 to 3, 1 to 4, 1 to 5, 1 to 6, or 1 to 7 of the first 7 nucleotides, and iii. Modifications at the 1st, 2nd, 3rd, 4th, 5th, 6th, and / or 7th nucleotides at the 5'-end, optionally with the modifications being consecutive, and including any one or more of the modifications. Embodiment 350 is the gRNA according to any one of Embodiments 161 to 349, which includes a 5'-end modification, and the 5'-end modification is i. internucleotide phosphorothioate (PS) bonds, ii. 2'-O-Me modified nucleotides, iii. 2'-O-moe modified nucleotides, iv. 2'-F modified nucleotides, v. inverted abasic modified nucleotides vi. ENA, UNA, and / or DNA, and vii. any one or more of their combinations. Embodiment 351 is the gRNA according to any one of Embodiments 161 to 350, which 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 ii. about 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, or 1 to 7 internucleotide PS bonds. Embodiment 352 is a gRNA according to any one of Embodiments 161 to 351, wherein the sgRNA includes a 5' terminal modification, the 5' terminal modification comprising at least one 2'-O-Me, 2'-O-moe, inverted debase, 2'-H, inosine, or 2'-F modified nucleotide. Embodiment 353 has a 5'-terminus modification that includes at least one PS bond. i. There is one PS bond, and the bond is located at nucleotide 1 of the guide region. ii. Two PS bonds are present, and the bonds are located at nucleotides 1 and 2 of the guide region. iii. A PS bond is present on one or more of nucleotides 1, 2, and 3 in the guide region. iv. A PS bond is present on one or more of the guide region nucleotides 1, 2, 3, and nucleotide 4. v. One or more PS bonds are present in any of the guide region nucleotides 1, 2, 3, 4, and nucleotide 5. vi. One or more PS bonds are present in any of the guide region 1, 2, 3, 4, 5, and nucleotide 6, or vii. The gRNA according to Embodiment 352, wherein one or more PS bonds are present on any of nucleotides 1, 2, 3, 4, 5, 6, and nucleotide 7 of the guide region. Embodiment 354 has a 5' terminal modification, i. Modifications of one or more nucleotides 1-7 in the variable region, wherein the modification is a PS bond, an inverted debasalized nucleotide, 2'-O-Me, 2'-O-moe, 2'-F, 2'-H, inosine, and / or a combination thereof. ii. PS binding to the first nucleotide of the guide region, and to an adjacent nucleotide, by choice, using 2'-O-Me, 2'-O-moe, 2'-F, 2'-H, inosine, or a combination thereof. iii. 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 combinations thereof, and optionally one or more PS bonds, iv. Modification of the first, second, and / or third nucleotides of the variable region by 2'-O-Me, 2'-O-moe, 2'-F, 2'-H, inosine, or combinations thereof, and optionally, one or more PS bonds. v. Modification of the first, second, third, and / or nucleotide 4 of the variable region by 2'-O-Me, 2'-O-moe, 2'-F, 2'-H, inosine, or combinations thereof, and optionally, one or more PS bonds, or vi. The gRNA according to Embodiment 352 or 353, comprising modifications to the first, second, third, fourth, and / or fifth nucleotides of the variable region by 2'-O-Me, 2'-O-moe, 2'-F, 2'-H, inosine, or a combination thereof, and optionally containing one or more PS bonds. Embodiment 355 includes a 5'-terminal modification, wherein the 5'-terminal modification is as follows: i. 5'-terminal modifications shown in any one of the following sequence numbers: 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 in nucleotides 1, 2, and 3 of the guide region, iii. 2'-OMe modified nucleotides in nucleotides 1, 2, and 3 of the guide region, and PS bonds between nucleotides 1 and 2, 2 and 3, and 3 and 4 of the guide region. iv. 2'-OMe modified nucleotides in guide regions 1, 2, 3, 4, and 5, v. 2'-OMe modified nucleotides in guide region 1, 2, 3, 4 and nucleotide 5, and PS bonds between nucleotides 1 and 2, 2 and 3, 3 and 4, 4 and 5, and 5 and 6 in guide region. vi. 2'O-moe modified nucleotides in nucleotides 1, 2, and 3 of the guide region. vii. 2'O-moe modified nucleotides in nucleotides 1, 2, and 3 of the guide region, and PS bonds between nucleotides 1 and 2, 2 and 3, and 3 and 4 of the guide region. viii. Inverted debasic modified nucleotide at nucleotide 1 in the guide region, ix. Inverted debasic 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. The gRNA according to any one of embodiments 161 to 354, comprising one or more PS bonds between nucleotides 1 and 2, 2 and 3, 3 and 4, 4 and 5, and 5 and 6 in the variable region. Embodiment 356 is a gRNA according to any one of Embodiments 161 to 355, wherein the gRNA is an sgRNA and the upper stem region includes at least one modification. Embodiment 357 has the following upper stem modification: i. One or more modifications from US1 to US12 in the upper stem region. ii. Modification of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or all 12 nucleotides in the upper stem region, and iii. The upper stem region contains one or more of the following modifications: approximately 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, or 1-12 nucleotides. This is the gRNA described in Embodiment 346. Embodiment 358 is characterized by an upper stem modification, i. 2'-O-Me modified nucleotide, ii. 2'-H modified nucleotides, iii. 2'-F modified nucleotides, and iv. Including one or more of those combinations, This is the gRNA described in Embodiment 356 or 357. Embodiment 359 is a gRNA according to any one of Embodiments 161 to 358, wherein the gRNA is an sgRNA containing one or more modifications in the hairpin 1 region. Embodiment 360 is the gRNA described in Embodiment 359, wherein the sgRNA includes modifications at H1-1. Embodiment 361 is a gRNA according to any one of Embodiments 161 to 360, wherein the gRNA is an sgRNA containing one or more modifications in the hairpin 2 region. Embodiment 362 is the gRNA described in Embodiment 361, wherein the sgRNA includes modifications at H2-1. Embodiment 363 is a gRNA according to any one of Embodiments 161 to 362, wherein the gRNA is an sgRNA that includes modifications in H1-1 to H1-12. Embodiment 364 is a gRNA according to any one of Embodiments 161 to 363, wherein the gRNA is an sgRNA that includes modifications at H2-1 to H2-15. Embodiment 365 is a gRNA according to any one of Embodiments 161 to 364, wherein the gRNA is an sgRNA having one or more modifications in each of the upper stem region, hairpin 1 region, and hairpin 2 region. Embodiment 366 is a gRNA according to any one of Embodiments 161 to 365, wherein the gRNA is an sgRNA containing modified nucleotides between the hairpin 1 region and the hairpin 2 region. Embodiment 367 is a gRNA according to any one of embodiments 161 to 366, which further comprises a lower stem region including modifications. Embodiment 368 is a gRNA according to any one of Embodiments 161 to 367, further comprising a 3' terminal 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 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 phosphorothioate (PS) bonds 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, which is an sgRNA further comprising a bulge region containing a modification. Embodiment 373 is the gRNA according to any one of Embodiments 161 to 372, which is an sgRNA further comprising a nexus region containing a modification. Embodiment 374 is an sgRNA comprising 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 an 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 SEQ ID NOs: 401 to 535, 601, 607 to 732, 801, 807 to 932, 1001, or 1007 to 1132, wherein 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. 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. 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 a gRNA according to any one of Embodiments 161 to 377, comprising a YA modification, wherein the modification alters the structure of the dinucleotide motif to reduce RNA endonuclease activity. Embodiment 379 is a gRNA according to any one of Embodiments 161 to 378, comprising a YA modification, the modification preventing recognition or cleavage of the YA site by RNase and / or stabilizing the RNA structure. Embodiment 380 includes YA modification, and the modification is 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 modification, d. Base modifications such as inosine, pseudouridine, and 5'-methylcytosine, and e. Containing one or more of the nucleoside bond modifications such as phosphorothioates, The gRNA is described in any one of embodiments 161 to 379. Embodiment 381 is a gRNA according to any one of Embodiments 1 to 380, wherein the gRNA includes a guide region comprising a modification at nucleotide 5, and optionally the guide region comprises 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. Embodiment 382 is a gRNA according to any one of Embodiments 1 to 381, wherein the gRNA includes a guide region comprising a modification at nucleotide 12, and optionally the guide region comprises 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. Embodiment 383 is a gRNA according to any one of Embodiments 1 to 382, ​​wherein the gRNA includes a guide region comprising a 2'-OMe modification at nucleotide 5 and / or nucleotide 12, and optionally the guide region comprising 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. Embodiment 384 is a gRNA according to any one of Embodiments 1 to 383, wherein the gRNA includes a guide region comprising a 2'-F modification at nucleotide 5 and / or nucleotide 12, and optionally the guide region comprises 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. Embodiment 385 is a gRNA according to any one of Embodiments 1 to 384, wherein the gRNA includes a guide region comprising a 2'-H modification at nucleotide 5 and / or nucleotide 12, and optionally the guide region comprises 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. Embodiment 386 is a gRNA according to any one of Embodiments 1 to 385, wherein the gRNA includes a guide region comprising a phosphorothioate modification on nucleotide 5 and / or nucleotide 12, and optionally the guide region comprising a 2'-OMe modification on nucleotides 1 to 4, a phosphorothioate modification on nucleotides 1 to 3 and 6 to 10, and / or a 2'-F modification on nucleotides 8 to 11, 13, 14, 17, and 18. Embodiment 387 is a case where gRNA is i. Nucleotides 8-10, ii. Nucleotides 8 and 9, iii. Nucleotides 8 and 10, or iv. Includes a guide region with modifications at nucleotides 9 and 10, The gRNA is one of any one of Embodiments 1 to 386, wherein the guide region optionally 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. Embodiment 388 is a case where gRNA is i. Nucleotides 8-10, ii. Nucleotides 8 and 9, iii. Nucleotides 8 and 10, iv. Nucleotides 9 and 10, or v. Includes a guide region containing a 2'-F modification at nucleotide 8, The gRNA is one of any one of Embodiments 1 to 387, wherein the guide region optionally 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. Embodiment 389 is a case where gRNA is i. Nucleotides 8-10, ii. Nucleotides 8 and 9, iii. Nucleotides 8 and 10, iv. Nucleotides 9 and 10, or v. Includes a guide region containing a 2'-F modification at nucleotide 8, The gRNA is one of any one of Embodiments 1 to 388, wherein nucleotides 8 to 10 are free of phosphorothioate modifications, and optionally, the guide region includes 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 11, 13, 14, 17, and 18. Embodiment 390 is a gRNA in which nucleotides 8-10 have 2'-F modifications, and i. Phosphothioate modification at nucleotide 1, 2, or 3 of nucleotides 8-10. ii. Phosphothioate modification at nucleotide 8, iii. Phosphothioate modification at nucleotide 9, iv. Phosphothioate modification at nucleotide 10, v. Phosphothioate modification at nucleotides 8 and 9, vi. Phosphothioate modifications at nucleotides 8 and 10, vii. Phosphothioate modification at nucleotides 9 and 10, or vii. Containing phosphorothioate modifications of nucleotides 8-10, The gRNA is one of any one of Embodiments 1 to 389, wherein the guide region optionally 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. Embodiment 391 is a case in which gRNA is i. 2'-F or phosphorothioate modification at nucleotides 5 and 6, ii. 2'-F modification in nucleotides 5 and 6, iii. Phosphothioate modifications in nucleotides 5 and 6, iv. 2'-F modification at nucleotide 5 and phosphorothioate modification at nucleotide 6, or v. Includes a guide region containing a 2'-F modification at nucleotide 6 and a phosphorothioate modification at nucleotide 5, The gRNA is one of any one of Embodiments 1 to 390, wherein the guide region optionally includes a 2'-OMe modification at nucleotides 1 to 4, a phosphorothioate modification at nucleotides 1 to 3 and 7 to 10, and / or a 2'-F modification at nucleotides 8 to 11, 13, 14, 17, and 18. Embodiment 392 is a gRNA according to any one of Embodiments 1 to 391, wherein the gRNA comprises a guide region containing at least 1, 2, 3, 4, 5, or 6 2'-F modifications on nucleotides 6 to 11, and optionally the guide region contains 2'-OMe modifications on nucleotides 1 to 4, phosphorothioate modifications on nucleotides 1 to 3, and / or 2'-F modifications on nucleotides 13, 14, 17, and 18. Embodiment 393 is a gRNA according to any one of Embodiments 1 to 392, wherein the gRNA includes a guide region comprising 2'-F modifications at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from nucleotides 1 to 4 and 6 to 11, and optionally the guide region comprising phosphorothioate modifications at nucleotides 1 to 3, and / or 2'-F modifications at nucleotides 13, 14, 17, and 18. Embodiment 394 is a gRNA according to any one of Embodiments 1 to 393, wherein the gRNA includes a guide region containing 2'-F modifications at nucleotides 6 to 11, and optionally the guide region contains 2'-OMe modifications at nucleotides 1 to 4, phosphorothioate modifications at nucleotides 1 to 3, and / or 2'-F modifications at nucleotides 13, 14, 17, and 18. Embodiment 395 is a gRNA according to any one of Embodiments 1 to 394, wherein the gRNA includes a guide region containing 2'-F modifications at nucleotides 1 to 4, and optionally the guide region contains 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. Embodiment 396 is a gRNA according to any one of Embodiments 1 to 395, wherein the gRNA includes a guide region that includes a 2'-F modification at nucleotide 9 and does not include a phosphorothioate modification at nucleotide 9, and 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 nucleotide 8 and 10, and / or a 2'-F modification at nucleotides 8, 10, 11, 13, 14, 17, and 18. Embodiment 397 is a gRNA according to any one of Embodiments 1 to 396, wherein the gRNA comprises a guide region that does not contain at least 1, 2, 3, 4, 5, 6, 7, or 8 2'-F modifications from nucleotides 8-11, 13, 14, 17, and 18, and optionally the guide region comprises a 2'-OMe modification at nucleotides 1-4, and / or a phosphorothioate modification at nucleotides 1-3 and 6-10. Embodiment 398 is a gRNA according to any one of Embodiments 1 to 397, wherein the gRNA includes a guide region that does not contain 2'-F modifications at nucleotides 8-11, 13, 14, 17, and 18, and optionally the guide region includes 2'-OMe modifications at nucleotides 1-4 and / or phosphorothioate modifications at nucleotides 1-3 and 6-10. Embodiment 399 is a gRNA according to any one of Embodiments 1 to 398, wherein the gRNA includes a guide region comprising at least one, two, three, or four 2'-OMe modifications on nucleotides 9, 11, 13, and 14, and optionally the guide region comprising 2'-OMe modifications on nucleotides 1 to 4, and / or phosphorothioate modifications on nucleotides 1 to 3 and 6 to 10. Embodiment 400 is a gRNA according to any one of Embodiments 1 to 399, wherein the gRNA includes a guide region comprising 2'-OMe modifications at nucleotides 9, 11, 13, and 14, and optionally the guide region comprises 2'-OMe modifications at nucleotides 1 to 4, and / or phosphorothioate modifications at nucleotides 1 to 3 and 6 to 10. Embodiment 401 is a gRNA according to any one of Embodiments 1 to 400, wherein the gRNA includes a guide region comprising a phosphorothioate modification at one or both of nucleotides 8 and nucleotide 10, and optionally the guide region comprises 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 8 to 11, 13, 14, 17, and 18. Embodiment 402 is a gRNA according to any one of Embodiments 1 to 401, comprising a guide region including modifications in at least one, two, three, four, five, six, seven, eight, nine, ten, one, one, one, two, one Embodiment 403 is a gRNA according to any one of Embodiments 1 to 402, wherein the gRNA includes a guide region with modifications at nucleotides 1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 13, 14, 17, and 18, and optionally the modifications are 2'-OMe, 2'-fluoro, or phosphorothioate modifications. Embodiment 404 is a gRNA according to any one of Embodiments 1 to 403, wherein the 2'-OMe modification is not present in the guide region at the nucleotides 6-11 and 13. Embodiment 405 is a gRNA according to any one of Embodiments 1 to 404, wherein the 2'-fluoro modification is absent in the guide regions at the nucleotides 1-7, 15, 16, and 19. Embodiment 406 is a gRNA according to any one of Embodiments 1 to 405, wherein the phosphorothioate modification is not present in the guide region at nucleotides 4, 5, 11-14, 17, and 18. Embodiment 407 is a gRNA according to any one of Embodiments 1 to 406, wherein the guide region includes an unmodified nucleotide 20. Embodiment 408 is a gRNA described in any one of Embodiments 1 to 407, wherein the guide region consists of 20 nucleotides. Embodiment 409 is a gRNA according to any one of Embodiments 1 to 408, wherein the guide region includes YA sites at nucleotides 5-6 and modifications at nucleotide 5. Embodiment 410 is a gRNA according to any one of Embodiments 1 to 409, wherein the guide region includes YA sites in nucleotides 12-13 and modifications of nucleotide 12. Embodiment 411 is a gRNA according to any one of Embodiments 1 to 410, wherein the guide region includes YA sites in nucleotides 15-16 and modification of nucleotide 15. Embodiment 412 is a gRNA according to any one of Embodiments 1 to 411, wherein the guide region includes YA sites in nucleotides 16-17 and modifications of nucleotide 16. Embodiment 413 is a gRNA according to any one of Embodiments 1 to 412, wherein the guide region includes YA sites in nucleotides 19-20 and modification of nucleotide 19. Embodiment 414 is a gRNA according to any one of Embodiments 1 to 413, wherein the guide region does not contain YA sites in nucleotides 5-6, and nucleotide 5 is unmodified. Embodiment 415 is a gRNA according to any one of Embodiments 1 to 414, wherein the guide region does not contain a YA site in nucleotides 12-13, and nucleotide 12 is unmodified. Embodiment 416 is a gRNA according to any one of Embodiments 1 to 415, wherein the guide region does not contain a YA site in nucleotides 15-16, and nucleotide 15 is unmodified. Embodiment 417 is a gRNA according to any one of Embodiments 1 to 416, wherein the guide region does not contain a YA site in nucleotides 16-17, and nucleotide 16 is unmodified. Embodiment 418 is a gRNA according to any one of Embodiments 1 to 417, wherein the guide region does not contain a YA site in nucleotides 19-20, and nucleotide 19 is unmodified. Embodiment 419 is a gRNA, which is as follows: a. 2'-OMe and phosphorothioate modifications in 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. Phosphothioate modification at nucleotide 6, f. Phosphothioate modification at nucleotide 7, g. 2'-fluoro and phosphorothioate modifications in nucleotide 8, 2'-fluoro and phosphorothioate modifications in nucleotide 9, i. 2'-fluoro and phosphorothioate modifications in nucleotide 10, 2'-fluoro modification at nucleotide 11, 2'-fluoro modification at k.nucleotide 13, l. 2'-fluoro modification at nucleotide 14, 2'-fluoro modification at m.nucleotide 17, and The gRNA is one of any one of Embodiments 1 to 418, and includes a guide region comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or all of the 2'-fluoro modifications at n.nucleotide 18. Embodiment 420 is a gRNA according to any one of Embodiments 1 to 419, wherein the guide region includes each of the modifications described in the previous embodiments. Embodiment 421 has the following guide region: i. When nucleotides 5 and 6 form the YA site, 2'-OMe modification in nucleotide 5, ii. When nucleotides 12 and 13 form the YA site, 2'-OMe modification in nucleotide 12, iii. When nucleotides 15 and 16 form the YA site, phosphorothioate modification at nucleotide 15, iv. When nucleotides 16 and 17 form the YA site, phosphorothioate modification in nucleotide 16, and If v. nucleotides 19 and 20 form a YA site, the gRNA is one of any one of embodiments 1 to 420, comprising at least one, two, three, or four of the phosphorothioates or 2'-fluoro modifications on nucleotide 19. Embodiment 422 is a gRNA according to any one of Embodiments 1 to 421, wherein the guide region includes YA sites at nucleotides 5-6 and a 2'-OMe modification at nucleotide 5. Embodiment 423 is a gRNA according to any one of Embodiments 1 to 422, wherein the guide region includes a YA site at nucleotides 12-13 and a 2'-OMe modification at nucleotide 12. Embodiment 424 is a gRNA according to any one of Embodiments 1 to 423, wherein the guide region includes YA sites at nucleotides 15-16 and a phosphorothioate modification at nucleotide 15. Embodiment 425 is a gRNA according to any one of Embodiments 1 to 424, wherein the guide region includes YA sites at nucleotides 16-17 and a phosphorothioate modification at nucleotide 16. Embodiment 426 is a gRNA according to any one of Embodiments 1 to 425, wherein the guide region includes YA sites at nucleotides 19-20 and a phosphorothioate modification at nucleotide 19. Embodiment 427 is a gRNA according to any one of Embodiments 1 to 426, wherein the guide region includes a 2'-fluoro modification at nucleotide 19. Embodiment 428 is a gRNA according to any one of Embodiments 1 to 427, wherein the guide region includes an unmodified nucleotide 15 or only a phosphorothioate modification at nucleotide 15. Embodiment 429 is a gRNA according to any one of Embodiments 1 to 428, wherein the guide region comprises an unmodified nucleotide 16 or only a phosphorothioate modification at the nucleotide 16. Embodiment 430 is a gRNA according to any one of Embodiments 1 to 429, wherein the gRNA is an sgRNA that includes 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 described in Embodiment 430, which has at least 5 to 10 consecutive nucleotides. Embodiment 432 has at least 5 to 10 missing nucleotides, i. Inside hairpin 1, ii. Within the "N" between hairpin 1 and hairpin 1 and hairpin 2, iii. In the two nucleotides located at hairpin 1 and immediately 3' to the side of hairpin 1, iv. Including at least a portion of hairpin 1, v. Inside hairpin 2, vi. Including at least a portion of hairpin 2, vii. Inside 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. Includes at least a portion of hairpin 1, includes "N" between hairpin 1 and hairpin 2, and includes at least a portion of hairpin 2, xi. Located within hairpin 1 or hairpin 2, optionally including "N" between hairpin 1 and hairpin 2. xii. Continuously, xiii. Consecutive, including "N" between hairpin 1 and hairpin 2, xiv. Continuously extending to at least a portion of hairpin 1 and a portion of hairpin 2, xv. Continuously extending to at least a portion of hairpin 1 and "N" between hairpin 1 and hairpin 2, or xvi. A gRNA according to Embodiment 430 or 431, which is continuous and extends to at least a portion of hairpin 1 and two nucleotides immediately 3' of hairpin 1. Embodiment 433 is a gRNA according to any one of Embodiments 430 to 432, wherein at least 5 to 10 nucleotides include 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 sgRNA includes modifications at least at H1-1 to H1-5 and H2-1 to H2-12. Embodiment 434 contains at least 5 to 10 nucleotides, i. Consists of 5 to 10 nucleotides, ii. Consists of 6 to 10 nucleotides, iii. Consists of 5 nucleotides, iv. Consists of 6 nucleotides, v. Consists of 7 nucleotides, vi. Consists of 8 nucleotides, vii. Consisting of 9 nucleotides, viii. Consisting of 10 nucleotides, ix. Consists of 5 to 10 consecutive nucleotides, Consists of x.6 to 10 consecutive nucleotides, xi. Consists of 5 consecutive nucleotides, xii. Consists of 6 consecutive nucleotides, xiii. Consists of 7 consecutive nucleotides, xiv. Consists of 8 consecutive nucleotides, xv. Consists of 9 consecutive nucleotides, or xvi. A gRNA consisting of 10 consecutive nucleotides, as described in any one of embodiments 430 to 433. Embodiment 435 is the gRNA according to Embodiment 434, wherein at least 5 to 10 nucleotides include 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 sgRNA includes modifications at least at H1-1 to H1-5 and H2-1 to H2-12. Embodiment 436 contains at least 5 to 10 nucleotides, i. Containing nucleotides 54-61 of sequence number 400, ii. Containing nucleotides 53-60 of sequence number 400, iii. Containing nucleotides 54-58 of sequence number 400, iv. Consists of nucleotides 54-61 of sequence number 400, v. Consists of nucleotides 53-60 of sequence number 400, or vi. A gRNA according to any one of embodiments 430 to 435, consisting of nucleotides 54 to 58 of sequence number 400. Embodiment 437 uses gRNAs with the following sequence numbers: 1-54, 201-254, 301-354, 401-535, 601, 607-732, 801, 807-932, 1001, 1007-1132, 1205-1212, 1322-1406, 1417-1501, 1511-1596, 3018-3059, 3063-3104, 3108-3149, 3153-3194, 3198-323 9, a gRNA according to any one of Embodiments 1 to 436, comprising modified and / or unmodified nucleotides in at least 15 of the nucleotides 1 to 20 from the 5' end of the 5' terminal, which matches the modification pattern in nucleotides 1 to 20 of any one of the gRNAs 3243 to 3284, 3295 to 3341, 3343 to 3385, or 3388 to 3430. Embodiment 438 uses gRNAs with sequence numbers 1-54, 201-254, 301-354, 401-535, 601, 607-732, 801, 807-932, 1001, 1007-1132, 1205-1212, 1322-1406, 1417-1501, 1511-1596, 3018-3059, 3063-3104, 3108-3149, 3153-3194, 3198-323 9, a gRNA according to any one of embodiments 1 to 437, comprising modified and / or unmodified nucleotides in at least 16 of the nucleotides 1 to 20 from the 5' end of the 5' end, which matches the modification pattern in nucleotides 1 to 20 of any one of the gRNAs 3243 to 3284, 3295 to 3341, 3343 to 3385, or 3388 to 3430. Embodiment 439 comprises a gRNA in which the gRNA includes at least 17 modified and / or unmodified nucleotides from nucleotides 1 to 20 from the 5' end of the 5' terminal that match the modification pattern in nucleotides 1 to 20 of the gRNA, and the gRNA is SEQ ID NOs: 1 to 54, 201 to 254, 301 to 354, 401 to 535, 601, 607 to 732, 801, 807 to 932, 1001, 1007 to 1 The gRNA is one of the gRNAs described in any one of Embodiments 1 to 438, which is one of the gRNAs described in any one of Embodiments 1 to 438, which is one of the gRNAs described in any one of Embodiments 1 to 438, which is one of the gRNAs described in any one of Embodiments 1 to 438, which is one of the gRNAs described in any one of Embodiments 1 to 438, which is one of the gRNAs described in any one of Embodiments 1 to 438, which is one of the gRNAs described in any one of Embodiments 1 to 438, which is one of the gRNAs described in any one of Embodiments 1 to 438, which is one of the gRNAs described in any one of Embodiments 1 to 438, which is one of the gRNAs described in any one of Embodiments 1 to 438, which is one of the gRNAs described in any one of Embodiments 1 to 438, which is one of the gRNAs described in any one of Embodiments 1 to 439 Embodiment 440 uses gRNAs with sequence numbers 1-54, 201-254, 301-354, 401-535, 601, 607-732, 801, 807-932, 1001, 1007-1132, 1205-1212, 1322-1406, 1417-1501, 1511-1596, 3018-3059, 3063-3104, 3108-3149, 3153-3194, 3198-323 9, a gRNA according to any one of embodiments 1 to 439, comprising modified and / or unmodified nucleotides in at least 18 of the nucleotides 1 to 20 from the 5' end of the 5' end, which matches the modification pattern in nucleotides 1 to 20 of any one of the gRNAs 3243 to 3284, 3295 to 3341, 3343 to 3385, or 3388 to 3430. Embodiment 441 uses gRNAs with the following sequence numbers: 1-54, 201-254, 301-354, 401-535, 601, 607-732, 801, 807-932, 1001, 1007-1132, 1205-1212, 1322-1406, 1417-1501, 1511-1596, 3018-3059, 3063-3104, 3108-3149, 3153-3194, 3198-323 9, a gRNA according to any one of Embodiments 1 to 440, comprising modified and / or unmodified nucleotides at least 19 of the nucleotides 1 to 20 from the 5' end of the 5' terminal, which matches the modification pattern at nucleotides 1 to 20 of any one of the gRNAs 3243 to 3284, 3295 to 3341, 3343 to 3385, or 3388 to 3430. Embodiment 442 uses gRNAs with sequence numbers 1-54, 201-254, 301-354, 401-535, 601, 607-732, 801, 807-932, 1001, 1007-1132, 1205-1212, 1322-1406, 1417-1501, 1511-1596, 3018-3059, 3063-3104, 3108-3149, 3153-3194, 3 The gRNA according to any one of Embodiments 1 to 441, comprising modified and / or unmodified nucleotides from the 5' end of the 5' terminal, which matches the modification pattern at nucleotides 1 to 20 of any one of the gRNAs 198 to 3239, 3243 to 3284, 3295 to 3341, 3343 to 3385, or 3388 to 3430. Embodiment 443 is a gRNA according to any one of Embodiments 1 to 442, wherein the gRNA has a modification pattern that matches at least 75% of the modification pattern of any one of the gRNAs with sequence numbers 1-54, 201-254, 301-354, 401-535, 601, 607-732, 801, 807-932, 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, or 3388-3430. Embodiment 444 includes one modification pattern from the gRNAs in Table 1, the modification patterns being SEQ ID NOs: 1-54, 201-254, 301-354, 401-535, 601, 607-732, 801, 807-932, 1001, 1007-1132, 1205-1212, 1322-1406, 1417-1501 The gRNA described in any one of Embodiments 1 to 443 is the same as any one of the gRNAs 1511-1596, 3018-3059, 3063-3104, 3108-3149, 3153-3194, 3198-3239, 3243-3284, 3295-3341, 3343-3385, or 3388-3430. Embodiment 445 is a gRNA according to any one of Embodiments 437 to 444, further comprising a sequence having at least 75% identity with the nucleotide 21-terminus sequence of the gRNA. Embodiment 446 is a gRNA according to any one of Embodiments 437 to 444, further comprising a sequence having at least 80% identity with the nucleotide 21-terminus sequence of the gRNA. Embodiment 447 is a gRNA according to any one of Embodiments 437 to 444, further comprising a sequence having at least 85% identity with the nucleotide 21-terminus sequence of the gRNA. Embodiment 448 is a gRNA according to any one of Embodiments 437 to 444, further comprising a sequence having at least 90% identity with the nucleotide 21-terminus sequence of the gRNA. Embodiment 449 is a gRNA according to any one of Embodiments 437 to 444, further comprising a sequence having at least 95% identity with the nucleotide 21-terminus sequence of the gRNA. Embodiment 450 is a gRNA according to any one of Embodiments 437 to 444, further comprising a sequence having at least 98% identity with the nucleotide 21-terminus sequence of the gRNA. Embodiment 451 is a gRNA according to any one of Embodiments 437 to 444, further comprising a sequence having 100% identity with the nucleotide 21-terminus sequence of the gRNA. Embodiment 452 is an LNP composition containing the gRNA described in any one of the prior embodiments. Embodiment 453 is a composition comprising a gRNA described in any one of Embodiments 1 to 451, associated with lipid nanoparticles (LNPs). Embodiment 454 is a gRNA according to any one of Embodiments 1 to 451 or a composition according to any one of Embodiments 452 to 453, further comprising a nuclease or mRNA encoding a nuclease. Embodiment 455 is the composition described in 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. aureus Cas9. Embodiment 458 is the composition according to any one of Embodiments 453 to 457, wherein the nuclease is nicasse or dCas. Embodiment 459 is a composition according to any one of Embodiments 453 to 458, wherein the nuclease is modified. Embodiment 460 is the composition according to Embodiment 459, wherein the modified nuclease contains a nuclear localization signal (NLS). Embodiment 461 is a composition according to any one of Embodiments 452 to 460, comprising mRNA encoding a nuclease. Embodiment 462 is the composition according to Embodiment 461, wherein the mRNA contains one of the sequences 3499-3527 or 3529-3546. Embodiment 463 is a pharmaceutical formulation comprising the gRNA described in any one of Embodiments 1 to 451 or the composition described in any one of Embodiments 452 to 462, and a pharmaceutically acceptable carrier. Embodiment 464 includes a Cas protein or a nucleic acid encoding a Cas protein, and the following: i. gRNA described in any one of Embodiments 1 to 451, ii. The composition according to any one of embodiments 452 to 462, and iii. A method for modifying target DNA, comprising delivering one or more of the pharmaceutical formulations of Embodiment 463 to cells. Embodiment 465 is the method of Embodiment 464, wherein the method results in a gene insertion or deletion. Embodiment 466 is the method of Embodiment 464 or Embodiment 465, further comprising delivering a template to a 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. Embodiment 467 is a gRNA according to any one of Embodiments 1 to 451, a composition of Embodiments 452 to 462, or a pharmaceutical formulation of Embodiment 463, for use in the preparation of a pharmaceutical for treating a disease or disorder. Embodiment 468 is the use of a gRNA described in any one of Embodiments 1 to 451, a composition of Embodiments 452 to 462, or a pharmaceutical formulation of Embodiment 463 in the manufacture of a pharmaceutical for treating a disease or disorder. [Brief explanation of the drawing]

[0009] [Figure 1A] The results of the in vivo editing % of the guide shown are displayed. [Figure 1B] The serum TTR results for the guide shown are presented. [Figure 2A] The results of the in vivo editing % of the guide shown are displayed. [Figure 2B] The serum TTR results for the guide shown are presented. [Figure 3A] The results of the in vivo editing % of the guide shown are displayed. [Figure 3B] The serum TTR results for the guide shown are presented. [Figure 3C] The results of the in vivo editing % of the guide shown are displayed. [Figure 3D] The serum TTR results for the guide shown are presented. [Figure 3E] The results of the in vivo edited % of the guideline in rats are shown. [Figure 3F] The results of the indicated guideline serum TTR in rats are shown. [Figure 4A] The results of the in vivo editing % of the guide shown are displayed. [Figure 4B] The serum TTR results for the guide shown are presented. [Figure 5] This shows the percentage of editing in neuro2A cells in vitro. [Figure 6A] The results of the in vivo editing % of the guide shown are displayed. [Figure 6B]The serum TTR results for the guide shown are presented. [Figure 7A] The results of the in vivo editing % of the guide shown are displayed. [Figure 7B] The serum TTR results for the guide shown are presented. [Figure 8A] The results of the in vivo editing % of the guide shown are displayed. [Figure 8B] The serum TTR results for the guide shown are presented. [Figure 8C] The result of in-vivo editing of the guide shown is displayed. [Figure 8D] The serum TTR results for the guide shown are presented. [Figure 9A] The guides shown indicate the edited percentages based on concentration in PHH(9A) cells. [Figure 9B] The guides shown indicate the edited percentages based on concentration in PCH(9B) cells. [Figure 9C] The guides shown indicate the edited percentages based on concentration in HepG2(9C) cells. [Figure 10A] An exemplary sgRNA (SEQ ID NO: 801, methylation not shown) in a possible secondary structure having labels indicating the individual nucleotides of the conserved region of the sgRNA, including the lower stem, bulge, upper stem, nexus (whose nucleotides can be designated N1-N18 in the 5'-3' direction, respectively), and hairpin regions including hairpin 1 and hairpin 2. The nucleotides between hairpin 1 and hairpin 2 are labeled with n. A guide region may be present on the sgRNA and is shown in this figure as "(N)x" before the conserved region of the sgRNA. [Figure 10B] Ten conserved YA regions in an exemplary sgRNA sequence (SEQ ID NO: 801, methylation not shown) are labeled with numbers 1 to 10. The numbers 25, 45, 50, 56, 64, 67, and 83 indicate the pyrimidine positions of YA regions 1, 5, 6, 7, 8, 9, and 10 in the sgRNA having a guide region indicated as (N)x, where x is, for example, 20 by choice. [Figure 11A]The results of a nuclease stability assay are shown, in which the indicated guide was incubated with 0.01 mg / mL human liver cytosol (HLC) to determine the cleavage site. FLP indicates the signal from the full-length product. [Figure 11B] The results of a nuclease stability assay are shown, in which the indicated guide was incubated with 0.01 mg / mL human liver cytosol (HLC) to determine the cleavage site. FLP indicates the signal from the full-length product. [Figure 11C] The results of a nuclease stability assay are shown, in which the indicated guide was incubated with 0.01 mg / mL human liver cytosol (HLC) to determine the cleavage site. FLP indicates the signal from the full-length product. [Figure 11D] The results of a nuclease stability assay are shown, in which the indicated guide was incubated with 0.01 mg / mL human liver cytosol (HLC) to determine the cleavage site. FLP indicates the signal from the full-length product. [Figure 11E] The results of a nuclease stability assay are shown, in which the indicated guide was incubated with 0.01 mg / mL human liver cytosol (HLC) to determine the cleavage site. FLP indicates the signal from the full-length product. [Figure 11F] Figures 11A–E illustrate the locations of the cleavage sites and possible secondary structures of sequence number 401 as mapped onto an exemplary guide sequence (not all modifications are shown). Blank triangles indicate YA cleavage sites in the guide region. Blacked triangles indicate YA cleavage sites in the conserved region. [Figure 12A] The results of a nuclease stability assay, in which the indicated guide was incubated with 0.01 mg / mL human liver cytosol (HLC) to determine the cleavage site, are shown. [Figure 12B] The results of a nuclease stability assay, in which the indicated guide was incubated with 0.01 mg / mL human liver cytosol (HLC) to determine the cleavage site, are shown. [Figure 12C]The results of a nuclease stability assay, in which the indicated guide was incubated with 0.01 mg / mL human liver cytosol (HLC) to determine the cleavage site, are shown. [Figure 12D] The results of a nuclease stability assay, in which the indicated guide was incubated with 0.01 mg / mL human liver cytosol (HLC) to determine the cleavage site, are shown. [Figure 12E] The results of a nuclease stability assay, in which the indicated guide was incubated with 0.01 mg / mL human liver cytosol (HLC) to determine the cleavage site, are shown. [Figure 12F] The results of a nuclease stability assay, in which the indicated guide was incubated with 0.01 mg / mL human liver cytosol (HLC) to determine the cleavage site, are shown. [Figure 12G] The results of a nuclease stability assay, in which the indicated guide was incubated with 0.01 mg / mL human liver cytosol (HLC) to determine the cleavage site, are shown. [Figure 13A] The results of a nuclease stability assay in which G010039 was incubated with 0.01 mg / mL (A) human liver cytosol (HLC) are shown. [Figure 13B] The results of a nuclease stability assay in which G010039 was incubated with 8.5 mg / mL (B) human liver cytosol (HLC) are shown. [Figure 14] The edited percentage results of experiments in which lipoplex containing the indicated guide was transfected into primary mouse hepatocytes (PMH) are shown. [Figure 15A] The results of experiments in which lipoplex containing the indicated guide was transfected into PMH are shown in edited percentages. [Figure 15B] The edited percentage results of experiments in which lipoplex containing the indicated guide was transfected into primary cynomolgus macaque liver cells (PCH) are shown. [Figure 15C] The edited percentage results of experiments in which lipoplex containing the indicated guide was transfected into primary human hepatocytes (PHH) are shown. [Figure 16A] This shows scattered plots and correlation values ​​in percentage results of experiments in which sgRNA was administered to mice in vivo or delivered to PMH by lipoplex transfection of sgRNA. [Figure 16B] This shows the correlation between in vitro and in vivo editing percentage results obtained by delivering sgRNA in LNPs to PHH. [Figure 16C] This shows the correlation between in vitro and in vivo editing percentage results obtained by delivering sgRNA in LNPs to PHH. [Figure 16D] This shows the correlation between in vitro and in vivo editing percentage results obtained by delivering sgRNA in LNPs to PHH. [Figure 16E] This shows the correlation between in vitro and in vivo editing percentage results obtained by delivering sgRNA in LNPs to PHH. [Figure 16F] This shows the correlation between in vitro and in vivo editing percentage results obtained by delivering sgRNA in LNPs to PHH. [Figure 16G] This shows a comparison of edited percentages using the indicated guide, delivered to PMH by lipoplex transfection (data in the left box), to PMH in LNP (data in the center box), or to mice in vivo (data in the right box). [Figure 16H] The following shows a comparison of edited percentages using the indicated guide, delivered to PMH in LNP (1 ng, 3 ng, 10 ng) or to mice in vivo (0.1 mpk, 0.3 mpk). [Figure 16I]Figure 16G shows the results replotted to illustrate the difference in editing between G000282 and G000211. The bar plot values ​​were created by dividing the editing % of the G000282 value by the editing % of the G000211 value to show the difference in editing multipliers. The indicated guides were delivered to PMH by lipoplex transfection (data in the left box), to PMH in LNP (data in the middle box), or to mice in vivo (data in the right box). [Figure 16J] The results from Figure 16H, replotted to show the difference in editing between G000283 and G000269, are shown. The bar plot values ​​were created by dividing the editing percentage of the G000283 value by the editing percentage of the G000269 value to show the difference in editing multiples. The indicated guides were delivered to PMH in LNP (data in the left box) or to mice in vivo (data in the right box). [Figure 17A] The results of the in vivo editing % of the guide shown are displayed. [Figure 17B] The serum TTR results for the guide shown are presented. [Figure 18A] The results of the in vivo editing % of the guide shown are displayed. [Figure 18B] The serum TTR results for the guide shown are presented. [Figure 18C] The results of the in vivo editing % of the guide shown are displayed. [Figure 18D] The serum TTR results for the guide shown are presented. [Figure 18E] The results of the in vivo editing % of the guide shown are displayed. [Figure 18F] The serum TTR results for the guide shown are presented. [Figure 19A] The results of the in vivo editing % of the guide shown are displayed. [Figure 19B] The serum TTR results for the guide shown are presented. [Figure 19C] The results of the in vivo editing % of the guide shown are displayed. [Figure 19D] The serum TTR results for the guide shown are presented. [Figure 20A]The results of the guide in vivo edited at the indicated concentration are shown. [Figure 20B] The guide serum TTR results at the indicated concentrations are shown. [Figure 20C] The results shown are the in vivo edited % results of the indicated guide at the indicated concentration. [Figure 20D] The serum TTR results for the indicated guide at the indicated concentrations are shown. [Figure 20E] The results shown are the in vivo edited % results of the indicated guide at the indicated concentration. [Figure 20F] The serum TTR results for the indicated guide at the indicated concentrations are shown. [Figure 21A] The results of the in vivo editing % of the guide shown are displayed. [Figure 21B] The serum TTR results for the guide shown are presented. [Figure 22A] The results of the in vivo editing % of the guide shown are displayed. [Figure 22B] The serum TTR results for the guide shown are presented. [Figure 23A] This indicates the frequency of editing the guide provided. [Figure 23B] This indicates the frequency of editing the guide provided. [Figure 24A] The results of the in vivo editing % of the guide shown are displayed. [Figure 24B] The serum TTR results for the guide shown are presented. [Figure 25A] The indel frequency versus guide concentration shown is presented. [Figure 25B] The indel frequency versus guide concentration shown is presented. [Figure 25C] The indel frequency versus guide concentration shown is presented. [Figure 25D] The indel frequency versus guide concentration shown is presented. [Figure 25E] The indel frequency versus guide concentration shown is presented. [Figure 26A] The indel frequency versus guide concentration shown is presented. [Figure 26B]The indel frequency versus guide concentration shown is presented. [Figure 26C] The indel frequency versus guide concentration shown is presented. [Figure 26D] The indel frequency versus guide concentration shown is presented. [Figure 26E] The indel frequency versus guide concentration shown is presented. [Figure 27A] The indel frequency versus guide concentration shown is presented. [Figure 27B] The indel frequency versus guide concentration shown is presented. [Figure 27C] The indel frequency versus guide concentration shown is presented. [Figure 27D] The indel frequency versus guide concentration shown is presented. [Figure 28A] The indel frequency versus guide concentration shown is presented. [Figure 28B] The indel frequency versus guide concentration shown is presented. [Figure 28C] The indel frequency versus guide concentration shown is presented. [Figure 28D] The indel frequency versus guide concentration shown is presented. [Figure 29A] The editing frequency of guides with the indicated dinucleotide modifications is shown (for a given 5' modification position, the position immediately immediately following it is also modified). [Figure 29B] The editing frequency of guides with the indicated dinucleotide modifications is shown (for a given 5' modification position, the position immediately immediately following it is also modified). [Figure 29C] This shows the editing frequency of guides with modifications shown in individual nucleotides. [Figure 29D] This shows the editing frequency of guides with modifications shown in individual nucleotides. [Figure 29E] This shows the editing frequency of guides with modifications shown in individual nucleotides. [Figure 29F]The editing frequency of guides with the indicated dinucleotide modifications is shown (for a given 5' modification position, the position immediately immediately following it is also modified). [Figure 30A] The influence scores of the modifications shown at guide positions 1-20 are displayed. [Figure 30B] The influence scores of the modifications shown at guide positions 1-20 are displayed. [Figure 30C] The influence scores of the modifications shown at guide positions 1-20 are displayed. [Figure 31A] This indicates the editing frequency of the displayed guides. Guides are grouped into boxes based on having similar storage area modification patterns. [Figure 31B] This indicates the editing frequency of the displayed guides. Guides are grouped into boxes based on having similar storage area modification patterns. [Figure 31C] This indicates the editing frequency of the displayed guides. Guides are grouped into boxes based on having similar storage area modification patterns. [Modes for carrying out the invention]

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

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

[0012] Certain gRNAs provided herein are modified single guide RNAs (sgRNAs) for use in gene editing methods. The sequences of manipulated and tested sgRNAs are shown in Table 1. Certain sgRNAs have specific modifications at the YA site in the sgRNA, including modifications at the crRNA and / or trRNA portions of the sgRNA.

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

[0014] This disclosure further provides the use of these gRNAs (e.g., sgRNAs, short sgRNAs, dgRNAs, or crRNAs) to alter the genome of a target nucleic acid in vitro (e.g., 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). This disclosure also provides methods for preventing or treating a disease in a subject by modifying a disease-related target gene. The disclosed gRNAs can be used in any cell type and any gene locus suitable for nuclease-mediated genome editing technology. Table 1 (array listing): [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] Table 1-5 Table 1-6 Table 1-7 Table 1-8 Table 1-9 Table 1-10 Table 1-11 Table 1-12 Table 1-13 Table 1-14 Table 1-15 Table 1-16 Table 1-17 Table 1-18 Table 1-19 Table 1-20 Table 1-21 Table 1-22 Table 1-23 Table 1-24 Table 1-25 Table 1-26 Table 1-27 Table 1-28 Table 1-29 Table 1-30 Table 1-31 Table 1-32 Table 1-33 Table 1-34 Table 1-35 Table 1-36 Table 1-37 Table 1-38 Table 1-39 Table 1-40 Table 1-41 Table 1-42 Table 1-43 Table 1-44 Table 1-45 Table 1-46 Table 1-47 Table 1-48 Table 1-49 Table 1-50 Table 1-51 Table 1-52 Table 1-53 Table 1-54 Table 1-55 Table 1-56 Table 1-57 Table 1-58 Table 1-59 Table 1-60 Table 1-61 Table 1-62 Table 1-63 Table 1-64 Table 1-65 Table 1-66

Table 1-67

Table 1-100

Table 1-110

Table 1-120

Table 1-150

Table 1-202

Table 1-204

Table 1-206

Table 1-210

Table 1-220

Table 1-234

Table 1-239

Table 1-251

Table 1-279

Table 1-289

Table 1-290

Table 1-301

Table 1-302

Table 1-307

Table 1-309

Table 1-319

Table 1-339

Table 1-340

Table 1-342

Table 1-343

Table 1-347

Table 1-349

Table 1-351

Table 1-357

Table 1-359

Table 1-360

Table 1-365

Table 1-368

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 bond; f: 2'-fluoro; (invd): inverted debase; moe: 2'-moe; e: ENA; d: deoxyribonucleotide (note that T is always a deoxyribonucleotide); x: UNA. Therefore, for example, mA represents 2'-O-methyladenosine, xA represents a UNA nucleotide with an adenine nucleic acid base, eA represents an ENA nucleotide with an adenine nucleic acid base, and dA represents adenosine deoxyribonucleotide.

[0017] sgRNA designations may be provided with one or more leading zeros immediately following the G. This does not affect the meaning of the designation. For example, G000282, G0282, G00282, and G282 refer to the same sgRNA. Similarly, crRNA and / or trRNA designations may be provided with one or more leading zeros immediately following the CR or TR, respectively, and this does not affect the meaning of the designation. For example, CR000100, CR00100, CR0100, and CR100 refer to the same crRNA, and TR000200, TR00200, TR0200, and TR200 refer to the same trRNA.

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

[0019] For sequence numbers 601 and 607-732, no guide region is shown, and the positions corresponding to the remaining region are each reduced by 20 compared to the positions given for sequence numbers 401-532. For sequence numbers 801 and 807-932, the spacer is of length x, and the positions corresponding to the remaining region are each reduced by 20 and increased by x compared to those given for sequence numbers 401-532.

[0020] definition As used herein, “editing efficiency,” “editing percentage,” or “editing percent” refers to the total number of sequence reads that result in the insertion or deletion of a nucleotide into the target region of interest, relative to the total number of sequence reads after cleavage by Cas RNP.

[0021] As used herein, the term “region” describes a conserved base of a nucleic acid. A region may also be referred to as a “module” or “domain.” Regions of sgRNA may perform specific functions, such as inducing the endonuclease activity of RNPs, as described, for example, in Briner AE et al., Molecular Cell 56:333-339 (2014). Exemplary regions of sgRNA are shown in Table 3.

[0022] As used herein, “hairpin” refers to a double helix of nucleic acid produced when a nucleic acid strand folds and forms a base pair with another section of the same strand. Hairpins can form structures including loops or U-shapes. In some embodiments, a hairpin may consist of an RNA loop. A hairpin can be formed by two complementary sequences in a single nucleic acid molecule bond, accompanied by molecular folding or wrinkling. In some embodiments, a hairpin includes a stem or stem-loop structure. As used herein, “hairpin region” refers to hairpin 1 and hairpin 2, as well as the “n” between hairpin 1 and hairpin 2 of the conserved portion of sgRNA.

[0023] As used herein, “ribonucleoprotein” (RNP) or “RNP complex” describes sgRNA in conjunction with a nuclease such as a Cas protein. In some embodiments, the RNP comprises Cas9 and gRNA (e.g., sgRNA, short sgRNA, dgRNA, or crRNA).

[0024] As used herein, “stem-loop” describes a secondary structure of nucleotides that forms a base-paired “stem” ending in a loop of unpaired nucleic acid. A stem can be formed if two regions of the same nucleic acid chain are at least partially complementary in the sequence when read in opposite directions. As used herein, “loop” describes a region of nucleotides that does not contain base pairs (i.e., is not complementary) and can cap a stem. A “tetra-loop” describes a loop of four nucleotides. As used herein, the upper stem of sgRNA may contain a tetra-loop.

[0025] "Guide RNA," "gRNA," and "guide" are used interchangeably herein to refer to either crRNA (also known as CRISPR RNA) or a combination of crRNA and trRNA (also known as tracrRNA). crRNA and trRNA may associate as a single-stranded RNA molecule (single guide RNA (sgRNA)) or as two separate RNA molecules (dual guide RNA (dgRNA)). "Guide RNA" or "gRNA" refers to each type. trRNA may be a naturally occurring sequence or a trRNA sequence that is modified or mutated compared to a naturally occurring sequence. Guide RNA may include modified RNA as described herein.

[0026] In some embodiments, the gRNA (e.g., sgRNA) includes a “guide region,” which may be referred to as a “spacer” or “spacer region,” as in Briner AE et al., Molecular Cell 56:333-339 (2014), for example, with respect to sgRNA (but applicable to all guide RNAs herein). 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 located immediately before the “sgRNA conserved portion” at its 5’ end, and in some embodiments, the sgRNA is a short sgRNA. Exemplary “sgRNA conserved portions” are shown in Table 2. 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 located at a given nucleotide position relative to the end of the guide region from the 5’ end. Such ranges of positions are referred to, for example, as "the 5th, 6th, 7th, 8th, 9th, or 10th nucleotides from the 5' end of the 5' end," where "end" in "end 5," etc., refers to most of the 3' nucleotides in the guide region. (Similarly, expressions such as "the 21st nucleotide of the gRNA" refer to the range from the 21st nucleotide from the 5' end of the gRNA's 5' end to the last nucleotide at the 3' end of the gRNA.) Furthermore, for example, the nucleotide that is the 6th nucleotide from the 5' end of a particular sgRNA segment is the 6th nucleotide of that segment, or "nucleotide 6" from the 5' end, e.g., XXXXXN, where N is the 6th nucleotide from the 5' end. The range of nucleotides located "before or after" the 6th nucleotide from the 5' end begins at the 6th nucleotide and continues down the chain toward the 3' end. Similarly, for example, the nucleotide that is the 5th nucleotide from the 3' end of the chain is the 5th nucleotide when counted from the 3' end of the chain, e.g., NXXXX.The numerical position or range within the guide region refers to the position determined from the 5' end unless a different reference point is specified; for example, "nucleotide 5" in the guide region is the 5th nucleotide from the 5' end.

[0027] In some embodiments, the gRNA contains 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 modifications (e.g., phosphorothioate, 2'-fluoro, 2'-OMe, etc.) as the nucleotides at the corresponding positions of the gRNA described herein, regardless of whether the nucleic acid bases at those positions are identical. For example, if in the first gRNA nucleotides 5 and 6 have 2'-OMe and phosphorothioate modifications, respectively, then this gRNA has the same modification pattern at nucleotides 5 and 6 as a second gRNA that also has 2'-OMe and phosphorothioate modifications at nucleotides 5 and 6, respectively, regardless of whether the nucleic acid bases at positions 5 and 6 are identical or different in the first and second gRNAs. However, a 2'-OMe modification at nucleotide 6 but not at nucleotide 7 does not have the same modification pattern as a 2'-OMe modification at nucleotide 7 but not at nucleotide 6. Similarly, a modification pattern that matches at least 75% of the modification patterns of the gRNA described herein means that at least 75% of the nucleotides have the same modifications as the corresponding positions of the gRNA described herein. The corresponding positions may be determined by pairwise or structural alignment.

[0028] Table 2 shows the "conserved region" of S. pyogenes Cas9 (also known as "spyCas9" or "spCas9"). The first row shows the nucleotide numbering, the second row shows the sequence (e.g., sequence number 400), and the third row shows the region.

[0029] As used herein, “short single guide RNA” (“short sgRNA”) is an sgRNA that includes a conserved portion of an sgRNA containing a hairpin region, the hairpin region lacking at least 5–10 nucleotides or 6–10 nucleotides. In some embodiments, the short sgRNA lacks at least nucleotides 54–58 (AAAAA) of the conserved portion of S. pyogenes Cas9 (“spyCas9”) sgRNA, as shown in Table 2. In some embodiments, the short sgRNA is a non-spyCas9 sgRNA lacking nucleotides corresponding to nucleotides 54–58 (AAAAA) of the conserved portion of spyCas9, as determined, for example, by pairwise or structural alignment. In some embodiments, the short sgRNA lacks at least nucleotides 54–61 (AAAAAGUG) of the conserved portion of spyCas9 sgRNA. In some embodiments, the short sgRNA lacks at least 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 from the conserved portion of nucleotides 53-60 (GAAAAAGU) or nucleotides 54-61 (AAAAAGUG) of the spyCas9 sgRNA, or the corresponding nucleotides of the conserved portion of the non-spyCas9 sgRNA, as determined, for example, by pairwise or structural alignment.

[0030] As used herein, “YA sites” refer to the 5'-pyrimidine-adenine-3' dinucleotide. For clarification, “YA sites” in the original sequence that are modified by base modification are still considered (modified) YA sites in the resulting sequence, regardless of the presence of the literal YA dinucleotide. “Conserved region YA sites” are located in the conserved region of the sgRNA. “Guide region YA sites” are located in the guide region of the sgRNA. Unmodified YA sites in the sgRNA may be susceptible to cleavage by RNase-A-like endonucleases, such as RNase A. In some embodiments, a 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 Figure 10B. Since the guide region can be any sequence containing any number of YA sites, exemplary guide region YA sites are not shown in Figure 10B. In some embodiments, the sgRNA contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 YA sites as shown in Figure 10B. In some embodiments, the sgRNA contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 YA sites at the following positions or subsets thereof: LS5-LS6, US3-US4, US9-US10, US12-B3, LS7-LS8, LS12-N1, N6-N7, N14-N15, N17-N18, and H2-2~H2-3. In some embodiments, the YA sites include modifications, meaning that at least one nucleotide of the YA site is modified. In some embodiments, the pyrimidine of the YA site (also referred to as the pyrimidine site) includes modifications (including modifications that alter the nucleoside bond immediately 3' of the sugar of the pyrimidine). In some embodiments, the adenine at the YA site (also referred to as the adenine position) includes modifications (including modifications that alter the nucleoside bond immediately 3' of the sugar of adenine). In some embodiments, the pyrimidine and adenine positions at the YA site include modifications.In some embodiments, the short sgRNA guide region or short sgRNA conserved region described herein includes one or more YA sites ("guide region YA site" or "conserved region YA site"). In some embodiments, the crRNA or trRNA described herein includes one or more YA sites.

[0031] As discussed herein, the nucleotide positions corresponding to those described for spyCas9 gRNA can be identified in other gRNAs with sequence and / or structural similarity by pairwise or structural alignment. Structural alignment is useful when molecules share similar structures despite significant sequence variations. For example, spyCas9 and Staphylococcus aureus Cas9 ("SaCas9") have different sequences but share significant structural alignment. See, for example, Figure 2(F) in Nishimasu et al., Cell 162(5):1113-1126 (2015). Structural alignment can be used to identify nucleotides in saCas9 or other sgRNAs that correspond to specific positions, such as nucleotides 54-58 (AAAAA), in the conserved portion of spyCas9 sgRNA.

[0032] Structural alignment involves identifying corresponding residues across two (or more) sequences by (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, as well as identifying the residue in the first sequence that is in the position most similar to the residue of interest in the second sequence. The corresponding residues are identified in several algorithms based on minimizing the distance between a given position in the superimposed structure (e.g., the 1st position of the nucleic acid base or the 1' carbon of the pentose ring in a polynucleotide, or the α-carbon in a polypeptide) (e.g., which pair of paired positions provides the least mean squared deviation for alignment). When identifying a position in a non-spyCas9 gRNA that corresponds to a position described for spyCas9 gRNA, the spyCas9 gRNA can be the "second" sequence. If the target non-spyCas9 gRNA does not have a known structure available but is more closely related to another non-spyCas9 gRNA with a known structure, it may be most effective to model the target non-spyCas9 gRNA using the known structure of the closely related non-spyCas9 gRNA and then compare that model to the spyCas9 gRNA structure to identify the desired corresponding residues in the target non-spyCas9 gRNA. Extensive literature exists on protein structural modeling and alignment, with representative disclosures including US6859736, US8738343, and those cited in Aslam et al., Electronic Journal of Biotechnology 20(2016)9-13. For considerations of structural modeling based on known related structures, see, for example, Bordoli et al., Nature Protocols 4(2009)1-13, and the references cited therein. For information on nucleic acid alignment, see also Figure 2(F) in Nishimasu et al., Cell 162(5):1113-1126(2015).

[0033] As used herein, “target sequence” refers to a sequence of nucleic acids in which the guide region induces a nuclease for cleavage. In some embodiments, the spyCas9 protein may be induced to the target sequence by the nucleotides present in the guide region. In some embodiments, the sgRNA does not contain a spacer region.

[0034] As used herein, “5' end” refers to the first nucleotide of a gRNA (including dgRNA (typically the 5' end of the crRNA of dgRNA), sgRNA, or short sgRNA), where the 5' position is not bound to another nucleotide.

[0035] As used herein, "5'-terminus modification" refers to a gRNA that includes a guide region having modifications in one or more of the approximately seven nucleotides at its 5' end, wherein the first nucleotide (from the 5' end) of the gRNA is optionally modified.

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

[0037] As used herein, "3'-terminal modification" refers to a gRNA having modifications in one or more of the approximately seven nucleotides at its 3' end, wherein, by arbitrary selection, the last nucleotide of the gRNA (i.e., the nucleotide closest to the 3' end) is modified. If a 3' tail is present, 1 to approximately 7 nucleotides may be present within the 3' tail. If a 3' tail is absent, 1 to approximately 7 nucleotides may be present within the conserved region of the sgRNA.

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

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

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

[0041] As used herein, “RNA-guided DNA conjugate” means a polypeptide or polypeptide complex having RNA and DNA-binding activity, or a DNA-binding subunit of such a complex, where the DNA-binding activity is sequence-specific and dependent on the RNA sequence. Exemplary RNA-guided DNA conjugates include Cas cleavage / nickases and their inactivated forms ("dCas DNA conjugates"). As used herein, “Cas nuclease,” also referred to as “Cas protein,” encompasses Cas cleavage agents, Cas nickases, and dCas DNA conjugates. Cas cleavases / 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, “Class 2 Cas nuclease” is a single-stranded polypeptide having RNA-induced DNA binding activity, such as Cas9 nuclease or Cpf1 nuclease. Class 2 Cas nucleases include Class 2 Cas cleavases and Class 2 Cas nickases (e.g., H840A, D10A, or N863A variants) that further have RNA-induced DNA cleavage or nickasing activity, as well as Class 2 dCas DNA binding agents whose cleavage / nickasing activity is inactivated. Class 2 Cas nucleases include, for example, Cas9, Cpf1, C2c1, C2c2, C2c3, HF Cas9 (e.g., N497A, R661A, Q695A, Q926A variants), HypaCas9 (e.g., N692A, M694A, Q695A, H698A variants), eSPCas9(1.0) (e.g., K810A, K1003A, R1060A variants), and eSPCas9(1.1) (e.g., K848A, K1003A, R1060A variants) proteins, as well as their modifications.The Cpf1 protein, Zetsche et al., Cell, 163:1-13 (2015), is homologous to Cas9 and contains a RuvC-like nuclease domain. Zetsche's Cpf1 sequence incorporates all of these by reference. See, for example, Zetsche's Tables S1 and S3. "Cas9" includes Spy Cas9, the Cas9 variants listed herein, and their equivalents. See, for example, Makarova et al., Nat Rev Microbiol, 13(11):722-36 (2015); Shmakov et al., Molecular Cell, 60:385-397 (2015).

[0042] As used herein, if the alignment of the first sequence to the second sequence shows that at least X% of the positions of the second sequence are matched by the first sequence, then the first sequence is considered to "contain a sequence having at least X% identity" with the second sequence. For example, the sequence AAGA contains a sequence that is 100% identical to the sequence AAG because the alignment gives 100% identity in that there is a match at all three positions of the second sequence. Differences between RNA and DNA (generally, the exchange of 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 (thymidine, uridine, or modified uridine, etc.) have the same complement (e.g., 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 complements). Therefore, for example, the sequence 5'-AXG, where X is any modified uridine, e.g., pseudouridine, N1-methylpseudridine, or 5-methoxyuridine, is considered 100% identical to AUG, in that both are perfectly 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. Those skilled in the art will understand what algorithm and parameter settings are appropriate for a given pair of sequences to be aligned. Generally, for sequences of similar length and expected identity of more than 50% for amino acids or more than 75% for nucleotides, the Needleman-Wunsch algorithm using the default settings of the Needleman-Wunsch algorithm interface provided by EBI on the www.ebi.ac.uk web server is generally appropriate.

[0043] "mRNA" is used herein to refer to a polynucleotide containing an open reading frame that is RNA or modified RNA and can be translated into a polypeptide (i.e., can function as a substrate for translation by ribosomes and aminoacylated tRNA). mRNA may contain a phosphate sugar backbone containing ribose residues or analogues, e.g., 2'-methoxyribose residues. In some embodiments, the sugars of the nucleic acid phosphate sugar backbone essentially consist 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 fewer than 30, 20, 10, 5, 4, 3, or 2 thymidine residues; or thymidine content less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1%). mRNA may contain modified uridines 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 the ORF, (a) using minimal uridine codons at all positions, and (b) encoding the same amino acid sequence as the given ORF. The minimum uridine codon(s) for a given amino acid is the codon(s) with the fewest uridine (usually 0 or 1, with the exception of the codon for phenylalanine, and the minimum uridine codon has 2 uridine). Modified uridine residues are considered equivalent to uridine for the purpose of evaluating the minimum uridine content.

[0045] As used herein, the “minimum uridine dinucleotide content” of a given ORF is the possible minimum uridine dinucleotide (UU) content of the ORF, which (a) uses the minimum uridine codon at all positions (as described above) and (b) encodes the same amino acid sequence as the given ORF. The uridine dinucleotide (UU) content can be expressed in absolute terms, either as an enumeration of UU dinucleotides in the ORF or as a rate-based percentage of the positions occupied by uridine in the uridine dinucleotide (for example, AUUAU has a 40% uridine dinucleotide content because two of its five positions are occupied by uridine in the uridine dinucleotide). Modified uridine residues are considered equivalent to uridine for the purpose of evaluating the minimum uridine dinucleotide content.

[0046] As used herein, the “minimum adenine content” of a given open reading frame (ORF) is the adenine content of the ORF, which (a) uses the minimum adenine codon at all positions and (b) encodes the same amino acid sequence as the given ORF. The minimum adenine codon(s) of a given amino acid is the codon(s) with 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 evaluating the minimum adenine content.

[0047] As used herein, the “minimum adenine dinucleotide content” of a given open reading frame (ORF) is the minimum possible adenine dinucleotide (AA) content of the ORF, which (a) uses the smallest adenine codon (as described above) at all positions and (b) 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 on a rate basis as the percentage of positions occupied by adenine of the adenine dinucleotide (for example, UAAUA has a 40% adenine dinucleotide content because two of its five 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.

[0048] As used herein, “Subject” refers to any member of the animal kingdom. In some embodiments, “Subject” refers to humans. In some embodiments, “Subject” refers to non-human animals. In some embodiments, “Subject” refers to primates. In some embodiments, “Subject” includes, but is not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In certain embodiments, non-human subjects are mammals (e.g., rodents, mice, rats, rabbits, monkeys, dogs, cats, sheep, cattle, primates, and / or pigs). In some embodiments, subjects may be transgenic animals, genetically modified animals, and / or clones. In certain embodiments of the present invention, subjects are adults, adolescents, or infants. In some embodiments, the terms “individual” or “patient” are used and are intended to be interchangeable with “subject.”

[0049] Types of modifications described herein Guide RNAs (e.g., sgRNA, short sgRNA, dgRNA, and crRNA) with modifications at various positions are disclosed herein. In some embodiments, the gRNA with modifications is modified by one or more of the following types of modifications:

[0050] 2'-O-methyl modification Modified sugars are thought to control nucleotide supple ring puckering, a physical property that affects oligonucleotide binding affinity to complementary chains, double-chain formation, and interaction with nucleases. Therefore, substitutions on the supple ring can alter the conformation and puckering of these sugars. For example, 2'-O-methyl (2'-OMe) modification can increase oligonucleotide binding affinity and nuclease stability, but the effect of any modification at a given position in the oligonucleotide needs to be determined empirically, as shown in the examples.

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

[0052] Ribonucleotides and modified 2'-O-methylribonucleotides can be described as follows: [ka]

[0053] 2'-O-(2-methoxyethyl) modification In some embodiments, the modification may be 2'-O-(2-methoxyethyl)(2'-O-moe). The modified 2'-O-moe ribonucleotide can be described as follows: [ka]

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

[0055] 2'-Fluorine modification Another chemical modification that has been shown to affect nucleotide sugar rings is halogen substitution. For example, 2'-fluoro(2'-F) substitutions on nucleotide sugar rings can increase oligonucleotide binding affinity and nuclease stability.

[0056] In this application, the terms “fA,” “fC,” “fU,” or “fG” may be used to represent a nucleotide substituted with 2'-F.

[0057] Ribonucleotides without a 2'-F substitution, and ribonucleotides with a 2'-F substitution, can be described as follows: [ka]

[0058] Phosphothioate modification A phosphorothioate (PS) linkage or bond refers to a bond in which sulfur is replaced, for example, by one uncrosslinked phosphate oxygen in a phosphodiester bond between nucleotides. When phosphorothioates are used to produce oligonucleotides, the modified oligonucleotide may also be called an S-oligo.

[0059] The asterisk "*" may be used to indicate PS modification. In this application, the terms A*, C*, U*, or G* may be used to indicate a nucleotide that is bonded to an adjacent (e.g., 3') nucleotide by a PS bond. Throughout this application, PS modifications are grouped by nucleotides in which the 3' carbon is bonded to a phosphorothioate, and therefore, a PS modification at position 1 means that the phosphorothioate is bonded to the 3' carbon of nucleotide 1 and the 5' carbon of nucleotide 2. Thus, when a YA site is indicated as "PS modified," the PS bond is between Y and A or between A and an adjacent nucleotide.

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

[0061] The diagram below shows the substitution of S- atoms with non-crosslinked phosphate oxygen, resulting in a PS bond instead of a phosphodiester bond: [ka]

[0062] Inverted debasement modification Debasic nucleotides refer to those lacking a nitrogen base. The following diagram describes oligonucleotides having a debasic site (in this case, shown as depurine; the debasic site may also be a depyrimidine site, and the description of the debasic site typically refers to a Watson-Crick base pair, while the depurine site refers to a site lacking a nitrogen base and will typically form a base pair with a pyrimidine site), where the base may be substituted at the 1' position of the furan ring with another site (e.g., a hydroxyl group or hydrogen, as shown below, which may form a ribose or deoxyribose site). [ka]

[0063] An inverted base refers to a base that has a bond that is the reverse of the usual 5'-to-3' bond (i.e., either a 5'-to-5' bond or a 3'-to-3' bond). For example, [ka]

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

[0065] Deoxyribonucleotide Deoxyribonucleotides (where the sugar is located at the 2'-deoxy position) are considered modifications in the context of gRNA, in that the nucleotide is modified from standard RNA by a proton substitution at the 2' hydroxyl position. Unless otherwise indicated, deoxyribonucleotide modifications at the U position in unmodified RNA can also include replacing the U nucleic acid base with T.

[0066] Bicyclic ribose analogs Exemplary bicyclic ribose analogs include locked nucleic acid (LNA), ENA, cross-linked nucleic acid (BNA), or other LNA-like modifications. In some examples, bicyclic ribose analogs have 2' and 4' positions linked via a linker. The linker is of the formula -X-(CH2) n -This may also be the case, where n is 1 or 2, X is O, NR, or S, and R is H or C 1-3Alkyl compounds, such as methyl compounds. Examples of bicyclic ribose analogs include LNA containing 2'-O-CH2-4' (oxy-LNA) (see WO98 / 39352 and WO99 / 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. al.,Biorg.Med.Chem.Lett.8:2219-2222(1998)).

[0067] ENA ENA modifications refer to nucleotides containing 2'-O,4'-C-ethylene modifications. Exemplary structures of ENA nucleotides are shown below, with wavy lines indicating connections to adjacent nucleotides (or, in some cases, terminal positions, where the 3' terminal nucleotide is an ENA nucleotide, the 3' position may contain a hydroxyl group rather than a phosphate group). For further discussion of ENA nucleotides, see, for example, Koizumi et al., Nucleic Acids Res. 31:3267-3273 (2003). [ka]

[0068] UNA UNA, or unlocked nucleic acid modification, refers to a nucleotide containing a 2',3'-seco-RNA modification, where the 2' and 3' carbons are not directly bonded to each other. An exemplary structure of a UNA nucleotide is shown below. The wavy lines indicate connections to or replacement of adjacent phosphates (or possibly terminal positions). For further discussion of UNA nucleotides, see, for example, Snead et al., Molecular Therapy 2:e103, doi:10.1038 / mtna.2013.36(2013). [ka]

[0069] Base modification Base modification is any modification that alters the structure of a nucleic acid base or its binding to its backbone, including isomerization (such as pseudouridine). In some embodiments, base modification includes inosine. In some embodiments, modification includes base modification that reduces RNA endonuclease activity, for example, by stabilizing the RNA structure (e.g., secondary structure) by interfering with RNase recognition of cleavage sites and / or reducing RNase accessibility of cleavage sites. 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, base modification can increase or decrease the melting temperature (Tm) of a nucleic acid, for example, 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 nucleoside bonds, modifications of bases (pyrimidine or adenine), such as chemical modifications, substitutions, or other modifications, and / or modifications of sugars (e.g., those at the 2' position of 2'-O-alkyl, 2'-F, 2'-moe, 2'-F arabinose, 2'-H (deoxyribose), etc.). In some embodiments, a “YA modification” is any modification that alters the structure of a dinucleotide motif to reduce RNA endonuclease activity, for example, by preventing recognition or cleavage of the YA site by RNases and / or by stabilizing the RNA structure (e.g., secondary structure) which reduces RNase access to the cleavage site. See Peacock et al., J Org Chem. 76:7295-7300 (2011); Behlke, Oligonucleotides 18:305-320 (2008); Ku et al., Adv. Drug Delivery Reviews 104:16-28 (2016); and Ghidini et al., Chem. Commun., 2013, 49, 9036. Peacock et al., Behlke, Ku, and Ghidini provide exemplary modifications suitable as YA modifications. Modifications known to those skilled in the art for reducing endonucleotide degradation are included. Exemplary 2'-ribose modifications affecting the 2'-hydroxyl group involved in RNase cleavage are 2'-H and 2'-O-alkyl, including 2'-O-Me. Modifications such as bicyclic ribose analogs of residues at the YA site, UNA, and modified nucleoside-interconnections can also be YA modifications. Exemplary base modifications that can stabilize the RNA structure are pseudouridine and 5-methylcytosine. In some embodiments, at least one nucleotide of the YA site is modified. In some embodiments, the pyrimidine of the YA site (also referred to as the "pyrimidine site") includes modifications (e.g., modifications altering the nucleoside bond at the 2' position, immediately 3' of the pyrimidine sugar, modifications of the pyrimidine base, and modifications of the ribose).In some embodiments, the adenine at the YA site (also referred to as the "adenine position") includes modifications (e.g., modifications altering the nucleoside bond immediately 3' of the pyrimidine sugar at the 2' position, modifications of the pyrimidine base, and modifications of the ribose). In some embodiments, the pyrimidine and adenine at 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] sgRNA domain / region Briner AE et al., Molecular Cell 56:333-339 (2014) describe the functional domains of sgRNA, referred to herein as "domains," including the "spacer" domain responsible for targeting, the "lower stem," "bulge," "upper stem" (which may include a tetraloop), the "nexus," and the "hairpin 1" and "hairpin 2" domains. See Briner et al., p. 334, Figure 1A.

[0074] Table 3 provides a schematic diagram of the sgRNA domains used herein. In Table 3, "n" between regions represents, for example, a variable number of nucleotides, such as 0-1, 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' end region In some embodiments, the sgRNA or short sgRNA contains nucleotides at its 5' end, as shown in Table 3. In some embodiments, the 5' end of the sgRNA or short sgRNA contains a spacer or guide region that functions to guide a Cas protein, such as the Cas9 protein, to a target nucleotide sequence. In some embodiments, the 5' end does not contain a guide region. In some embodiments, the 5' end contains a spacer and additional nucleotides that do not function to guide the Cas protein to the target nucleotide region.

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

[0077] In some embodiments, the lower stem region contains 1 to 12 nucleotides; for example, in one embodiment, the lower stem region contains LS1 to LS12. In some embodiments, the lower stem region contains fewer nucleotides than shown in Table 3. In some embodiments, the lower stem region contains more nucleotides than shown in Table 3. If the lower stem region contains fewer or more nucleotides than shown in the schematic diagram 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 complementarity of the lower stem in the nucleic acid sequence results in a secondary structure of the stem in the sgRNA or short sgRNA (e.g., the regions may form base pairs with each other). In some embodiments, the lower stem regions may not be perfectly complementary to each other when read in the opposite direction.

[0079] Bulge In some embodiments, the sgRNA or short sgRNA includes a bulge region containing six nucleotides B1–B6. When viewed linearly, the bulge region is separated into two regions. See Table 3. In some embodiments, the bulge region contains six nucleotides, with the first two nucleotides followed by the upper stem region, followed by the last four 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 diagram in Table 3, the modification pattern must be maintained, as will be apparent to those skilled in the art.

[0080] In some embodiments, the presence of a bulge results in directional curling between the upper and lower stem modules in 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 includes 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 diagram of Table 3, the modification pattern must be maintained, 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 of the upper stem in the nucleic acid sequence results in a secondary structure of the stem in the sgRNA or short sgRNA (e.g., the regions may form base pairs with each other). In some embodiments, the upper stem regions may not be perfectly 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 includes 18 nucleotides. In some embodiments, the nexus region includes nucleotides N1-N18 as 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 diagram of Table 3, the modification pattern must be maintained, 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 a secondary structure of stem and / or stem-loop in the sgRNA or short sgRNA (for example, certain nucleotides in the nexus region may form base pairs with each other). In some embodiments, the nexus regions may not be perfectly complementary to each other when read in the opposite direction.

[0087] hairpin In some embodiments, the sgRNA or short sgRNA includes one or more hairpin regions. In some embodiments, the hairpin region is located downstream (e.g., 3' side) of the nexus region. In some embodiments, the nucleotide region immediately downstream of the nexus region is referred to as "hairpin 1" or "H1". In some embodiments, the region from nucleotide 3' to hairpin 1 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 either hairpin 1 or hairpin 2.

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

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

[0090] In some embodiments, one or more nucleotides are present between hairpin 1 region and hairpin 2 region. The one or more nucleotides between hairpin 1 region and hairpin 2 region may be modified or unmodified. In some embodiments, hairpin 1 and hairpin 2 are separated by a single 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. If the hairpin region contains fewer or more nucleotides than shown in the schematic diagram of Table 3, the modification pattern must be maintained, as will be apparent to those skilled in the art.

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

[0092] In some embodiments, the sgRNA or short sgRNA includes replacing hairpin 1 with a nucleotide "n", where "n" is an integer from 1 to 50, 40, 30, 20, 15, 10, 5, 4, 3, and 2. In some embodiments, the hairpin 1 region of the sgRNA is replaced with two nucleotides.

[0093] 3' end An sgRNA or short sgRNA has a 3' end, which is the last nucleotide of the sgRNA. The 3' end 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 includes a 3' end region, in which case the last nucleotide of the 3' end is the 3' end. In some embodiments, the 3' tail contains 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, for example. 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. [Table 2] [Table 3]

[0094] gRNA containing modifications, including modifications at the YA site. In some embodiments, the gRNAs described herein (e.g., sgRNAs, short sgRNAs, dgRNAs, or crRNAs) include modifications at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more YA sites (e.g., in the conserved region and / or guide region), and / or modifications such as YA modifications at nucleotide 6 from the 5' end of the 5' end or one or more nucleotides located thereafter. In some embodiments, the pyrimidines of the YA sites include modifications (including modifications that alter the nucleoside bond immediately 3' of the sugar of the pyrimidine). In some embodiments, the adenines of the YA sites include modifications (including modifications that alter the nucleoside bond immediately 3' of the sugar of the adenine). In some embodiments, the pyrimidines and adenines of the YA sites include modifications such as sugar, base, or nucleoside bond modifications. The YA modifications may be any of the types of modifications described herein. In some embodiments, the YA modification comprises one or more of phosphorothioates, 2'-OMe, or 2'-fluoro compounds. In some embodiments, the YA modification comprises a pyrimidine modification comprising one or more of phosphorothioates, 2'-OMe, or 2'-fluoro compounds. In some embodiments, the YA modification comprises a bicyclic ribose analog (e.g., LNA, BNA, or ENA) within an RNA double-stranded region containing one or more YA sites. In some embodiments, the YA modification comprises a bicyclic ribose analog (e.g., LNA, BNA, or ENA) within an RNA double-stranded region containing a YA site, and the YA modification is distal to the YA site.

[0095] Any of the embodiments described above may 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' terminal is free of 2'-fluoro modification, and / or (ii) at least one of nucleotides 6-10 from the 5' end of the 5' terminal is free of phosphorothioate bond, and (i) at least one of nucleotides 7-10 from the 5' end of the 5' terminal is free of 2'-OMe modification, (ii) nucleotide 20 from the 5' end of the 5' terminal is free of 2'-OMe modification, and / or (iii) or the guide RNA includes 2'-fluoro modification in one or more of nucleotides 1-20 from the 5' end of the 5' terminal, and at least one of nucleotides 11, 12, 13, 14, 17, or nucleotide 18 from the 5' end of the 5' terminal is free of 2'-fluoro modification, and optionally, nucleotide 12 from the 5' end of the 5' terminal is free of 2'-fluoro modification. Such embodiments may be further or, to the extent feasible, combined with any one or more other embodiments described herein.

[0096] Modification of the guide region, including modification of the YA site. In some embodiments, the guide region includes one or more modifications, optionally including YA site modifications. In some embodiments, the guide region includes one, two, three, four, five or more YA sites ("guide region YA sites") that may include YA modifications. In some embodiments, one or more YA sites located from the 5' end to the 5, 6, 7, 8, 9, or 10 end of the 5' end ("5 end", etc., refers to position 5 relative to the 3' end of the guide region, i.e., the nucleotide furthest 3' in the guide region) include YA modifications. In some embodiments, two or more YA sites located from the 5' end to the 5, 6, 7, 8, 9, or 10 end of the 5' end include YA modifications. In some embodiments, three or more YA sites located from the 5' end to the 5, 6, 7, 8, 9, or 10 end of the 5' end include YA modifications. In some embodiments, four or more YA sites located from the 5' end to the 5, 6, 7, 8, 9, or 10 end of the 5' end contain YA modifications. In some embodiments, five or more YA sites located from the 5' end to the 5, 6, 7, 8, 9, or 10 end of the 5' end contain YA modifications. The modification guide region YA sites contain YA modifications.

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

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

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

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

[0101] In some embodiments, the guide region includes 1 to 14 modifications from nucleotides 1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 13, 14, 17, and 18 of the guide region. Such modifications may be 2'-OMe, 2'-fluoro, 2'-H, inosine, or phosphorothioate modifications, or a combination thereof. For example, 2'-OMe modifications may be present in any or all of nucleotides 1-4 and 12, phosphorothioate modifications may be present in any or all of nucleotides 1-3 and 6-10, and / or 2'-fluoro modifications may be present in any or all of nucleotides 8-11, 13, 14, 17, and 18. In negative terms, 2'-OMe modifications may be excluded from nucleotides 6-11 and 13, 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, nucleotides are modified in a manner dependent on the YA site, for example, if the YA site is located on any of nucleotides 5-6, 12-13, 15-16, 16-17, or 19-20, then at least one nucleotide of the YA site is modified, for example, at least a pyrimidine of the YA site is modified, and 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 at the YA site; the modification at nucleotide 12 is 2'-OMe if the modification is a pyrimidine at the YA site; the modification at nucleotide 15 is a phosphorothioate if the modification is a pyrimidine at the YA site; the modification at nucleotide 16 is a phosphorothioate if the modification is a pyrimidine at the YA site; and / or the modification at nucleotide 19 is a phosphorothioate if the modification is a pyrimidine at the YA site. Thus, recognizing that the YA site cannot be present at both positions 15-16 and 16-17, it is possible for up to four modifications to be present, subject to the presence of the YA site.In alternative embodiments, the modification at nucleotide 19 may instead be a 2'-fluoro. This can be present in a manner dependent on the YA site, or 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 phosphorothioates, for example, by nucleotides that are pyrimidines of the YA site located at nucleotides 15-16 or 16-17. In some embodiments, nucleotides 15 and 16 contain unmodified ribose and / or unmodified nucleic acid bases. In some embodiments, nucleotide 5 is unmodified or modified only by 2'-OMe if it is a pyrimidine of the YA site. In some embodiments, nucleotide 12 is unmodified or modified only by 2'-OMe if it is a pyrimidine of the YA site. In some embodiments, nucleotide 20 (or the 3' terminal nucleotide of the guide region) is unmodified. In any of the prior embodiments, the guide region may consist of 20 nucleotides.

[0102] In some embodiments, the gRNA includes a guide region with modifications at one or more of nucleotides 5 and / or 12. The modifications at nucleotides 5 and / or 12 may be independently selected from the modifications described herein, e.g., 2'-OMe, 2'-F, phosphorothioate, and 2'-H (deoxyribonucleotide). Such modifications may be combined with other modification patterns or nucleotide modifications described herein, as shown, for example, in the gRNA described herein. Specific examples of such embodiments are described herein, for example, in the embodiments of the particular number 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, phosphorothioate modifications at nucleotides 1-3 and 6-10, and / or 2'-F modifications at nucleotides 8-11, 13, 14, 17, and 18.

[0103] In some embodiments, the gRNA includes a guide region in which any one, two, or all of nucleotides 8-10 are modified. Such modifications may be combined with other modification patterns or nucleotide modifications described herein, as shown, for example, in the gRNA described herein. The modifications may be independently selected from the modifications described herein, e.g., 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 do not include phosphorothioate modifications. In some embodiments, any one, two, or all of nucleotides 8-10 include 2'-F modifications and phosphorothioate modifications. Specific examples of such embodiments are described herein, for example, in the embodiments of a particular number 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 following: 2'-OMe modifications in nucleotides 1-4, phosphorothioate modifications in nucleotides 1-3 and 6-7, and / or 2'-F modifications in nucleotides 11, 13, 14, 17, and 18.

[0104] In some embodiments, the gRNA includes a guide region with modifications on either or both of nucleotides 5 and 6. The modifications may be independently selected from the modifications described herein, e.g., 2'-F modifications and phosphorothioate modifications, or combinations thereof. In some embodiments, either or both of nucleotides 5 and 6 include a 2'-F modification. In some embodiments, either or both of nucleotides 5 and 6 include a 2'-F modification but do not include a phosphorothioate modification. In some embodiments, either or both of nucleotides 5 and 6 include both a 2'-F modification and a phosphorothioate modification. Specific examples of such embodiments are described herein, for example, in the embodiments of the specific number 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 on nucleotides 1-4, phosphorothioate modifications on nucleotides 1-3 and 7-10, and / or 2'-F modifications on nucleotides 8-11, 13, 14, 17, and 18.

[0105] In some embodiments, the gRNA includes a guide region containing modifications at at least one, two, three, four, five, or six of nucleotides 6-11. These modifications may be independently selected from the modifications described herein, e.g., 2'-F modifications. In some embodiments, the 2'-F modifications at one, two, three, four, five, or six of nucleotides 6-11 are combined with another suitable modification, such as a phosphorothioate modification, at one or more positions containing the 2'-F modification. Specific examples of such embodiments are described herein, for example, in the embodiments of the specific number 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, phosphorothioate modifications at nucleotides 1-3, and / or 2'-F modifications at nucleotides 13, 14, 17, and 18.

[0106] In some embodiments, the gRNA includes a guide region containing modifications at at least one, two, three, four, five, six, seven, eight, nine, or ten of nucleotides 1-4 and 6-11. These modifications may be independently selected from the modifications described herein, e.g., 2'-F modifications. In some embodiments, the 2'-F modifications at one, two, three, four, five, six, seven, eight, nine, or ten of nucleotides 1-4 and 6-11 are combined with another suitable modification, such as a phosphorothioate modification, at one or more of the positions containing the 2'-F modifications. Specific examples of such embodiments are described herein, for example, in the embodiments of the specific number 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 phosphorothioate modifications at nucleotides 1-3 and / or the 2'-F modifications at nucleotides 13, 14, 17, and 18.

[0107] In some embodiments, the gRNA includes a guide region containing 2'-OMe modifications at at least one, two, three, or four of nucleotides 9, 11, 13, and 14. In some embodiments, the 2'-OMe modifications at at least one, two, three, or four of nucleotides 9, 11, 13, and 14 are combined with another suitable modification, such as a phosphorothioate modification, at one or more of the positions containing the 2'-OMe modifications. Specific examples of such embodiments are described herein, for example, in the embodiments of the specific number 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 and / or phosphorothioate modifications at nucleotides 1-3 and 6-10.

[0108] In some embodiments, the modified guide region YA includes the modifications described for the YA region.

[0109] Additional embodiments of guide region YA site modification are shown in the summary above. Any embodiments described elsewhere in this disclosure may be combined with any of the prior embodiments to the extent feasible.

[0110] Modification of the YA region in the preserved area The preserved regions YA parts 1 to 10 are shown in Figure 1B. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 preserved regions YA parts include modifications.

[0111] In some embodiments, the storage region YA sites 1, 8, or 1 and 8 include YA modifications. In some embodiments, the storage region YA sites 1, 2, 3, 4, and 10 include YA modifications. In some embodiments, YA sites 2, 3, 4, 8, and 10 include YA modifications. In some embodiments, the storage region YA sites 1, 2, 3, and 10 include YA modifications. In some embodiments, YA sites 2, 3, 8, and 10 include YA modifications. In some embodiments, YA sites 1, 2, 3, 4, 8, and 10 include YA modifications. In some embodiments, 1, 2, 3, 4, 5, 6, 7, or 8 additional storage region YA sites include YA modifications.

[0112] In some embodiments, 1, 2, 3, or 4 of the preserved region YA sites 2, 3, 4, and 10 include YA modifications. In some embodiments, 1, 2, 3, 4, 5, 6, 7, or 8 additional preserved region YA sites include YA modifications.

[0113] In some embodiments, the modified preservation region YA includes the modifications described for the YA region.

[0114] Further embodiments of the modification of the YA site in the preserved region are shown in the summary above. Any embodiments described elsewhere in this disclosure may be combined with any of the prior embodiments to the extent feasible.

[0115] Modification of terminal nucleotides In some embodiments, the 5' and / or 3' terminal regions of the gRNA (e.g., sgRNA, short sgRNA, dgRNA, or crRNA) are modified.

[0116] 3' end region modification In some embodiments, nucleotides 1, 2, 3, 4, 5, 6, or 7 in the 3' terminal region are modified. Throughout, this modification may be referred to as a “3' terminal modification”. In some embodiments, the terminal (i.e., last) nucleotides 1, 2, 3, 4, 5, 6, or 7 in the 3' terminal region include two or more modifications. In some embodiments, at least one of the terminal (i.e., last) nucleotides 1, 2, 3, 4, 5, 6, or 7 in the 3' terminal region is modified. In some embodiments, at least two of the terminal (i.e., last) nucleotides 1, 2, 3, 4, 5, 6, or 7 in the 3' terminal region are modified. In some embodiments, at least three of the terminal (i.e., last) nucleotides 1, 2, 3, 4, 5, 6, or 7 in the 3'' terminal region are modified. In some embodiments, the modification includes a PS bond. In some embodiments, the modification of the 3' terminal region is a 3' protective end modification. In some embodiments, the 3' terminal modification includes a 3' protective end modification.

[0117] In some embodiments, the 3'-terminal modification includes a modified nucleotide selected from 2'-O-methyl (2'-O-Me) modified nucleotides, 2'-O-(2-methoxyethyl) (2'-O-moe) modified nucleotides, 2'-fluoro (2'-F) modified nucleotides, internucleotide phosphorothioate (PS) bonds, inverted debasic modified nucleotides, ENA, UNA, 2'-H(DNA), or combinations thereof.

[0118] In some embodiments, the 3'-terminal modification includes a 2'-O-methyl (2'-O-Me) modified nucleotide.

[0119] In some embodiments, the 3'-terminal modification includes or further includes a 2'-fluoro(2'-F) modified nucleotide.

[0120] In some embodiments, the 3' terminal modification includes an internucleotide phosphorothioate (PS) bond.

[0121] In some embodiments, the 3'-terminus modification includes or further includes an inverted debasitized nucleotide.

[0122] In some embodiments, the 3'-terminus modification includes or further includes ENA.

[0123] In some embodiments, the 3'-terminus modification includes or further includes a UNA.

[0124] In some embodiments, the 3'-terminus modification includes or further includes 2'-H(DNA).

[0125] In some embodiments, the 3'-terminus modification includes or further includes one or more modifications from the last 7, 6, 5, 4, 3, 2, or 1 nucleotide. In some embodiments, the 3'-terminus modification includes or further includes one modified nucleotide. In some embodiments, the 3'-terminus modification includes or further includes two modified nucleotides. In some embodiments, the 3'-terminus modification includes or further includes three modified nucleotides. In some embodiments, the 3'-terminus modification includes or further includes four modified nucleotides. In some embodiments, the 3'-terminus modification includes or further includes five modified nucleotides. In some embodiments, the 3'-terminus modification includes or further includes six modified nucleotides. In some embodiments, the 3'-terminus modification includes or further includes seven modified nucleotides.

[0126] In some embodiments, the 3'-terminus modification includes or further includes modifications of 1 to 7 or 1 to 5 nucleotides.

[0127] In some embodiments, the 3' terminal modification includes, or further includes, a modification of 1, 2, 3, 4, 5, 6, or 7 nucleotides at the 3' end of the gRNA.

[0128] In some embodiments, the 3' terminal modification includes, or further includes, modifications of approximately 1-3, 1-5, 1-6, or 1-7 nucleotides at the 3' end of the gRNA.

[0129] In some embodiments, the 3'-terminal modification includes or further includes one or more of the following: internucleotide phosphorothioate (PS) bonds, 2'-O-Me modified nucleotides, 2'-O-moe modified nucleotides, 2'-F modified nucleotides, inverted debasalized nucleotides, ENAs, UNAs, and combinations thereof.

[0130] In some embodiments, the 3' terminal modification includes or further includes 1, 2, 3, 4, 5, 6, or 7 internucleotide PS bonds.

[0131] In some embodiments, the 3'-terminal modification comprises or further comprises at least one 2'-O-Me, 2'-O-moe, inverted debase, or 2'-F modified nucleotide.

[0132] In some embodiments, the 3'-terminus modification includes one or more PS bonds, the bond located between the last nucleotide and the second to last nucleotide. In some embodiments, the 3'-terminus modification includes two or more PS bonds between the last three nucleotides. In some embodiments, the 3'-terminus modification includes four or more PS bonds between the last four nucleotides.

[0133] In some embodiments, the 3'-terminus modification includes or further includes a PS bond between any one or more of the last four nucleotides. In some embodiments, the 3'-terminus modification includes or further includes a PS bond between any one or more of the last five nucleotides. In some embodiments, the 3'-terminus modification includes or further includes a PS bond between any one or more of the last two, three, four, five, six, or seven nucleotides.

[0134] In some embodiments, the 3'-terminal modification includes or further includes one or more modifications of the last 1 to 7 nucleotides, the modifications being PS-linked, inverted debasalized nucleotides, 2'-O-Me, 2'-O-moe, 2'-F, or a combination thereof.

[0135] In some embodiments, the 3'-terminal modification includes or further includes a modification of the last nucleotide having 2'-O-Me, 2'-O-moe, 2'-F, or a combination thereof, as well as, optionally, one or two PS bonds to the nucleotide adjacent to the 3' tail and / or to the first nucleotide.

[0136] In some embodiments, the 3'-terminal modification includes or further includes a modification of the last and / or second-to-last nucleotide by 2'-O-Me, 2'-O-moe, 2'-F, or a combination thereof, and optionally one or more PS bonds.

[0137] In some embodiments, the 3'-terminal modification includes or further includes modifications of the last nucleotide, the second-to-last nucleotide, and / or the third-to-last nucleotide by 2'-O-Me, 2'-O-moe, 2'-F, or a combination thereof, and optionally one or more PS bonds.

[0138] In some embodiments, the 3'-terminal modification includes or further includes modifications of the last, second-to-last, third-to-last, and / or fourth-to-last nucleotide by 2'-O-Me, 2'-O-moe, 2'-F, or a combination thereof, and optionally one or more PS bonds.

[0139] In some embodiments, the 3'-terminal modification includes or further includes modifications of the last, second-to-last, third-to-last, fourth-to-last, and / or fifth-to-last nucleotide by 2'-O-Me, 2'-O-moe, 2'-F, or a combination thereof, and optionally one or more PS bonds.

[0140] In some embodiments, an sgRNA or short sgRNA with a 3' terminal modification includes or further includes a 3' tail, the 3' tail containing one or more modifications of any of the nucleotides present in the 3' tail. In some embodiments, the 3' tail is fully modified. In some embodiments, the 3' tail contains 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, with one or more of these nucleotides optionally modified.

[0141] In some embodiments, sgRNAs or short sgRNAs containing a 3' terminal modification are provided, the 3' terminal modification being one of the 3' terminal modifications shown in any one of SEQ ID NOs: 1 to 132. In some embodiments, sgRNAs containing a 3' protective terminal modification are provided.

[0142] In some embodiments, an sgRNA or short sgRNA is provided that includes a 3' terminal modification, the 3' terminal modification comprising (i) a 2'-OMe modified nucleotide at the last nucleotide of the sgRNA or the conserved region of the sgRNA, (ii) three consecutive 2'O-moe modified nucleotides immediately 5' to the 2'-OMe modified nucleotide, and (iii) three consecutive PS bonds between the last three nucleotides.

[0143] In some embodiments, an sgRNA or short sgRNA is provided that includes a 3' terminal modification, the 3' terminal modification comprising (i) five consecutive 2'-OMe modified nucleotides from the last nucleotide of the sgRNA or the conserved region of the sgRNA, and (ii) three PS bonds between the last three nucleotides.

[0144] In some embodiments, sgRNA or short sgRNAs containing 3' terminal modifications are provided, which include inverted base-modified nucleotides at the last nucleotide of the sgRNA or conserved region of the sgRNA.

[0145] In some embodiments, sgRNA or short sgRNAs are provided that include a 3' terminal modification, the 3' terminal modification comprising (i) an inverted debasic 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.

[0146] In some embodiments, sgRNA or short sgRNAs are provided that include a 3' terminal modification, the 3' terminal modification comprising (i) 15 consecutive 2'-OMe modified nucleotides from the last nucleotide of the conserved region of the sgRNA, (ii) 5 consecutive 2'-F modified nucleotides immediately 5' to the 2'-OMe modified nucleotide, and (iii) 3 PS bonds between the last 3 nucleotides.

[0147] In some embodiments, an sgRNA or short sgRNA is provided that includes a 3' terminal modification, the 3' terminal modification comprising (i) alternating 2'-OMe modified nucleotides and 2'-F modified nucleotides in the last 20 nucleotides of the sgRNA or the conserved region of the sgRNA, and (ii) three PS bonds between the last three nucleotides.

[0148] In some embodiments, sgRNA or short sgRNAs containing a 3' terminal modification are provided, the 3' terminal modification comprising (i) two or three consecutive 2'-OMe modified nucleotides, and (ii) three PS bonds between the last three nucleotides.

[0149] In some embodiments, sgRNA or short sgRNAs are provided that include a 3' terminal modification, the 3' terminal modification comprising a single PS bond between the last nucleotide and the last adjacent nucleotide.

[0150] In some embodiments, the sgRNA or short sgRNA includes 5' and 3' terminal modifications.

[0151] 3' tail In some embodiments, the sgRNA includes a 3' end portion containing a 3' tail following the 3' end of the conserved portion of the sgRNA. In some embodiments, the 3' tail contains 1 to about 20 nucleotides, 1 to about 15 nucleotides, 1 to about 10 nucleotides, 1 to about 5 nucleotides, 1 to about 4 nucleotides, 1 to about 3 nucleotides, and 1 to about 2 nucleotides. In some embodiments, the 3' tail contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In some embodiments, the 3' tail contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In some embodiments, the 3' tail contains 1 nucleotide. In some embodiments, the 3' tail contains 2 nucleotides. In some embodiments, the 3' tail contains 3 nucleotides. In some embodiments, the 3' tail contains 4 nucleotides. In some embodiments, the 3' tail contains approximately 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.

[0152] In some embodiments, the 3' tail contains 1 to 20 nucleotides and follows the 3' end of the conserved portion of the sgRNA.

[0153] In some embodiments, the 3' tail includes or further includes one or more of the following: protective end modifications, internucleotide phosphorothioate (PS) bonds, 2'-O-Me modified nucleotides, 2'-O-moe modified nucleotides, 2'-F modified nucleotides, inverted debasal modified nucleotides, and combinations thereof.

[0154] In some embodiments, the 3' tail comprises or further comprises one or more internucleotide phosphorothioate (PS) bonds. In some embodiments, the 3' tail comprises or further comprises one or more 2'-O-Me modified nucleotides. In some embodiments, the 3' tail comprises or further comprises one or more 2'-O-moe modified nucleotides. In some embodiments, the 3' tail comprises or further comprises one or more 2'-F modified nucleotides. In some embodiments, the 3' tail comprises or further comprises one or more inverted debasic modified nucleotides. In some embodiments, the 3' tail comprises or further comprises one or more protective end modifications. In some embodiments, the 3' tail comprises or further comprises one or more combinations of internucleotide phosphorothioate (PS) bonds, 2'-O-Me modified nucleotides, 2'-O-moe modified nucleotides, 2'-F modified nucleotides, and inverted debasic modified nucleotides.

[0155] In some embodiments, the sgRNA does not contain a 3' tail.

[0156] 5' end region modification In some embodiments, the 5' terminal region is modified, for example, the first 1, 2, 3, 4, 5, 6, or 7 nucleotides of the gRNA (e.g., sgRNA, short sgRNA, or crRNA) are modified. Throughout, this modification may be referred to as a “5' terminal modification”. In some embodiments, the first 1, 2, 3, 4, 5, 6, or 7 nucleotides of the 5' terminal region (i.e., the 1, 2, 3, 4, 5, 6, or 7 nucleotides from the 5' end of the 5' terminal) include two or more modifications. In some embodiments, at least one of the terminal (i.e., first) 1, 2, 3, 4, 5, 6, or 7 nucleotides of the 5' end of the 5' terminal is modified. In some embodiments, at least two of the terminal nucleotides from the 5' end of the 5' terminal are modified. In some embodiments, at least three of the terminal nucleotides from the 5' end of the 5' terminal are modified. In some embodiments, the modification includes a PS bond. In some embodiments, the modification of the 5' terminal region is a 5' protective end modification. In some embodiments, the 5' terminal modification includes a 5' protective end modification.

[0157] In some embodiments, both the 5' and 3' terminal regions of the sgRNA or short sgRNA are modified (e.g., including the first and last nucleotides of the gRNA). In some embodiments, only the 5' terminal region of the sgRNA or short sgRNA is modified. In some embodiments, only the 3' terminal region (plus or minus 3' tail) of the conserved portion of the sgRNA or short sgRNA is modified.

[0158] In some embodiments, the gRNA (e.g., sgRNA, short sgRNA, or crRNA) includes modifications on 1, 2, 3, 4, 5, 6, or 7 of the first 7 nucleotides from the 5' end of the 5' terminal of the gRNA. In some embodiments, the gRNA (e.g., sgRNA, short sgRNA, or crRNA) includes modifications on 1, 2, 3, 4, 5, 6, or 7 of the 7 terminal nucleotides from the 3' end of the 3' terminal. In some embodiments, 2, 3, or 4 of the first 4 nucleotides from the 5' end of the 5' terminal and / or 2, 3, or 4 of the 4 terminal nucleotides from the 3' end of the 3' terminal are modified. In some embodiments, 2, 3, or 4 of the first 4 nucleotides from the 5' end of the 5' terminal are linked by phosphorothioate (PS) bonds.

[0159] In some embodiments, 5'-terminus and / or 3'-terminus modifications include 2'-O-methyl (2'-O-Me) or 2'-O-(2-methoxyethyl) (2'-O-moe) modifications. In some embodiments, modifications include 2'-fluoro (2'-F) modifications to the nucleotide. In some embodiments, modifications include internucleotide phosphorothioate (PS) bonds. In some embodiments, modifications include inverted debasalized nucleotides. In some embodiments, modifications include protective end modifications. In some embodiments, modifications include two or more modifications selected from protective end modifications, 2'-O-Me, 2'-O-moe, 2'-fluoro (2'-F), internucleotide phosphorothioate (PS) bonds, 2'-H(DNA), ENA, UNA, and inverted debasalized nucleotides. In some embodiments, equivalent modifications are included.

[0160] In some embodiments, the gRNA (e.g., sgRNA, short sgRNA, or crRNA) contains one or more phosphorothioate (PS) bonds between the first 1, 2, 3, 4, 5, 6, or 7 nucleotides at the 5' end. In some embodiments, the sgRNA contains one or more PS bonds between the last 1, 2, 3, 4, 5, 6, or 7 nucleotides at the 3' end. In some embodiments, the sgRNA or short sgRNA contains one or more PS bonds both between the last 1, 2, 3, 4, 5, 6, or 7 nucleotides at the 3' end and between the first 1, 2, 3, 4, 5, 6, or 7 nucleotides from the 5' end at the 5' end. In some embodiments, in addition to PS bonds, the 5' and 3' terminal nucleotides may contain 2'-O-Me, 2'-O-moe, or 2'-F modified nucleotides.

[0161] In some embodiments, the sgRNA includes a 5' terminal modification, for example, the first nucleotide of the guide region is modified. In some embodiments, the sgRNA includes a 5' terminal modification, and the first nucleotide of the guide region includes a 5' protective terminal modification.

[0162] In some embodiments, the 5'-terminal modification includes 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 debasitized nucleotide, an ENA, an UNA, a 2'H(DNA), or a combination thereof.

[0163] In some embodiments, the 5'-terminal modification includes or further includes a 2'-O-methyl (2'-O-Me) modified nucleotide.

[0164] In some embodiments, the 5'-terminal modification includes or further includes a 2'-fluoro(2'-F) modified nucleotide.

[0165] In some embodiments, the 5' terminal modification includes an internucleotide phosphorothioate (PS) bond.

[0166] In some embodiments, the 5'-terminus modification includes or further includes an inverted debasitized nucleotide.

[0167] In some embodiments, the 5'-terminus modification includes or further includes ENA.

[0168] In some embodiments, the 5'-terminus modification includes or further includes a UNA.

[0169] In some embodiments, the 5'-terminus modification includes or further includes 2'-H(DNA).

[0170] In some embodiments, the 5'-terminal modification includes or further includes one or more modifications of nucleotides 1-7 in the guide region of the gRNA (e.g., sgRNA, short sgRNA, or crRNA). In some embodiments, the 5'-terminal modification includes or further includes one modified nucleotide. In some embodiments, the 5'-terminal modification includes or further includes two modified nucleotides. In some embodiments, the 5'-terminal modification includes or further includes three modified nucleotides. In some embodiments, the 5'-terminal modification includes or further includes four modified nucleotides. In some embodiments, the 5'-terminal modification includes or further includes five modified nucleotides. In some embodiments, the 5'-terminal modification includes or further includes six modified nucleotides. In some embodiments, the 5'-terminal modification includes or further includes seven modified nucleotides.

[0171] In some embodiments, the 5' terminal modification includes or further includes modifications of 1-7, 1-5, 1-4, 1-3, or 1-2 nucleotides.

[0172] In some embodiments, the 5'-terminus modification includes or further includes modifications of 1, 2, 3, 4, 5, 6, or 7 nucleotides from the 5' end. In some embodiments, the 5'-terminus modification includes or further includes modifications of approximately 1–3, 1–4, 1–5, 1–6, or 1–7 nucleotides from the 5' end.

[0173] In some embodiments, the 5'-terminus modification includes or further includes a modification at the first nucleotide from the 5' end of the gRNA (e.g., sgRNA, short sgRNA, or crRNA). In some embodiments, the 5'-terminus modification includes or further includes modifications at the first and second nucleotides from the 5' end of the gRNA (e.g., sgRNA, short sgRNA, or crRNA). In some embodiments, the 5'-terminus modification includes or further includes modifications from the first, second, and third nucleotides at the 5' end of the gRNA (e.g., sgRNA, short sgRNA, or crRNA). In some embodiments, the 5'-terminus modification includes or further includes modifications at the first, second, third, fourth, and fifth nucleotides from the 5' end of the gRNA (e.g., sgRNA, short sgRNA, or crRNA). In some embodiments, the 5' terminal modification includes or further includes modifications at the 1st, 2nd, 3rd, 4th, 5th, and nucleotide 6 from the 5' end of the gRNA (e.g., sgRNA, short sgRNA, or crRNA). In some embodiments, the 5' terminal modification includes or further includes modifications at the 1st, 2nd, 3rd, 4th, 5th, 6th, and nucleotide 7 from the 5' end of the gRNA (e.g., sgRNA, short sgRNA, or crRNA).

[0174] In some embodiments, the 5'-terminal modification includes or further includes internucleotide phosphorothioate (PS) bonds and / or 2'-O-Me modified nucleotides and / or 2'-O-moe modified nucleotides and / or 2'-F modified nucleotides and / or inverted debasic modified nucleotides, and / or combinations thereof.

[0175] In some embodiments, the 5'-terminus modification includes at least 1, 2, 3, 4, 5, 6, and / or 7 internucleotide PS bonds. In some embodiments, the 5'-terminus modification includes or further includes about 1-2, 1-3, 1-4, 1-5, 1-6, or 1-7 internucleotide PS bonds.

[0176] In some embodiments, the 5' terminal modification includes or includes at least one PS bond, and if one PS bond is present, the bond is located between nucleotides 1 and 2 of the guide region.

[0177] In some embodiments, the 5' terminal modification includes or further includes at least two PS bonds, the bonds located between nucleotides 1 and 2 and between 2 and 3 in the guide region.

[0178] In some embodiments, the 5'-terminus modification includes or further includes one or more PS bonds between creotides 1 and 2, between 2 and 3, and between 3 and 4.

[0179] In some embodiments, the 5' terminal modification includes or further includes one or more PS bonds between nucleotides 1 and 2, 2 and 3, 3 and 4, and 4 and 5 in the guide region.

[0180] In some embodiments, the 5' terminal modification includes or further includes one or more PS bonds between nucleotides 1 and 2, 2 and 3, 3 and 4, 4 and 5, and 5 and 6 in the guide region.

[0181] In some embodiments, the 5' terminal modification includes or further includes one or more PS bonds between nucleotides 1 and 2, 2 and 3, 3 and 4, 4 and 5, 5 and 6, and 7 and 8 in the guide region.

[0182] In some embodiments, the 5' terminal modification includes or further includes one or more modifications of nucleotides 1-7 in the guide region, the modifications being PS bonds, inverted debasalized nucleotides, 2'-O-Me, 2'-O-Moe, 2'-F, and / or combinations thereof.

[0183] In some embodiments, the 5'-terminus modification includes or further includes modification of the first nucleotide of the guide region by 2'-O-Me, 2'-O-moe, 2'-F, or a combination thereof, and a PS bond to an adjacent nucleotide of optional choice.

[0184] In some embodiments, the 5'-terminus modification includes modification of the first and / or second nucleotides of the guide region by 2'-O-Me, 2'-O-moe, 2'-F, or a combination thereof, and optionally includes or further includes one or more PS bonds between the first and second nucleotides and / or between the second and third nucleotides.

[0185] In some embodiments, the 5'-terminus modification includes modifications of the first, second, and / or third nucleotides of the variable region by 2'-O-Me, 2'-O-moe, 2'-F, or a combination thereof, and optionally includes or further includes one or more PS bonds between the first and second nucleotides, between the second and third nucleotides, and / or between the third and fourth nucleotides.

[0186] In some embodiments, the 5'-terminus modification includes modifications of the first, second, third, and / or fourth nucleotides of the variable region by 2'-O-Me, 2'-O-moe, 2'-F, or a combination thereof, and optionally includes or further includes one or more PS bonds between the first and second nucleotides, between the second and third nucleotides, between the third and fourth nucleotides, and / or between the fourth and fifth nucleotides.

[0187] In some embodiments, the 5'-terminus modification includes modifications of the first, second, third, fourth, and / or fifth nucleotides of the variable region by 2'-O-Me, 2'-O-moe, 2'-F, or a combination thereof, and optionally includes or further includes one or more PS bonds between the first and second nucleotides, between the second and third nucleotides, between the third and fourth nucleotides, between the fourth and fifth nucleotides, and / or between the fifth and sixth nucleotides.

[0188] In some embodiments, gRNAs (e.g., sgRNAs, short sgRNAs, or crRNAs) containing 5' terminal modifications are provided, where the 5' terminal modifications include those indicated in any one of SEQ ID NOs: 401-532, 1001, or 1007-1132. In some embodiments, gRNAs (e.g., sgRNAs, short sgRNAs, or crRNAs) containing 5' terminal modifications are provided, where the 5' terminal modifications include those indicated in nucleotides 1-3 of any one of SEQ ID NOs: 401-532, 1001, or 1007-1132. In some embodiments, gRNAs (e.g., sgRNAs, short sgRNAs, or crRNAs) containing 5' terminal modifications are provided, where the 5' terminal modifications include those indicated in nucleotides 1-4 of any one of SEQ ID NOs: 401-532, 1001, or 1007-1132. In some embodiments, gRNAs (e.g., sgRNAs, short sgRNAs, or crRNAs) containing 5' terminal modifications are provided, where the 5' terminal modifications include those indicated on nucleotides 1-5 of any one of sequence numbers 401-532, 1001, or 1007-1132. In some embodiments, gRNAs (e.g., sgRNAs, short sgRNAs, or crRNAs) containing 5' terminal modifications are provided, where the 5' terminal modifications include those indicated on nucleotides 1-6 of any one of sequence numbers 401-532, 1001, or 1007-1132. In some embodiments, gRNAs (e.g., sgRNAs, short sgRNAs, or crRNAs) containing 5' terminal modifications are provided, where the 5' terminal modifications include those indicated on nucleotides 1-7 of any one of sequence numbers 401-532, 1001, or 1007-1132.

[0189] In some embodiments, the gRNA (e.g., sgRNA, short sgRNA, or crRNA) includes a 5' terminal modification, which includes a 5' protective terminal modification. In some embodiments, a gRNA (e.g., sgRNA, short sgRNA, or crRNA) including a 5' terminal modification is provided, where the 5' terminal modification includes 2'-OMe modified nucleotides at nucleotides 1, 2, and 3 of the guide region.

[0190] In some embodiments, gRNAs (e.g., sgRNAs, short sgRNAs, or crRNAs) are provided that include 5' terminal modifications, the 5' terminal modifications including 2'-OMe modified nucleotides at nucleotides 1, 2, and 3 of the guide region, as well as PS bonds between nucleotides 1 and 2, 2 and 3, and 3 and 4 of the guide region.

[0191] In some embodiments, gRNAs (e.g., sgRNAs, short sgRNAs, or crRNAs) containing 5' terminal modifications are provided, where the 5' terminal modifications include 2'-OMe modified nucleotides at nucleotides 1, 2, 3, and 4 of the guide region and nucleotide 5.

[0192] In some embodiments, gRNAs (e.g., sgRNAs, short sgRNAs, or crRNAs) are provided that include 5' terminal modifications, the 5' terminal modifications including 2'-OMe modified nucleotides in guide regions 1, 2, 3, 4, and 5, and PS bonds between nucleotides 1 and 2, 2 and 3, 3 and 4, 4 and 5, and 5 and 6 in guide regions.

[0193] In some embodiments, gRNAs (e.g., sgRNAs, short sgRNAs, or crRNAs) containing 5' terminal modifications are provided, where the 5' terminal modifications include 2'O-moe modified nucleotides in guide regions 1, 2, and 3.

[0194] In some embodiments, gRNAs (e.g., sgRNAs, short sgRNAs, or crRNAs) are provided that include 5' terminal modifications, the 5' terminal modifications including 2'O-moe modified nucleotides at nucleotides 1, 2, and 3 of the guide region, as well as PS bonds between nucleotides 1 and 2, 2 and 3, and 3 and 4 of the guide region.

[0195] In some embodiments, gRNAs (e.g., sgRNAs, short sgRNAs, or crRNAs) containing 5' terminal modifications are provided, where the 5' terminal modification includes an inverted debasalized nucleotide at nucleotide 1 of the guide region.

[0196] In some embodiments, agRNAs (e.g., sgRNAs, short sgRNAs, or crRNAs) containing 5' terminal modifications are provided, the 5' terminal modifications including an inverted debasalized nucleotide at nucleotide 1 of the guide region, and 2'-OMe modified nucleotides at nucleotides 1, 2, and 3 of the guide region.

[0197] In some embodiments, agRNAs (e.g., sgRNAs, short sgRNAs, or crRNAs) having 5' terminal modifications are provided, the 5' terminal modifications including an inverted debasalized nucleotide at nucleotide 1 of the guide region, 2'-OMe modified nucleotides at nucleotides 1, 2, and 3 of the guide region, and PS bonds between nucleotides 1 and 2, 2 and 3, 3 and 4, 4 and 5, and 5 and 6 of the guide region.

[0198] In some embodiments, sgRNAs or short sgRNAs are provided that include 5'-terminated and 3'-terminated modifications. Any of the 5'-terminated modifications disclosed herein above and / or otherwise can be combined with 3'-terminated modifications such as those represented in sequence listings and / or discussed below.

[0199] In some embodiments, the sgRNA or short sgRNA includes modified nucleotides at the 5' and 3' ends, as well as modified nucleotides in one or more other regions as listed in Table 3.

[0200] In some embodiments, the sgRNA or short sgRNA contains modified nucleotides that are not present at the 5' or 3' end. Exemplary patterns of modifications are shown below and in Table 1.

[0201] Modifications to stabilize the secondary structure In some embodiments, gRNA (e.g., sgRNA, short sgRNA, or crRNA) includes modifications that stabilize a secondary structure, such as a double-stranded region. The increase in secondary structure stability can be determined empirically, for example, by melting temperature analysis. To simplify the analysis, the secondary structure element intended for stabilization can be isolated from the rest of the sgRNA structure and tested. The increase in secondary structure stability can also be determined by a decrease in the accessibility of the endonuclease cleavage site (e.g., YA site), where the modification does not alter the primary structure of the endonuclease cleavage site but occurs in the secondary structure containing the endonuclease cleavage site, or does not affect that secondary structure. This is referred to as having a distal effect on the endonuclease cleavage site. In some embodiments, the endonuclease cleavage site is located in the lower stem. In some embodiments, the endonuclease cleavage site is the conserved region YA site 1. In some embodiments, the endonuclease cleavage site is the conserved region YA site 2. In some embodiments, the endonuclease cleavage site is the conserved region YA site 3. In some embodiments, the endonuclease cleavage site is the conserved region YA site 10. In some embodiments, the modification is a bicyclic ribose analog modification, e.g., locked nucleic acid (LNA) or LNA-like modification. In some embodiments, the modification is an ENA modification. In some embodiments, nucleotide LS8 includes a modification that stabilizes the secondary structure. In some embodiments, nucleotide LS11 includes a modification that stabilizes the secondary structure. In some embodiments, one or both of nucleotides LS8 and LS11 collectively include one or more modifications (e.g., two modifications), such as an ENA modification, that stabilize the secondary structure. See the discussions in Examples G10008 and G10038.

[0202] Additional modifications In some embodiments, the gRNA (e.g., sgRNA, short sgRNA, or crRNA) includes at least 15 modified and / or unmodified nucleotides from nucleotides 1 to 20 of the 5' end of the 5' terminal, matching the modification pattern of nucleotides 1 to 20 of the gRNA described herein, for example, in Table 1. In some embodiments, the gRNA (e.g., sgRNA, short sgRNA, or crRNA) includes at least 16 modified and / or unmodified nucleotides from nucleotides 1 to 20 of the 5' end of the 5' terminal, matching the modification pattern of nucleotides 1 to 20 of the gRNA described herein, for example, in Table 1. In some embodiments, the gRNA (e.g., sgRNA, short sgRNA, or crRNA) includes at least 17 modified and / or unmodified nucleotides from nucleotides 1 to 20 of the 5' end of the 5' terminal, matching the modification pattern of nucleotides 1 to 20 of the gRNA described herein, for example, in Table 1. In some embodiments, the gRNA (e.g., sgRNA, short sgRNA, or crRNA) includes modified and / or unmodified nucleotides in at least 18 of the nucleotides 1-20 from the 5' end of the 5' terminal, matching the modification pattern in nucleotides 1-20 of the gRNA described herein, for example, in Table 1. In some embodiments, the gRNA (e.g., sgRNA, short sgRNA, or crRNA) includes modified and / or unmodified nucleotides in at least 19 of the nucleotides 1-20 from the 5' end of the 5' terminal, matching the modification pattern in nucleotides 1-20 of the gRNA described herein, for example, in Table 1. In some embodiments, the gRNA (e.g., sgRNA, short sgRNA, or crRNA) includes modified and / or unmodified nucleotides in the nucleotides 1-20 from the 5' end of the 5' terminal, matching the modification pattern in nucleotides 1-20 of the gRNA described herein, for example, in Table 1. In some embodiments, the gRNA includes a modification pattern that matches at least 75% of the modification pattern of the gRNA described herein, for example, in Table 1.In some embodiments, the gRNA includes a modification pattern that matches at least 80% of the gRNA modification patterns described herein, for example, in Table 1. In some embodiments, the gRNA includes a modification pattern that matches at least 85% of the gRNA modification patterns described herein, for example, in Table 1. In some embodiments, the gRNA includes a modification pattern that matches at least 90% of the gRNA modification patterns described herein, for example, in Table 1. In some embodiments, the gRNA includes a modification pattern that matches at least 95% of the gRNA modification patterns described herein, for example, in Table 1. In some embodiments, the gRNA includes a modification pattern that matches at least 98% of the gRNA modification patterns described herein, for example, in Table 1. In some embodiments, the gRNA includes a modification pattern that matches the gRNA modification patterns described herein, for example, in Table 1. In some embodiments, the sgRNA or short sgRNA includes modifications in one or more of the regions indicated as modified in Table 1. In some embodiments, the sgRNA or short sgRNA includes modifications in any of the positions indicated as modified in Table 1. In some embodiments, the sgRNA or short sgRNA includes one of the modifications shown in Table 1. Additional modifications are shown in the summary section above, and these may be combined, to the extent feasible, with modifications disclosed elsewhere in this specification, such as YA site modifications.

[0203] In some embodiments, an sgRNA or short sgRNA is provided that includes an upper stem modification, the upper stem modification comprising one or more modifications from US1 to US12 in the upper stem region.

[0204] In some embodiments, an sgRNA or short sgRNA is provided that includes an upper stem modification, the upper stem modification comprising modification of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides in the upper stem region.

[0205] In some embodiments, an sgRNA or short sgRNA is provided that includes an upper stem modification, the upper stem modification comprising approximately 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, or 1-12 nucleotide modifications in the upper stem region.

[0206] In some embodiments, an sgRNA or short sgRNA is provided that includes an upper stem modification, the upper stem modification containing one, two, three, four, or five YA modifications at the YA site. In some embodiments, an sgRNA or short sgRNA is provided that includes an upper stem modification, the upper stem modification containing at least one, two, three, four, or five YA modifications. In some embodiments, an sgRNA or short sgRNA is provided that includes an upper stem modification, the upper stem modification containing one YA modification. In some embodiments, an sgRNA or short sgRNA is provided that includes an upper stem modification, the upper stem modification containing two YA modifications. In some embodiments, the upper stem modification containing three YA modifications. In some embodiments, one or more YA modifications are located at the YA site. In some embodiments, one or more YA modifications are located distal to the YA site.

[0207] In some embodiments, an sgRNA or short sgRNA is provided that includes an upper stem modification, the upper stem modification comprising a 2'-O-Me modified nucleotide. In some embodiments, an sgRNA or short sgRNA is provided that includes an upper stem modification, the upper stem modification comprising a 2'-O-moe modified nucleotide. In some embodiments, an sgRNA or short sgRNA is provided that includes an upper stem modification, the upper stem modification comprising a 2'-F modified nucleotide.

[0208] In some embodiments, sgRNAs or short sgRNAs containing upper stem modifications are provided, where the upper stem modifications include 2'-O-Me modified nucleotides, 2'-O-moe modified nucleotides, 2'-F modified nucleotides, and / or combinations thereof.

[0209] In some embodiments, the sgRNA or short sgRNA includes an upper stem modification as shown in any one of the sequences in Table 1. In some embodiments, such an upper stem modification is combined with a 5' protective end modification, as shown for the corresponding sequence in Table 1, for example. In some embodiments, such an upper stem modification is combined with a 3' protective end modification, as shown for the corresponding sequence in Table 1, for example. In some embodiments, such an upper stem modification is combined with a 5' and a 3' end modification, as shown for the corresponding sequence in Table 1.

[0210] In some embodiments, the sgRNA or short sgRNA includes a 5' end modification and an upper stem modification. In some embodiments, the sgRNA or short sgRNA includes a 3' end modification and an upper stem modification. In some embodiments, the sgRNA or short sgRNA includes a 5' end modification, a 3' end modification, and an upper stem modification.

[0211] In some embodiments, the sgRNA or short sgRNA includes modifications in the hairpin region. In some embodiments, the hairpin region modification is located at hairpin 1. In some embodiments, the hairpin region modification is located at hairpin 2. In some embodiments, the modifications are located at hairpins 1 and 2, and optionally, the "n" between hairpins 1 and 2 is also modified. In some embodiments, the hairpin region modification includes at least one modified nucleotide selected from 2'H modified nucleotides (DNA), PS modified nucleotides, YA modified nucleotides, 2'-O-methyl (2'-O-Me) modified nucleotides, 2'-fluoro (2'-F) modified nucleotides, and / or combinations thereof.

[0212] In some embodiments, an sgRNA or short sgRNA containing a hairpin modification is provided, where the hairpin modification comprises one, two, or three YA modifications at the YA site. In some embodiments, an sgRNA or short sgRNA containing a hairpin modification is provided, where the hairpin modification comprises at least one, two, three, four, five, or six YA modifications. In some embodiments, an sgRNA or short sgRNA containing a hairpin modification is provided, where the hairpin modification comprises one YA modification. In some embodiments, an sgRNA or short sgRNA containing a hairpin modification is provided, where the hairpin modification comprises two YA modifications. In some embodiments, the hairpin modification comprises three YA modifications. In some embodiments, one or more YA modifications are located at the YA site. In some embodiments, one or more YA modifications are distal to the YA site.

[0213] In some embodiments, the modification includes or further includes a 2'-O-methyl (2'-O-Me) modified nucleotide.

[0214] In some embodiments, the hairpin modification includes or further includes a 2'-fluoro(2'-F) modified nucleotide.

[0215] In some embodiments, the sgRNA or short sgRNA includes modifications at the 3' end and modifications in the hairpin region.

[0216] In some embodiments, the sgRNA or short sgRNA includes modifications at the 5' end and modifications in the hairpin region.

[0217] In some embodiments, the sgRNA or short sgRNA includes modifications in the upper stem and the hairpin region.

[0218] In some embodiments, the sgRNA or short sgRNA includes a hairpin modification shown in any one of the sequences in Table 1. In some embodiments, such a hairpin modification is combined with a 5' end modification shown for the corresponding sequence in Table 1. In some embodiments, such a hairpin modification is combined with a 3' end modification shown for the corresponding sequence in Table 1. In some embodiments, such a hairpin modification is combined with both a 5' end modification and a 3' end modification shown for the corresponding sequence in Table 1.

[0219] In some embodiments, the sgRNA or short sgRNA includes 3' terminal modifications, modifications in the hairpin region, upper stem modifications, and 5' terminal modifications.

[0220] In some embodiments, an sgRNA or short sgRNA having one or more modifications of the YA site is, for example, a short sgRNA described herein that has at least 5 to 10 missing nucleotide hairpin regions relative to the hairpin region defined herein or shown in Table 2. Such an sgRNA may have any of the features described herein with respect to sgRNA, for example, in the sections on the summary and detailed description of short sgRNAs above.

[0221] Exemplary modified sgRNA In some embodiments, the sgRNAs described herein include or consist of any of the sequences shown in Table 1. Furthermore, sgRNAs containing any modification of any of the sequences shown in Table 1 and identified by sequence number in Table 1 are included. That is, the nucleotides may be identical or different, but the modification pattern shown may be identical or similar to the modification pattern of the guide sequences in Table 1. The modification pattern includes the relative position and identity of the modifications in the sgRNA (e.g., 5' terminal region, lower stem region, bulge region, upper stem region, nexus region, hairpin 1 region, hairpin 2 region, 3' tail region).

[0222] In some embodiments, the modification pattern includes at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% of any one modification of the sequences shown in the columns of sequences in Table 1, or across at least one or more regions of the sequences. In some embodiments, the modification pattern is at least 50%, about 55%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, and about 99% identical to any one modification pattern of the sequences shown in the columns of sequences in Table 1. In some embodiments, the modification pattern is at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% identical across one, two, three, four, five, six, seven, or eight regions of the array shown in Table 1, e.g., the 5' terminal region, the lower stem region, the bulge region, the upper stem region, the nexus region, the hairpin 1 region, the hairpin 2 region, and / or the 3' terminal region.

[0223] For example, in some embodiments, sgRNAs are included in which the modification pattern is at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% identical to the modification pattern of the sequence across the 5' terminal region. In some embodiments, sgRNAs are included in which the modification pattern is at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% identical across the lower stem. In some embodiments, sgRNAs are included in which the modification pattern is at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% identical across the bulge. In some embodiments, sgRNAs are included in which the modification pattern is identical for at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% across the upper stem. In some embodiments, sgRNAs are included in which the modification pattern is identical for at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% across the nexus. In some embodiments, sgRNAs are included in which the modification pattern is identical for at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% across hairpin 1. In some embodiments, sgRNAs are included in which the modification pattern is identical by at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% across hairpin 2. In some embodiments, sgRNAs are included in which the modification pattern is identical by at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% across the 3' end. In some embodiments, the modification pattern differs from the modification pattern of the sequence in Table 1 or a region of such a sequence (e.g., 5' end, lower stem, bulge, upper stem, nexus, hairpin 1, hairpin 2, 3' end) at 0, 1, 2, 3, 4, 5, or 6 nucleotides.In some embodiments, the sgRNA includes modifications at nucleotides 0, 1, 2, 3, 4, 5, or 6 that differ from the modifications of the sequence shown in Table 1. In some embodiments, the sgRNA includes modifications at nucleotides 0, 1, 2, 3, 4, 5, or 6 that differ from the modifications of the sequence region shown in Table 1 (e.g., 5' end, lower stem, bulge, upper stem, nexus, hairpin 1, hairpin 2, 3' end).

[0224] In some embodiments, the modification includes a 2'-O-methyl (2'-O-Me) modified nucleotide. In some embodiments, the modification includes a 2'-O-(2-methoxyethyl) (2'-O-moe) modified nucleotide. In some embodiments, the modification includes a 2'-fluoro (2'-F) modified nucleotide. In some embodiments, the modification includes an internucleotide phosphorothioate (PS) bond. In some embodiments, the sgRNA includes YA modification.

[0225] In some embodiments, the sgRNA comprises a 5'-terminal modification, a 3'-terminal modification, or both a 5'-terminal modification and a 3'-terminal modification, and further comprises a YA modification. In some embodiments, the 5'-terminal modification comprises a protective terminal modification. In some embodiments, the 5'-terminal modification comprises an internucleotide phosphorothioate (PS) bond. In some embodiments, the 5'-terminal modification comprises a 2'-O-methyl (2'-O-Me), 2'-O-(2-methoxyethyl) (2'-O-moe), and / or a 2'-fluoro (2'-F) modified nucleotide. In some embodiments, the 5'-terminal modification comprises at least one phosphorothioate (PS) bond, and one or more of the 2'-O-methyl (2'-O-Me), 2'-O-(2-methoxyethyl) (2'-O-moe), and / or a 2'-fluoro (2'-F) modified nucleotide. Terminal modifications may include phosphorothioate (PS), 2'-O-methyl (2'-O-Me), 2'-O-(2-methoxyethyl) (2'-O-moe), and / or 2'-fluoro (2'-F) modifications. Equivalent terminal modifications are also encompassed by the embodiments described herein. In some embodiments, the sgRNA includes terminal modifications in combination with modifications in one or more regions of the sgRNA.

[0226] This includes modified sgRNAs that include combinations of the 5' end modification, 3' end modification, upper stem modification, hairpin modification, and 3' end modification described above. Exemplary modified sgRNAs are listed below.

[0227] In some embodiments, sgRNAs are provided that include or consist of one of the sequences described in SEQ ID NOs: 401-535, 601, 607-732, 801, 807-932, 1001, or 1007-1132.

[0228] In some embodiments, an sgRNA is provided containing one of the modified sequences of SEQ ID NOs. 601 or 607–732, the sgRNA further comprising a guide region complementary to the target sequence, which guides Cas9 to its target for cleavage. In some cases, an sgRNA is provided containing a nucleic acid having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, or 70% identity with one of the nucleic acids of SEQ ID NOs. 401–535, 601, 607–732, 801, 807–932, 1001, or 1007–1132, the modification pattern being identical to the modification patterns shown in the reference sequence identifiers in Table 1. In some embodiments, the sgRNA further comprising three phosphorothioate (PS) bonds binding the first four nucleotides at the 5' end and three PS bonds binding the last four nucleotides at the 3' end.

[0229] In some embodiments, the sgRNA includes modifications on one, two, three, or four of the first four nucleotides at the 5' end. In some embodiments, the first three or four nucleotides at the 5' end and the last three or four nucleotides at the 3' end are modified. In some embodiments, the first four nucleotides at the 5' end and the last four nucleotides at the 3' end are linked by phosphorothioate (PS) bonds. In some embodiments, the modification includes 2'-O-Me. In some embodiments, the modification includes 2'-F. In some embodiments, the modification includes 2'-O-moe.

[0230] In some embodiments, the sgRNA includes modifications on one, two, three, or four of the first four nucleotides at the 5' end, if the nucleotides mentioned are present in the sgRNA. In some embodiments, the sgRNA includes modifications on one, two, three, or four of the last four nucleotides at the 3' end (the 3' tail or conserved portion of the sgRNA). In some embodiments, the first four nucleotides at the 5' end and the last four nucleotides at the 3' end are joined by PS bonds, and the first three nucleotides at the 5' end and the last three nucleotides at the 3' end include 2'-O-Me or 2'-O-moe modifications.

[0231] In some embodiments, the first four nucleotides at the 5' end and the last four nucleotides at the 3' end are linked by PS bonds, and the first three nucleotides at the 5' end and the last three nucleotides at the 3' end contain 2'-F modifications.

[0232] In some embodiments, if the nucleotides mentioned are present in the sgRNA, the sgRNA is provided and LS1, LS6, LS7, LS8, LS11, and LS12 are modified with 2'-O-Me. In some embodiments, each of the nucleotides in the bulge region of the sgRNA is modified with 2'-O-Me. In some embodiments, each of the nucleotides in the upper stem region of the sgRNA is modified with 2'-O-Me. In some embodiments, N16, N17, and N18 in the nexus region of the sgRNA are modified with 2'-O-Me. In some embodiments, each of the nucleotides in the hairpin 1 region of the sgRNA is modified with 2'-O-Me. In some embodiments, each of the nucleotides in the hairpin 2 region of the sgRNA is modified with 2'-O-Me.

[0233] In some embodiments, the sgRNA includes 2'-O-Me modified nucleotides in the following nucleotides: the first three nucleotides at the 5' end, LS1, LS6, LS7, LS8, LS11, and LS12; B1 and B2 in the bulge region; each of the nucleotides in the upper stem region of the sgRNA; N16, N17, and N18 in the nexus region; each of the nucleotides in the hairpin 1 region; each of the nucleotides in the hairpin 2 region; and the last four nucleotides at the 3' end.

[0234] In some embodiments, the sgRNA further includes three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end and three PS bonds connecting the last four nucleotides at the 3' end. In some embodiments, the sgRNA further includes 2'-O-Me or 2'-F modified nucleic acids in the first three nucleotides at the 5' end and 2'-O-Me or 2'-F modified nucleic acids in the last four nucleotides at the 3' end. In some embodiments, LS9 and LS10 are modified with 2'-F. In some embodiments, N15, N16, N17, and N18 are modified with 2'-F. In some embodiments, H2-9, H2-10, H2-11, H2-12, H2-13, HS-14, and H2-15 are modified with 2'-F. In some embodiments, the second to last, third to last, and fourth to last nucleotides at the 3' end are modified with 2'-F.

[0235] In some embodiments, an sgRNA is provided comprising 2'-F modified nucleic acids at the following nucleotides: LS9 and LS10 in the lower stem region, N15, N16, N17, and N18 in the nexus region, and H2-9, H2-10, H2-11, H2-12, H2-13, HS-14, and H2-15 in the hairpin 2 region. In some embodiments, the sgRNA further comprises 2'-F modified nucleotides at the second to last, third to last, and fourth to last nucleotides at the 3' end. In some embodiments, the sgRNA further comprises three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end, and three PS bonds connecting the last four nucleotides at the 3' end. In some embodiments, the sgRNA further comprises 2'-O-Me or 2'-F modified nucleic acids in the first three nucleotides at the 5' end, and 2'-O-Me or 2'-F modified nucleic acids in three of the last four nucleotides at the 3' end.

[0236] In some embodiments, an sgRNA is provided comprising 2'-O-Me modified nucleotides in the first three nucleotides at the 5' end, 2'-O-Me modified nucleotides at LS1 and LS6, 2'-O-Me modified nucleotides at US1 to US12, 2'-O-Me modified nucleotides at H1-1 to H1-12, 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2, 2'-O-Me modified nucleotides at H2-1 to H2-15, and 2'-O-Me modified nucleotides in the last four nucleotides at the 3' end. In some embodiments, the sgRNA further comprises three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end, and three PS bonds connecting the last four nucleotides at the 3' end.

[0237] In some embodiments, an sgRNA is provided comprising 2'-O-Me modified nucleotides in the first three nucleotides at the 5' end, 2'-F modified nucleotides in LS1-LS6, 2'-O-Me modified nucleotides in US1-US12, 2'-O-Me modified nucleotides in H1-1-H1-12, 2'-O-Me modified nucleotides in "n" between hairpin 1 and hairpin 2, 2'-O-Me modified nucleotides in H2-1-H2-15, and 2'-O-Me modified nucleotides in the last four nucleotides at the 3' end. In some embodiments, the sgRNA further comprises three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end, and three PS bonds connecting the last four nucleotides at the 3' end.

[0238] In some embodiments, an sgRNA is provided comprising 2'-O-Me modified nucleotides in the first three nucleotides at the 5' end, 2'-F modified nucleotides in LS2-LS5, 2'-O-Me modified nucleotides in LS1 and LS6, 2'-O-Me modified nucleotides in US1-US12, 2'-O-Me modified nucleotides in H1-1-H1-12, 2'-O-Me modified nucleotides in "n" between hairpin 1 and hairpin 2, 2'-O-Me modified nucleotides in H2-1-H2-15, and 2'-O-Me modified nucleotides in the last four nucleotides at the 3' end. In some embodiments, the sgRNA further comprises three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end and three PS bonds connecting the last four nucleotides at the 3' end.

[0239] In some embodiments, an sgRNA is provided comprising 2'-O-Me modified nucleotides in the first three nucleotides at the 5' end, 2'-O-Me modified nucleotides in US1-US12, 2'-O-Me modified nucleotides in LS7, LS8, LS11, and LS12, 2'-O-Me modified nucleotides in H1-1-H1-12, 2'-O-Me modified nucleotides in "n" between hairpin 1 and hairpin 2, 2'-O-Me modified nucleotides in H2-1-H2-15, and 2'-O-Me modified nucleotides in the last four nucleotides at the 3' end. In some embodiments, the sgRNA further comprises three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end, and three PS bonds connecting the last four nucleotides at the 3' end.

[0240] In some embodiments, an sgRNA is provided comprising 2'-O-Me modified nucleotides in the first three nucleotides at the 5' end, 2'-O-Me modified nucleotides in US1-US12, 2'-O-Me modified nucleotides in LS8, LS10, and LS12, 2'-OF modified nucleotides in LS7, LS9, and LS11, 2'-O-Me modified nucleotides in H1-1-H1-12, 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2, 2'-O-Me modified nucleotides in H2-1-H2-15, and 2'-O-Me modified nucleotides in the last four nucleotides at the 3' end. In some embodiments, the sgRNA further comprises three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end and three PS bonds connecting the last four nucleotides at the 3' end.

[0241] In some embodiments, an sgRNA is provided comprising 2'-O-Me modified nucleotides in the first three nucleotides at the 5' end, 2'-O-Me modified nucleotides in LS1, LS6, LS7, LS8, LS11, and LS12, 2'-O-Me modified nucleotides in US1 to US12, 2'-O-Me modified nucleotides in H1-1 to H1-12, 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2, 2'-O-Me modified nucleotides in H2-1 to H2-15, and 2'-O-Me modified nucleotides in the last four nucleotides at the 3' end. In some embodiments, the sgRNA further comprises three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end and three PS bonds connecting the last four nucleotides at the 3' end.

[0242] In some embodiments, an sgRNA is provided comprising 2'-O-Me modified nucleotides in the first three nucleotides at the 5' end, 2'-O-Me modified nucleotides in LS1, LS6, LS7, LS8, LS11, and LS12, 2'-F modified nucleotides in LS9 and LS10, 2'-O-Me modified nucleotides in US1 to US12, 2'-O-Me modified nucleotides in H1-1 to H1-12, 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2, 2'-O-Me modified nucleotides in H2-1 to H2-15, and 2'-O-Me modified nucleotides in the last four nucleotides at the 3' end. In some embodiments, the sgRNA further comprises three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end and three PS bonds connecting the last four nucleotides at the 3' end.

[0243] In some embodiments, an sgRNA is provided comprising 2'-O-Me modified nucleotides in the first three nucleotides at the 5' end, 2'-O-Me modified nucleotides in US1-US12, 2'-O-Me modified nucleotides in H1-1-H1-12, 2'-O-Me modified nucleotides between hairpin 1 and hairpin 2, 2'-O-Me modified nucleotides in H2-1-H2-8, 2'-F modified nucleotides in H2-9-H2-15, 2'-F modified nucleotides in the second to last, third to last, and fourth to last nucleotides at the 3' end, and a 2'-O-Me modified nucleotide in the last nucleotide at the 3' end. In some embodiments, the sgRNA further comprises three phosphorothioate (PS) bonds connecting the first four nucleotides at the 5' end, and three PS bonds connecting the last four nucleotides at the 3' ...

Claims

1. A guide RNA (gRNA) which is a short single guide RNA (short sgRNA) comprising a guide region and a conserved portion of sgRNA, wherein the conserved portion comprises a lower stem region which is nucleotides 1-6 and 25-30 of SEQ ID NO: 400, an upper stem region which is nucleotides 9-20 of SEQ ID NO: 400, a bulge region which is nucleotides 7-8 and 21-24 of SEQ ID NO: 400, a nexus region which is nucleotides 31-48 of SEQ ID NO: 400, and a hairpin region which is a sequence lacking 5-10 nucleotides from nucleotides 49-76 of SEQ ID NO: 400, and the short sgRNA comprises a 5'-terminal modification or a 3'-terminal modification, and further comprises 2'-O-methyl (2'-OMe) modified nucleotides in at least 10 nucleotides in the upper stem region.

2. The gRNA according to claim 1, wherein the short sgRNA includes a 5' terminal modification.

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

4. The gRNA according to claim 1, wherein the short sgRNA includes a 5' terminal modification and a 3' terminal modification.

5. The gRNA according to any one of claims 1 to 4, wherein the short sgRNA includes a 3' tail.

6. (i) Modification in the hairpin region, (ii) 3' terminal modification and modification in the hairpin region, (iii) 3'-terminal modification, modification in the hairpin region, and 5'-terminal modification, (iv) 5' terminal modification and modification in the hairpin region, Or, a combination of two or more of (i) to (iv). gRNA according to any one of claims 1 to 5, comprising:

7. The hairpin region includes hairpin 1, which is nucleotides 49-60 of SEQ ID NO: 400; hairpin 2, which is nucleotides 62-76 of SEQ ID NO: 400; and nucleotide N between hairpin 1 and hairpin 2 (where "N" is nucleotide 61 of SEQ ID NO: 400). The aforementioned 5 to 10 missing nucleotides are i. Inside hairpin 1, ii. Within the "N" between hairpin 1 and hairpin 1 and hairpin 2, iii. Within the two nucleotides of hairpin 1 and the 3' side immediately to the side of hairpin 1, or iv. Including at least a portion of hairpin 1, The gRNA according to any one of claims 1 to 6.

8. (i) The modification in the hairpin region includes 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'-fluoro(2'-F) modified nucleotide, (iv) The 3' or 5' terminal modification comprises or further comprises an internucleotide phosphorothioate (PS) bond, (v) The 3' or 5' terminal modification comprises or further comprises an inverted debasicized nucleotide, (vi) The modification in the hairpin region comprises or further comprises a 2'-O-methyl (2'-OMe) modified nucleotide. (vii) The modification in the hairpin region comprises or further comprises a 2'-fluoro(2'-F) modified nucleotide. Alternatively, a combination of two or more of (i) to (vii), The gRNA according to any one of claims 1 to 7.

9. The gRNA according to any one of claims 1 to 8, wherein the 5 to 10 nucleotides include 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 includes modifications at least in nucleotides 49 to 53 and 62 to 73 of SEQ ID NO:

400.

10. Table 1 includes any of the chemical modification patterns of sequence numbers 206, 207, 215, 216, 223, 224, 228, 231, 241, 242, 245-248, 250, 251, 253, 254, 306, 307, 315, 316, 323, 324, 328, 331, 341, 342, 345-348, 350, 351, 353, or 354, or The chemical modification pattern includes one of the chemical modification patterns shown in Table 1, which has at least 90% identity with any one of the following: SEQ ID NOs: 206, 207, 215, 216, 223, 224, 228, 231, 241, 242, 245-248, 250, 251, 253, 254, 306, 307, 315, 316, 323, 324, 328, 331, 341, 342, 345-348, 350, 351, 353, or 354. The gRNA according to claim 1.

11. The guide region comprises at least 20 nucleotides and is one of the following sequence numbers shown in Table 1: SEQ ID NOs: 1-54, 301-354, 401-535, 1001, 1007-1132, 1205-1212, 1322-1406, 1417-1501, 1511-1596, 3018-3059, 3063-3104, 3108-3149, 3153-3194, 3198-3239, 32 The gRNA according to any one of claims 1 to 10, comprising modified and / or unmodified nucleotides at least 15, 16, 17, 18, 19, or 20 of nucleotides 1 to 20, which match the modification pattern at nucleotides 1 to 20 of any one gRNA from 43 to 3284, 3295 to 3341, 3343 to 3385, or 3388 to 3430.

12. The gRNA according to any one of claims 1 to 11, wherein the conserved portion of the gRNA includes a modified sequence having at least 90% identity with any one of sequence numbers 206, 207, 215, 216, 223, 224, 228, 231, 241, 242, 245-248, 250, 251, 253, or 254 shown in Table 1.

13. The gRNA according to any one of claims 1 to 12, wherein the gRNA is a short sgRNA having a chemical modification pattern that is at least 99, 98, 97, 96, 95, or 90% identical to the sequence of sequence number 206 or 306 shown in Table 1.

14. The gRNA according to any one of claims 1 to 13, wherein the gRNA is a short sgRNA containing the chemical modification pattern of SEQ ID NO: 206 or 306 as shown in Table 1.

15. An LNP composition comprising the gRNA described in any one of claims 1 to 14.

16. A composition comprising the gRNA according to any one of claims 1 to 14, further comprising lipid nanoparticles (LNPs).

17. A composition comprising a gRNA according to any one of claims 1 to 14 or the composition according to claim 15 or 16, further comprising a nuclease or mRNA encoding a nuclease, wherein the nuclease is S. pyogenes Cas9.

18. The composition according to claim 17, wherein the nuclease is nicasse or dCas.

19. The composition according to claim 17 or 18, wherein the mRNA comprises one of the sequences 3499 to 3527 or 3529 to 3546.

20. A pharmaceutical formulation comprising gRNA according to any one of claims 1 to 14 or a composition according to any one of claims 15 to 19, and a pharmaceutically acceptable carrier.

21. A method for modifying target DNA in vitro or ex vivo, (a) S. pyogenes Cas9 protein or nucleic acid encoding S. pyogenes Cas9 protein, i. The gRNA according to any one of claims 1 to 14, ii. The composition according to claim 15 or 16, and iii. A pharmaceutical formulation comprising the gRNA according to any one of claims 1 to 14 or the composition according to claim 15 or 16, and a pharmaceutically acceptable carrier. One or more of the following and To deliver to cells, or (b) i. The composition according to any one of claims 17 to 19, and ii. A pharmaceutical formulation comprising the composition according to any one of claims 17 to 19 and a pharmaceutically acceptable carrier. Delivering one or more of the following to the cells The method, including the method described above.

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 or 22, further comprising delivering a template to the cells, wherein at least a portion of the template is incorporated into target DNA at or near a double-strand break site induced by the Cas9 protein.

24. An agent for modifying target DNA, comprising S. pyogenes Cas9 protein or a nucleic acid encoding S. pyogenes Cas9 protein and the gRNA described in any one of claims 1 to 14.

25. The agent according to claim 24, wherein the agent causes an insertion or deletion in a gene.

26. The agent according to claim 24 or 25, further comprising a template, wherein at least a portion of the template is incorporated into target DNA at or near a double-strand break site induced by the Cas9 protein.

27. A gRNA according to any one of claims 1 to 14, a composition according to claims 15 to 19, or a pharmaceutical preparation according to claim 20, for use in the preparation of a pharmaceutical for treating a disease or disorder.

28. A gRNA according to any one of claims 1 to 14, a composition according to any one of claims 15 to 19, or a pharmaceutical formulation according to claim 20, for use in modifying target DNA.

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