Modified guide RNA

Abstract

Since RNA can be degraded in cells and serum by endonuclease or exonuclease cleavage, this invention provides improved methods and compositions to prevent such degradation, improve gRNA stability, and enhance gene editing efficiency. [Solution] A single guide RNA (sgRNA) is provided, comprising a 5' terminal modification and one or more modifications in one or more of a. upper stem region, b. hairpin 1 region, and c. hairpin 2 region, wherein the 5' terminal modification includes at least two phosphorothioate (PS) ligations in the first seven nucleotides at the 5' end of the 5' terminal.

Claims

1. A single guide RNA (sgRNA) with 5' terminal modification, a. Upper stem region, b. Hairpin 1 region, and c. Including one or more modifications in one or more of the hairpin 2 regions, The aforementioned 5' terminal modification is found in small amounts among the first seven nucleotides at the 5' end of the 5' terminal. sgRNA containing at least two phosphorothioate (PS) ligatures.

2. At least one modification involves a 2'-O-methyl (2'-O-Me) modified nucleotide. , the sgRNA according to claim 1.

3. Claims that at least one modification comprises a 2'-fluoro(2'-F) modified nucleotide. sgRNA according to claim 1 or claim 2.

4. If at least one modification involves a phosphorothioate (PS) bond between nucleotides, The sgRNA described in any one of the requests 1 to 3.

5. The upper stem region includes one or more modifications, according to any one of claims 1 to 4. The sgRNA described.

6. The sgRNA according to claim 5, comprising modifications in US1 to US12.

7. The hairpin region includes one or more modifications, according to any one of claims 1 to 6. The sgRNA described.

8. The sgRNA according to claim 7, comprising modification in H1-1.

9. The hairpin 2 region includes one or more modifications, according to any one of claims 1 to 8. The sgRNA described.

10. The sgRNA according to claim 9, comprising modification in H2-1.

11. The s according to any one of claims 1 to 10, including modifications in H1-1 to H1-12. gRNA.

12. The s according to any one of claims 1 to 11, including modifications in H2-1 to H2-15. gRNA.

13. Within each of the upper stem region, the hairpin 1 region, and the hairpin 2 region The sgRNA according to any one of claims 1 to 12, comprising one or more modifications.

14. Claims 1 to 13, wherein a modified nucleotide is included between hairpin region 1 and hairpin region 2. The sgRNA listed in any one of the items.

15. The sg according to any one of claims 1 to 14, further comprising a lower stem region including modification. RNA.

16. The sgR according to any one of claims 1 to 15 further comprises a 3' terminal region including modifications. NA.

17. The sgRNA according to claim 16, further comprising a 3' terminal modification in the 3' terminal.

18. At least two of the last four nucleotides at the 3' end of the 3' terminus The sgRNA according to claim 17, wherein the two parts are modified.

19. At least two of the last four nucleotides at the 3' end of the 3' terminus Claim 17, wherein each is modified with 2'-O-Me, 2'-F, or 2'-O-moe. The sgRNA described above.

20. One or multiple of the last four nucleotides at the 3' end of the 3' terminus Any one of claims 17 to 19, further comprising phosphorothioate (PS) bonds between the numbers. The sgRNA described in the section.

21. sgRN according to any one of claims 1 to 20, further comprising a bulge region including modification A.

22. The sgR according to any one of claims 1 to 21 further comprises a nexus region including modifications. NA.

23. At least the first three nucleotides at the 5' end of the 5' terminus, and The last three nucleotides at the 3' end of the 3' terminus are modified, claim The sgRNA described in any one of items 1 to 22.

24. The first four nucleotides at the 5' end of the 5' terminus, and the 3' terminus. The last four nucleotides at the 3' end are linked by a phosphorothioate (PS) bond. The sgRNA according to any one of claims 1 to 23, which is more ligated.

25. The sgRNA according to claim 24, wherein the terminal modification includes 2'-O-Me.

26. The sgRNA according to claim 24, wherein the terminal modification includes 2'-F.

27. The first four nucleotides at the 5' end of the 5' terminus and the 3' terminus The last four nucleotides at the 3' end are linked by PS bonds, and The first three nucleotides at the 5' end of the 5' terminus and the nucleotides before the 3' terminus Claims 1 to 1, wherein the last three nucleotides at the 3' end contain a 2'-O-Me modification. The sgRNA described in any one of item 26.

28. The first four nucleotides at the 5' terminus and the last four nucleotides at the 3' terminus The nucleotides are linked by PS bonds, and the first three at the 5' terminus The nucleotides and the last three nucleotides at the 3' terminus are 2'-O-M Any of claims 1 to 26, comprising the modifications e, 2'-F, and / or 2'-O-moe. The sgRNA described in item 1.

29. LS1, LS6, LS7, LS8, LS11, and / or LS12 are 2'-O-M The sgRNA according to any one of claims 1 to 28, modified with e.

30. Each of the nucleotides in the bulge region is modified with 2'-O-Me. The sgRNA according to any one of claims 1 to 29.

31. At least 50% of the nucleotides in the bulge region are modified with 2'-O-Me. The sgRNA according to any one of claims 1 to 29.

32. Each of the nucleotides in the upper stem region is modified with 2'-O-Me. The sgRNA according to any one of claims 1 to 31.

33. N16, N17, and / or N18 in the aforementioned nexus region are modified with 2'-O-Me. The sgRNA according to any one of claims 1 to 32.

34. N15, N16, N17, and / or N18 in the nexus region are modified. The sgRNA according to any one of claims 1 to 32.

35. The modification in the nexus region is selected from 2'-O-Me and 2'F, according to the claim. sgRNA according to claim 33 or claim 34.

36. N16, N17, and N18 are connected by a PS bond, any of claims 32 to 35 The sgRNA described in item 1.

37. Each of the nucleotides in the hairpin 1 region is modified with 2'-O-Me. The sgRNA according to any one of claims 1 to 36.

38. Each of the nucleotides in the hairpin 2 region is modified with 2'-O-Me. The sgRNA according to any one of claims 1 to 37.

39. A single guide RNA (sgRNA) having a 2'-O-Me modified nucleotide Lower position: a. The first three nucleotides at the 5' end of the 5' terminus, b. LS1, LS6, LS7, LS8, LS11, and / or in the lower stem region LS12 and, c. B1 and / or B2 in the bulge region; d. Each nucleotide in the upper stem region, e. N16, N17, and / or N18 in the nexus region, f. Each nucleotide in hairpin region 1, g. Each nucleotide in the hairpin 2 region, h. The last four nucleotides at the 3' terminus and the sgRNA contained therein.

40. The sgRNA according to claim 39, wherein B3 to B6 are modified with 2'-O-Me.

41. The three nucleotides that link the first four nucleotides at the 5' end of the 5' terminus Holothioate (PS) bond and the last four nu at the 3' end of the 3' terminus The sgRNA according to claim 39, further comprising three PS bonds that link the creotide.

42. LS9 and LS10 are modified, for example, with 2'-F or 2'-OMe, The sgRNA according to any one of claims 1 to 41.

43. N15, N16, N17, and N18 are corrected, for example, with 2'-F or 2'-OMe. An sgRNA according to any one of claims 1 to 42, which is decorated.

44. H2-9, H2-10, H2-11, H2-12, H2-13, H2-14, and H sgRN according to any one of claims 1 to 43, wherein 2-15 is modified with 2'-F A.

45. The second to last, third to last, and fourth to last nuclei at the 3' terminus sgRN according to any one of claims 1 to 44, wherein the rheotide is modified with 2'-F A.

46. A single guide RNA (sgRNA) in which a 2'-F modified nucleotide is located at the following position Place: a. LS9 and LS10 in the lower stem region, b. N15, N16, N17, and N18 in the nexus region, c. H2-9, H2-10, H2-11, H2-12, H2-13 in hairpin region 2 sgRNA, which is contained in H2-14 and H2-15.

47. The second to last, third to last, and fourth to last nucleos at the 3' terminus. The sgRNA according to claim 46, further comprising a 2'-F modified nucleotide in the tide.

48. The three nucleotides that link the first four nucleotides at the 5' end of the 5' terminus Holothioate (PS) bond and the last four nu at the 3' end of the 3' terminus The claim 46 or 47 further comprises three PS bonds linking the creotide. sgRNA.

49. In the first three nucleotides at the 5' end of the 5' terminus, 2'-O-M Further comprising e or 2'-F modified nucleotide, and the 3' terminus of the 3' terminus In the last four nucleotides, three of them are modified with 2'-O-Me or 2'-F nucleotides. The sgRNA according to any one of claims 46 to 48, further comprising ocide.

50. It is a single guide RNA (sgRNA), a. 2'-O-Me modification in the first three nucleotides at the 5' end of the 5' terminus Cleotide and, b. 2'-O-Me modified nucleotides in US1 to US12, c. 2'-O-Me modified nucleotides in H1-1 to H1-12, d. 2'-O-Me modified nucleotides in H2-1 to H2-15, e. 2'-O-Me modification at the last four nucleotides at the 3' end of the 3' terminus sgRNA containing decorative nucleotides.

51. LS1 and / or LS6 further comprises a 2'-O-Me modified nucleotide, The sgRNA according to claim 50.

52. A claim further comprising a 2'-O-Me modified nucleotide between hairpin 1 and hairpin 2. The sgRNA according to item 50 or claim 51.

53. It is a single guide RNA (sgRNA), a. 2'-O-Me repair in the first three nucleotides at the 5' end of the 5' terminus Decorative nucleotides, b. 2'-F modified nucleotides in LS1 to LS6, c. 2'-O-Me modified nucleotides in US1 to US12, d. 2'-O-Me modified nucleotides in H1-1 to H1-12, e. The 2'-O-Me modified nucleotide between hairpin 1 and hairpin 2, f. 2'-O-Me modified nucleotides in H2-1 to H2-15, g. 2'-O-Me modification at the last four nucleotides at the 3' end of the 3' terminus sgRNA containing decorative nucleotides.

54. It is a single guide RNA (sgRNA), a. 2'-O-Me repair in the first three nucleotides at the 5' end of the 5' terminus Decorative nucleotides, b. 2'-F modified nucleotides in LS2 to LS5, c. 2'-O-Me modified nucleotides in LS1 and LS6, d. 2'-O-Me modified nucleotides in US1 to US12, e. 2'-O-Me modified nucleotides in H1-1 to H1-12, f. The 2'-O-Me modified nucleotide between hairpin 1 and hairpin 2, g. 2'-O-Me modified nucleotides in H2-1 to H2-15, h. 2'-O-Me modification at the last four nucleotides at the 3' end of the 3' terminus. sgRNA containing decorative nucleotides.

55. It is a single guide RNA (sgRNA), a. 2'-O-Me repair in the first three nucleotides at the 5' end of the 5' terminus Decorative nucleotides, b. 2'-O-Me modified nucleotides in US1 to US12, c. 2'-O-Me modified nucleos in LS7, LS8, LS11, and LS12 Chido and, d. 2'-O-Me modified nucleotides in H1-1 to H1-12, e. The 2'-O-Me modified nucleotide between hairpin 1 and hairpin 2, f. 2'-O-Me modified nucleotides in H2-1 to H2-15, g. 2'-O-Me modification at the last four nucleotides at the 3' end of the 3' terminus sgRNA containing decorative nucleotides.

56. It is a single guide RNA (sgRNA), a. 2'-O-Me repair in the first three nucleotides at the 5' end of the 5' terminus Decorative nucleotides, b. 2'-O-Me modified nucleotides in US1 to US12, c. 2'-O-Me modified nucleos in LS7, LS8, LS11, and LS12 Chido and, d. 2'-F modified nucleotides in LS9 and LS10, e. 2'-O-Me modified nucleotides in H1-1 to H1-12, f. The 2'-O-Me modified nucleotide between hairpin 1 and hairpin 2, g. 2'-O-Me modified nucleotides in H2-1 to H2-15, h. 2'-O-Me modification at the last four nucleotides at the 3' end of the 3' terminus. sgRNA containing decorative nucleotides.

57. It is a single guide RNA (sgRNA), a. 2'-O-Me repair in the first three nucleotides at the 5' end of the 5' terminus Decorative nucleotides, b. 2'-O-Me modified nucleotides in US1 to US12, c. 2'-O-Me modified nucleotides in LS8, LS10, and LS12, d. 2'-O-F modified nucleotides in LS7, LS9, and LS11, e. 2'-O-Me modified nucleotides in H1-1 to H1-12, f. The 2'-O-Me modified nucleotide between hairpin 1 and hairpin 2, g. 2'-O-Me modified nucleotides in H2-1 to H2-15, h. 2'-O-Me modification at the last four nucleotides at the 3' end of the 3' terminus. sgRNA containing decorative nucleotides.

58. It is a single guide RNA (sgRNA), a. 2'-O-Me repair in the first three nucleotides at the 5' end of the 5' terminus Decorative nucleotides, b. 2'-O- in LS1, LS6, LS7, LS8, LS11, and LS12 Me-modified nucleotides and c. 2'-O-Me modified nucleotides in US1 to US12, d. 2'-O-Me modified nucleotides in H1-1 to H1-12, e. The 2'-O-Me modified nucleotide between hairpin 1 and hairpin 2, f. 2'-O-Me modified nucleotides in H2-1 to H2-15, g. 2'-O-Me modification at the last four nucleotides at the 3' end of the 3' terminus sgRNA containing decorative nucleotides.

59. It is a single guide RNA (sgRNA), a. 2'-O-Me repair in the first three nucleotides at the 5' end of the 5' terminus Decorative nucleotides, b. 2'-O- in LS1, LS6, LS7, LS8, LS11, and LS12 Me-modified nucleotides and c. 2'-F modified nucleotides in LS9 and LS10, d. 2'-O-Me modified nucleotides in US1 to US12, e. 2'-O-Me modified nucleotides in H1-1 to H1-12, f. The 2'-O-Me modified nucleotide between hairpin 1 and hairpin 2, g. 2'-O-Me modified nucleotides in H2-1 to H2-15, h. 2'-O-Me modification at the last four nucleotides at the 3' end of the 3' terminus. sgRNA containing decorative nucleotides.

60. It is a single guide RNA (sgRNA), a. 2'-O-Me in the first three nucleotides at the 5' end of the 5' end Modified nucleotides and b. 2'-O-Me modified nucleotides in US1 to US12, c. 2'-O-Me modified nucleotides in H1-1 to H1-12, d. The 2'-O-Me modified nucleotide between hairpin 1 and hairpin 2, e. 2'-O-Me modified nucleotides in H2-1 to H2-8, f. 2'-F modified nucleotides in H2-9 to H2-15, g. The second to last, third to last, and fourth to last nuclei at the 3' terminus. 2'-F modified nucleotides in rheotides, h. 2'-O-Me modified nucleus at the last nucleotide at the 3' end of the 3' terminus sgRNA containing rheotide.

61. It is a single guide RNA (sgRNA), a. 2'-O-Me repair in the first three nucleotides at the 5' end of the 5' terminus Decorative nucleotides, b. 2'-O-Me modified nucleotides in US1 to US12, c. In H1-2, H1-4, H1-6, H1-8, H1-10, and H1-12 2'-O-Me modified nucleotides, d. In H1-1, H1-3, H1-5, H1-7, H1-9, and H1-11 2'-F modified nucleotides, e. The 2'-F modified nucleotide between hairpin 1 and hairpin 2, f. H2-2, H2-4, H2-6, H2-8, H2-10, H2-12; and H2 -14 2'-F modified nucleotide and, g. H2-1, H2-3, H2-5, H2-7, H2-9, H2-11; H2-13, and the 2'-O-Me modified nucleotide in H2-15, h. At the second to last and fourth to last nucleotides at the 3' terminus 2'-F modified nucleotides, i. At the third to last and last nucleotide at the 3' end of the 3' terminus sgRNA containing a 2'-O-Me modified nucleotide.

62. The three nucleotides that link the first four nucleotides at the 5' end of the 5' terminus Holothioate (PS) bond and the last four nu at the 3' end of the 3' terminus The present invention further comprises three PS bonds linking the creotide, according to any one of claims 50 to 61. The sgRNA described.

63. At least one of the first seven nucleotides at the 5' end of the 5' terminus The present invention further comprises a phosphorothioate (PS) bond, as described in any one of claims 50 to 61. sgRNA.

64. It is a single guide RNA (sgRNA), a. 2'-O-Me modified nucleotides LS8, LS10, LS12, H1-2, H1- 4, H1-6, H1-8, H1-10, H1-12, H2-1, H2-3, H2-5, H 2-7, H2-9, H2-11, H2-13, and H2-15, b. LS7, LS9, LS11; H1-1, H1-3, H1-5, H1-7, H1-9 , H1-11, H1-13, H2-2, H2-4, H2-6, H2-8, H2-10, H sgRNA containing 2'-F modified nucleotides in 2-12 and H2-14. 。

65. Three phosphorothiocyanates link the first four nucleotides at the 5' end of the 5' terminus. eth (PS) bond and the last four nucleotides at the 3' end of the 3' terminus The sgRNA according to claim 64, further comprising three PS bonds linking the dots.

66. a. At the 3' terminus, at the last and third-to-last nucleotides, 2 '-O-Me modified nucleotides, b. The second to last and fourth to last nucleotides at the 3' end of the 3' terminus The 2'-F modified nucleotide in the present, further comprising the present in claim 64 or claim 65. The sgRNA listed.

67. sgRNA containing any of sequence numbers 228-332, including the modifications shown in Table 4.

68. Table 4 includes the modified sequences 235-240, 265-285, and 309-329. sgRNA containing any of the following.

69. sgRNA containing sequence number 240.

70. sgRNA containing sequence number 240, which includes the modifications shown in Table 4.

71. sgRNA containing SEQ ID NO: 242, which includes the modifications shown in Table 4.

72. As depicted in Table 4, sgRNA containing the modification of sequence number 242.

73. sgRNA containing sequence number 358.

74. Any one of sequence numbers 235-240, 265-285, and 309-329 For each nucleic acid, at least 99, 98, 97, 96, 95, 94, 93, 92, 91, Containing nucleic acids with 90%, 85%, 80%, 75%, or 70% identity, as referenced in Table 4. The modifications at each nucleotide of the sgRNA corresponding to the nucleotide of the sequence identifier are shown in Table 4. sgRNA that is identical or equivalent to the modification shown in the reference sequence identifier in [the specified location].

75. The three phosphorothioates linking the first four nucleotides at the 5' terminus (PS) bond and three Ps linking the last four nucleotides at the 3' terminus The sgRNA according to any one of claims 66 to 74, further comprising an S bond.

75. Further, at least three PS bonds linking the nucleotides in the hairpin 1 region The sgRNA according to any one of claims 1 to 74, which is included in the above.

76. Further, at least three PS bonds linking the nucleotides in the hairpin 2 region The sgRNA according to any one of claims 1 to 75, which is included in the above.

77. Further, the at least three PS bonds linking the nucleotides in the upper stem region The sgRNA according to any one of claims 1 to 76, which is included in the above.

78. It forms a ribonucleoprotein complex with Cas9 of S. pyogenes. The sgRNA described in any one of the requests 1 to 77.

79. Guide RNA containing a 2'-O-Me modification at each nucleotide in the upper stem region. 。

80. The following nucleotides: US1, US2, US3, US4, US5, US6, US7, U S8, US9, US10, US11, and US12, specifically 1, 2, 3, or 4, 5 , 6, 7, 8, 9, 10, or 11 guide RNs containing 2'-O-Me modifications A.

81. 50% or more of the nucleotides in hairpin 1 region and hairpin 2 region A guide RNA containing a 2'-O-Me modification.

82. Each nucleotide in the upper stem region contains a 2'-O-Me modification, and is hairpin 50% or more of the nucleotides in region 1 and hairpin region 2 are 2' A guide RNA containing -O-Me modification.

83. Each nucleotide starting with H2-1 or H2-2 and ending at the 3' terminus A guide RNA containing or consisting of a 2'-O-Me modification.

84. LS1 and / or LS6 contain or consist of a 2'-O-Me modification, Furthermore, a guide RNA that does not contain modifications in LS2 to LS5.

85. Two or more of LS8, LS9, LS10, LS11, and / or LS12 A guide RNA containing more than 2'-O-Me modified nucleotides.

86. Claim 85, wherein at least LS8 and LS10 are modified with 2'-O-Me The included guide RNA.

87. 50% or more of the nucleotides in the nexus region are 2'-O-M The guide RNA according to any one of claims 79 to 86, further comprising e-modification.

88. At least 55%, 60%, 65%, and 70% of the nucleotides in the nexus region 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% The guide RNA according to claim 87, which is modified with 2'-O-Me.

89. Guy DRNA.

90. 2'-O-Me modification at four or more nucleotides in the nexus region A guide RNA containing this RNA.

91. At least one, two, or three of the nucleotides N2 to N6 in the aforementioned nexus region, The guide RNA according to claim 90, wherein four or five of the RNA molecules are modified with 2'-O-Me. 。

92. Ten or more nucleotides in the nexus region have a 2'-O-Me modification. Guide RNA containing decorative nucleotides.

93. The guide according to claim 92, wherein at least N2 to N6 are modified with 2'-O-Me. RNA.

94. 50% or more of the nucleotides in the bulge region are modified with 2'-O-Me. A guide RNA containing this RNA.

95. At least 55%, 60%, 65%, and 70% of the nucleotides in the bulge region, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% are 2 The guide RNA according to claim 94, which is modified with '-O-Me'.

96. Claim 94, wherein B2, B3, and / or B4 are modified with 2'-O-Me The included guide RNA.

97. Three or more nucleotides in the bulge region are 2'-O-Me modified Guide RNA containing creotides.

98. Claim 97, wherein B2, B3, and / or B4 are modified with 2'-O-Me The included guide RNA.

99. Having the modification pattern of the guide RNA according to any one of claims 1 to 98 A guide RNA containing the nucleotide of sequence number 357.

100. Having the modification pattern of the guide RNA according to any one of claims 1 to 99 A guide RNA containing the nucleotide of sequence number 356.

101. The first three, four, or five nucleotides at the end of the 5' terminus of the 5' terminus The claim according to any one of claims 79 to 100, further comprising a 2'-O-Me modification in the do. The guide RNA.

102. The last three, last four, or last five elements at the end of the 3' terminus of the 3' terminus region. Any of claims 79 to 100 further comprising a 2'-O-Me modification in the nucleotide. The guide RNA described in item 1.

103. The last nucleotide at the 3' end of the 3' terminal region is not modified. The guide RNA described in item 102.

104. The second to last and third last nuclei at the end of the 3' terminal region The guide RNA according to claim 102, wherein only the rheotide is modified.

105. The second to last, third to last, and fourth to last nuclei in the aforementioned 3' terminal region The guide RNA according to claim 102, wherein only the rheotide is modified.

106. The first three, four, or five nucleotides at the 5' end of the 5' terminus The guide RNA according to any one of claims 79 to 100, further comprising a PS bond in between.

107. The first four nucleotides at the 5' end of the aforementioned 5' terminus are linked by a PS bond. The guide RNA according to claim 106.

108. The last three, four, or five nucleotides at the 3' end of the 3' terminus The guide RNA according to any one of claims 79 to 100, further comprising a PS bond in between.

109. The last four nucleotides at the 3' end of the aforementioned 3' terminus are linked by PS bonds. The guide RNA according to claim 108.

110. The first three or four nucleotides at the 5' end of the 5' terminus, and the preceding The last three or four nucleotides at the 3' terminus are 2'-O-M A guide RNA according to any one of claims 79 to 100, comprising e-modification.

111. The first three or four nucleotides at the 5' end of the 5' terminus are PS-bound. They are connected by and the last three or four at the end of the 3' end The nucleotides are linked by PS bonds, according to any one of claims 79 to 100. The guide RNA described.

112. The first three or four nucleotides at the 5' end of the 5' terminus are 2'-O -Includes Me modification and is linked by PS bonding, and the 3' terminus of the 3' terminus The last three or four nucleotides at the end contain a 2'-O-Me modification, and PS A guide RNA according to any one of claims 79 to 100, which is linked by binding.

113. At least one of the nucleotides LS8, LS9, LS10, LS11, and LS12 The guide according to any one of claims 79 to 100, wherein two of the modifications include 2'-O-Me. RNA.

114. Claims 79-1, wherein at least LS8 and LS10 are modified with 2'-O-Me A guide RNA as described in any one of the items 00.

115. At least 50% of the nucleotides in the nexus region are modified with 2'-O-Me. A guide RNA according to any one of claims 79 to 100.

116. At least 55%, 60%, 65%, and 70% of the nucleotides in the nexus region 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% The guide RNA according to claim 115, which is modified with 2'-O-Me.

117. The guide according to claim 115, wherein at least N2 to N6 are modified with 2'-O-Me DRNA.

118. At least 10 of the nucleotides in the nexus region are 2'-O-Me A modified guide RNA according to any one of claims 79 to 100.

119. The guide according to claim 115, wherein at least N2 to N6 are modified with 2'-O-Me DRNA.

120. At least 50% of the nucleotides in the bulge region are modified with 2'-O-Me. The guide RNA according to any one of claims 79 to 100.

121. At least 55%, 60%, 65%, and 70% of the nucleotides in the bulge region, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% are 2 The guide RNA according to claim 120, which is modified with '-O-Me'.

122. Claim 121, wherein B2, B3, and / or B4 are modified with 2'-O-Me The guide RNA described.

123. At least three of the nucleotides in the bulge region are modified with 2'-O-Me The guide RNA according to any one of claims 79 to 100.

124. Claim 123, wherein B2, B3, and / or B4 are modified with 2'-O-Me. The guide RNA described.

125. A guide RNA according to any one of claims 79 to 124, which is an sgRNA.

126. A guide RNA according to any one of claims 79 to 125, which is a dgRNA.

127. At least 55 of the nucleotides in the hairpin 1 region and the hairpin 2 region ％、６０％、６５％、７０％、７５％、８０％、８５％、９０％、９５％、９６％、９７ Claim 81 or 82, wherein %, 98%, or 99% is modified with 2'-OMe. The included guide RNA.

128. It forms a ribonucleoprotein complex with Cas9 of S. pyogenes. A guide RNA described in any one of the requests 79 to 127.

129. It is a single guide RNA (sgRNA), a. The first nucleotide at the 5' end of the 5' terminus, b. Each nucleotide in the upper stem region, c. Each nucleotide in hairpin region 1, d. The nucleotide between hairpin 1 and hairpin 2, e. Each nucleotide in the hairpin 2 region, f. The last four nucleotides at the 3' end of the 3' terminus, and 2'-O-M sgRNA containing or consisting of e-modified nucleotides.

130. The PS bond connecting the first four nucleotides at the 5' end of the 5' terminus and PS bond connecting the last four nucleotides at the 3' end of the 3' terminus The sgRNA according to claim 129, further comprising:

131. The modifier patterns shown in sequence numbers 350, 351, 352, or 353, as depicted in Table 4 sgRNA, which has a nucleotide.

132. It is a single guide RNA (sgRNA), a. The first three, four, five, or seven nucleotides at the 5' end of the 5' terminus. and, b. Each nucleotide in the upper stem region, c. Each nucleotide in hairpin region 1, d. The nucleotide between hairpin 1 and hairpin 2, e. Each nucleotide in the hairpin 2 region, f. The last four nucleotides at the 3' end of the 3' terminus, and 2'-O-M sgRNA containing or consisting of e-modified nucleotides.

133. It is a single guide RNA (sgRNA), a. The first three, four, five, or seven nucleotides at the 5' end of the 5' terminus. and, b. Nucleotides US1, US2, US3, US4, US5, US6, US7, US8 , US9, US10, US11 and US12 respectively, c. Navates H1-1, H1-2, H1-3, H1-4, H1-5, H1-6, H Each of 1-7, H1-8, H1-9, H1-10, H1-11, and H1-12 、 d. The nucleotide between hairpin 1 and hairpin 2, e. κB1 H2-1, H2-2, H2-3, H2-4, H2-5, H2-6, H 2-7, H2-8, H2-9, H2-10, H2-11, H2-12, H2-13, H2 -14 and H2-15 respectively, f. The last four nucleotides at the 3' end of the 3' terminus, and 2'-O-M sgRNA containing or consisting of e-modified nucleotides.

134. The PS bond connecting the first four nucleotides at the 5' end of the 5' terminus and PS bond connecting the last four nucleotides at the 3' end of the 3' terminus The sgRNA according to any one of claims 129 to 133, further comprising:

135. Modification patterns for sequence numbers 350, 351, 352, or 353 as shown in Table 4 sgRNA possessing this property.

136. It forms a ribonucleoprotein complex with Cas9 of S. pyogenes. The sgRNA described in any one of the requests 129-135.

137. crispr RNA (crRNA), The following areas, namely, a. The first five nucleotides at the 5' end of the 5' terminus, b. Lower stem region and c. Bulge region and, d. Upper stem region; and e. The last five nucleotides at the 3' end of the crRNA, and one of them or crRNA containing one or more modifications within multiple regions.

138. Further includes a 5'-terminus modification, wherein the 5'-terminus modification is at the 5' end of the 5' terminus. Claims that the first seven nucleotides contain one or more phosphorothioate linkages. crRNA as described in 136.

139. Further includes a 5'-terminus modification, wherein the 5'-terminus modification is at the 5' end of the 5' terminus. The first seven nucleotides contain at least two phosphorothioate links, claim crRNA as described in 136.

140. At least one modification involves a 2'-O-methyl (2'-O-Me) modified nucleotide. crRNA according to any one of claims 137 to 139.

141. Claims that at least one modification comprises a 2'-fluoro(2'-F) modified nucleotide. crRNA as described in any one of items 137-140.

142. If at least one modification involves a phosphorothioate (PS) bond between nucleotides, The crRNA described in any one of the requests 137-141.

143. The first three nucleotides at the 5' end of the 5' terminus and the 3' terminus Claims 137-14, wherein the last three nucleotides at the 3' end are modified. crRNA as described in any one of item 2.

144. The first four nucleotides at the 5' end of the 5' terminus, and the 3' terminus. The last four nucleotides at the 3' end are linked by a phosphorothioate (PS) bond. The crRNA according to any one of claims 137 to 143, which is more ligated.

145. The crRNA according to claim 143, wherein the modification includes 2'-O-Me.

146. The crRNA according to claim 143, wherein the modification includes 2'-F.

147. The first four nucleotides at the 5' end of the 5' terminus and the 3' terminus The last four nucleotides at the 3' end are linked by PS bonds, and The first three nucleotides at the 5' end of the 5' terminus and the nucleotides before the 3' terminus Claim 13, wherein the last three nucleotides at the 3' end include a 2'-O-Me modification. crRNA as described in any one of items 7 to 146.

148. The first four nucleotides at the 5' end of the 5' terminus and the 3' terminus The last four nucleotides at the 3' end are linked by PS bonds, and The first three nucleotides at the 5' end of the 5' terminus and the nucleotides before the 3' terminus Claims 137-1, wherein the last three nucleotides at the 3' end contain a 2'-F modification. crRNA as described in any one of item 46.

149. Any of claims 137 to 148, wherein LS1 and LS6 are modified with 2'-O-Me crRNA as described in item 1.

150. Each nucleotide in the upper stem region is modified with 2'-O-Me, claim crRNA as described in any one of items 137 to 149.

151. crispr RNA (crRNA), a. LS1 and LS6 in the lower stem region, b. Each nucleotide in the upper stem region and a 2'-O-Me modified nucleotide crRNA, including

152. Three phosphorothiocyanates link the first four nucleotides at the 5' end of the 5' terminus. EAT (PS) bond and the last four nucleotides at the 3' end of the 3' terminus. The crRNA according to claim 151, further comprising three PS bonds.

153. In the first three nucleotides at the 5' end of the 5' terminus, 2'-O-M Further comprising e or 2'-F modified nucleotide, and the 3' terminus of the 3' terminus The last three nucleotides are 2'-O-Me or 2'-F modified nucleotides. The crRNA according to claim 151 or claim 152, further comprising:

154. Claims 137-153, wherein LS1, LS2, and LS6 are modified with 2'-F crRNA as described in any one of the items.

155. Claims 137-15, wherein each nucleotide in the bulge region is modified with 2'-F. crRNA as described in any one of item 4.

156. crispr RNA (crRNA), a. LS1, LS2, and LS6 in the lower stem region, b. Each nucleotide in the bulge region contains a 2'-F modified nucleotide, c rRNA.

157. The three nucleotides that link the first four nucleotides at the 5' end of the 5' terminus Holothioate (PS) bond and the last four nu at the 3' end of the 3' terminus Any one of claims 137 to 156 further comprises three PS bonds linking the creotide. The crRNA described in the section.

158. In the first three nucleotides at the 5' end of the 5' terminus, 2'-O-M Further comprising e or 2'-F modified nucleotide, and the 3' terminus of the 3' terminus The last three nucleotides are 2'-O-Me or 2'-F modified nucleotides. The crRNA according to any one of claims 137 to 157, further comprising:

159. crRNA containing one of the sequence numbers 1 to 187, which include the modifications shown in Table 4.

160. Table 4 contains modified sequence numbers 19-31, 53-73, 104-130, and 161- crRNA containing one of the 187.

161. Among sequence numbers 19-31, 53-73, 104-130, and 161-187 For each of the differences, at least 99, 98, 97, 96, 95, 94, 93, 92, 91 , containing nucleic acids having 90, 85, 80, 75, or 70% identity, as shown in Table 4. Table 4 shows the modifications at each nucleotide of the crRNA corresponding to the nucleotide of the column identifier. crRNA that is identical or equivalent to the modification shown in the reference sequence identifier.

162. The three nucleotides that link the first four nucleotides at the 5' end of the 5' terminus Holothioate (PS) bond and the last four nu at the 3' end of the 3' terminus Any one of claims 159 to 161 further comprises three PS bonds linking the creotide. The crRNA described in the section.

163. The crRNA combined with trRNA is the Ca of S. pyogenes. Forms a ribonucleoprotein complex with s9, as described in any one of claims 137 to 162. crRNA.

164. tracr RNA (trRNA), The following areas: a. The first five nucleotides at the 5' end of the 5' terminus, b. Upper stem region and, c. Bulge region and, d. Lower stem region and e. Nexus region and, f. Hairpin 1 region and g. Hairpin 2 region and h. The last five nucleotides at the 3' end of the 3' terminus, and one or more of them A trRNA containing one or more modifications within a numerical region.

165. At least one modification involves a 2'-O-methyl (2'-O-Me) modified nucleotide. , the trRNA according to claim 164.

166. Claims that at least one modification comprises a 2'-fluoro(2'-F) modified nucleotide. The trRNA according to claim 164 or claim 165.

167. If at least one modification involves a phosphorothioate (PS) bond between nucleotides, The trRNA described in any one of the requests 164-166.

168. The first four nucleotides at the 5' end of the 5' terminus, and the 3' terminus. The last four nucleotides at the 3' end are linked by a phosphorothioate (PS) bond. The trRNA according to any one of claims 164 to 167, which is more ligated.

169. The first three nucleotides at the 5' end of the 5' terminus and the 3' terminus Claims 164-16, wherein the last three nucleotides at the 3' end are modified. The trRNA described in any one of item 8.

170. The trRNA according to claim 169, wherein the modification includes 2'-O-Me.

171. The trRNA according to claim 169, wherein the modification includes 2'-F.

172. The first four nucleotides at the 5' end of the 5' terminus and the 3' terminus The last four nucleotides at the 3' end are linked by PS bonds, and The first three nucleotides at the 5' end of the 5' terminus and the nucleotides before the 3' terminus Claim 16, wherein the last three nucleotides at the 3' end include a 2'-O-Me modification. The trRNA described in any one of items 4 to 171.

173. The first four nucleotides at the 5' end of the 5' terminus and the 3' terminus The last four nucleotides at the 3' end are linked by PS bonds, and The first three nucleotides at the 5' end of the 5' terminus and the nucleotides before the 3' terminus Claims 164-1, wherein the last three nucleotides at the 3' end contain a 2'-F modification. The trRNA described in any one of item 71.

174. Claim 1, in which each nucleotide in the upper stem region is modified with 2'-O-Me The trRNA described in any one of items 64 to 173.

175. Claim 164, wherein B1 and B2 in the bulge region are modified with 2'-O-Me trRNA as described in any one of items ~174.

176. N3, N4, N5, N15, N16, N17, and / or N in the aforementioned nexus region The method according to any one of claims 164 to 175, wherein 18 is modified with 2'-O-Me trRNA.

177. Claim 1, in which each nucleotide in the hairpin 1 region is modified with 2'-O-Me The trRNA described in any one of items 64 to 176.

178. Claim 1, in which each nucleotide in the hairpin 2 region is modified with 2'-O-Me The trRNA described in any one of items 64 to 177.

179. tracr RNA (trRNA), a. Each nucleotide in the upper stem, b. B1 and / or B2 within the bulge region, c. N3, N4, N5, N15, N16, N17, and / or N in the nexus region 18 and, d. Each nucleotide in the hairpin 1 region, e. Each nucleotide in the hairpin 2 region and a 2'-O-Me modified nucleotide trRNA, which includes this.

180. Three phosphorothiocyanates link the first four nucleotides at the 5' end of the 5' terminus. EAT (PS) bond and the last four nucleotides at the 3' end of the 3' terminus. The trRNA according to claim 179, further comprising three PS bonds.

181. In the first three nucleotides at the 5' end of the 5' terminus, 2'-O-M Further comprising e or 2'-F modified nucleotide, and the 3' terminus of the 3' terminus The last three nucleotides further modify the nucleic acid with 2'-O-Me or 2'-F modification. Including the trRNA described in claim 179 or claim 180.

182. Claims 164- The trRNA described in any one of item 181.

183. LS1, LS3, and LS5 are modified with 2'-F, and LS2, LS4, In any one of claims 164 to 182, the and LS6 is modified with 2'-O-Me. The trRNA described.

184. The three nucleotides that link the first four nucleotides at the 5' end of the 5' terminus Holothioate (PS) bond and the last four nu at the 3' end of the 3' terminus Any one of claims 164 to 183 further comprises three PS bonds linking the creotide. The trRNA described in the section.

185. In the first three nucleotides at the 5' end of the 5' terminus, 2'-O-M Further comprising e or 2'-F modified nucleotide, and the 3' terminus of the 3' terminus The last three nucleotides are 2'-O-Me or 2'-F modified nucleotides. The trRNA according to any one of claims 164 to 184, further comprising:

186. A trRNA containing one of the sequence numbers 188-227, including the modifications shown in Table 4.

187. For any one of sequence numbers 188 to 227, at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, or 70% identity It contains nucleic acids, and each trRNA corresponding to the nucleotide of the reference sequence identifier in Table 4 The modification in the nucleotide is the same as the modification shown in the reference sequence identifier in Table 4. These are equivalent, trRNA.

188. Three phosphorothiocyanates link the first four nucleotides at the 5' end of the 5' terminus. EAT (PS) bond and the last four nucleotides at the 3' end of the 3' terminus. trR according to any one of claims 186 to 187, further comprising three PS bonds. NA.

189. The trRNA combined with crRNA is the Ca of S. pyogenes. Forms a ribonucleoprotein complex with s9, as described in any one of claims 164 to 188. trRNA.

190. A dual guide containing crRNA and trRNA, wherein the crRNA is as shown in Table 4. It contains one of the sequence numbers 1 to 187 described, and the trRNA is as shown in Table 4 A dual guide containing one nucleic acid from sequence numbers 188-227 as described in Do.

191. crRNA according to any one of claims 137 to 163 and claims 164 to 189 A dual guide containing the trRNA described in any one of the items.

192. The crRNA and unmodified trRNA described in any one of claims 137 to 163 are included. Hmm, dual guides.

193. The collection includes unmodified crRNA and the trRNA described in any one of claims 137 to 189. Hmm, dual guides.

194. It forms a ribonucleoprotein complex with Cas9 of S. pyogenes. A dual guide as described in any one of the requirements 190 to 193.

195. LN comprising sgRNA according to any one of claims 1 to 78 and 129 to 136 P composition.

196. gRN comprising or consisting of the RNA described in any one of claims 1 to 193 An LNP composition containing A.

197. Any one of claims 1 to 78 and 129 to 136, associated with lipid nanoparticles (LNPs) A composition comprising the sgRNA described in the section.

198. cr according to any one of claims 137 to 163, associated with lipid nanoparticles (LNPs) A composition containing RNA.

199. The tr according to any one of claims 164 to 189, associated with lipid nanoparticles (LNPs). A composition containing RNA.

200. The sgRNA according to any one of claims 1 to 78 and 129 to 136, The gRNA described in any one of claims 79 to 128, or any one of claims 137 to 163 The crRNA described in the item, the trRN described in any one of claims 164 to 189 A, the dgRNA according to any one of claims 190 to 194, or claim 195 The composition comprises the composition described in any one of the items ~199, and includes a nuclease or the nuclease A composition further comprising mRNA encoding ze.

201. The composition according to claim 200, wherein the nuclease is a Cas protein.

202. The composition according to claim 201, wherein the Cas protein is Cas9.

203. Claim 202 states that the Cas9 is Cas9 of S. pyogenes. The composition of the listed items.

204. The present invention according to any one of claims 200 to 203, wherein the nuclease is nicasse. composition.

205. The combination according to any one of claims 200 to 204, wherein the nuclease is modified. Finished product.

206. Claim 205, wherein the modified nuclease includes a nuclear localization signal (NLS). The composition described.

207. Any one of claims 200 to 206, comprising mRNA encoding the nuclease. The composition described above.

208. sgRNA according to any one of claims 1 to 78 and 129 to 136, claim 79 gRNA as described in any one of claims ~128, or as described in any one of claims 137 to 163. crRNA, trRNA according to any one of claims 164 to 189, claim 190 dgRNA as described in any one of claims 194, or any one of claims 195 to 207. A pharmaceutical preparation comprising the composition described in the section and a pharmaceutically acceptable carrier.

209. A method for modifying target DNA, Cas protein or nucleic acid encoding Cas protein, and the following: a. The sgRNA according to any one of claims 1 to 78 and 129 to 136; b. The gRNA according to any one of claims 79 to 128; c. The crRNA according to any one of claims 137 to 163; d. The trRNA according to any one of claims 164 to 189; e. The dgRNA according to any one of claims 190 to 194; f. The composition according to any one of claims 195 to 207; or g. Delivering one or more of the pharmaceutical formulations described in claim 208 to cells A method that includes doing something.

210. The method according to claim 209, which results in an insertion or deletion in a gene.

211. The process further includes delivering a template to the cells, wherein at least a portion of the template is Casta At or near protein-induced double-strand break sites, the target DNA The method according to claim 209, which is incorporated into the interior.

212. Claims 1 to 78 for use in the preparation of a drug for treating a disease or disorder. and the sgRNA described in any one of items 129 to 136.

213. Claims 737 to for use in the preparation of a drug for treating a disease or disorder crRNA as described in any one of item 163.

214. Claims 164 to for use in the preparation of a drug for treating a disease or disorder The trRNA described in any one of item 189.

215. For use in the preparation of a drug for treating a disease or disorder, claims 164 to A combination of the trRNA described in any one of claims 189, of claims 137 to 163 crRNA as described in item 1.

216. Claim 208, for use in the preparation of a drug for treating a disease or disorder The pharmaceutical preparation described.

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