Methods and compositions for directed genome editing

Abstract

The present invention provides compositions and methods for increasing the editing efficiency of target nucleic acids. [Solution] The composition may comprise a guide nucleic acid, Cas9 nickase, or reverse transcriptase. The reverse transcriptase may be fused to Cas9 nickase. The reverse transcriptase may heterodimerize with Cas9 nickase. The reverse transcriptase may bind to the guide nucleic acid. The reverse transcriptase may be engineered to increase its processing capacity. The guide nucleic acid may be engineered to facilitate the synthesis or editing of sequences. The guide nucleic acid, Cas9 nickase, and reverse transcriptase may be engineered to fit within an AAV vector. The guide nucleic acid may include a region that binds to another region on the guide nucleic acid to improve gene editing.

Claims

1. It has a low deoxynucleoside triphosphate (dNTP) concentration and is suitable for gene editing. A method for increasing gene editing efficiency in cells containing A polymerase, wherein the method is By increasing the intracellular dNTP concentration compared to the baseline dNTP concentration... A method that includes [this].

2. Increasing the dNTP concentration within the cell results in the deoxynucleoin concentration within the cell. The method according to claim 1, comprising inhibiting triphosphohydrolase ditriphosphate.

3. The deoxynucleotide triphosphate triphosphohydrolase has a SAM domain and H The method according to claim 2, comprising D-domain-containing protein 1 (SAMHD1).

4. Inhibiting SAMHD1 allows SAMHD1 to come into contact with the Vpx protein. The method according to claim 3, which includes expressing the Vpx protein in the cell. method.

5. Inhibiting SAMHD1 allows SAMHD1 to come into contact with the BGLF4 protein. Claim 3, which includes either doing so or expressing the BGLF4 protein in the cell. Methods used.

6. Inhibiting SAMHD1 causes the mRNA encoding SAMHD1 to be mR Contacting NA with microRNA or siRNA that hybridizes, or before Claim 3 includes expressing the microRNA or siRNA within the cell. Method of description.

7. Inhibiting SAMHD1 brings SAMHD1 into contact with the small molecule SAMHD1 inhibitor. The method according to claim 3, which includes causing to do so.

8. Increasing the dNTP concentration within the cell allows nucleosides or nucleosides to be introduced into the cell. The procedure includes administering a rheoside, wherein the nucleoside or nucleotide is optionally selected Deoxynucleoside (dN), deoxynucleoside monophosphate (dNMP), or nucleoside The method according to claim 1, comprising rheoside triphosphate (NTP).

9. Administering nucleosides or nucleotides to the aforementioned cells is a prior procedure for a subject including the aforementioned cells. The method according to claim 8, comprising administering the nucleoside or nucleotide.

10. The method according to claim 9, wherein the administration is by oral or injection.

11. Increasing the dNTP concentration within the cells delivers dNTP synthase to the cells. The method according to claim 1, including reaching

12. The method according to claim 11, wherein the dNTP synthase comprises a kinase.

13. The kinases mentioned above include nucleoside kinases, deoxynucleoside kinases, and deoxynu Claims comprising cleoside monophosphate kinase or deoxynucleotide diphosphate kinase. The method described in item 12.

14. The method according to claim 1, wherein the DNA polymerase comprises a reverse transcriptase.

15. The aforementioned cells contain Cas9 programmable nuclease, guide nucleic acid, or a combination thereof. The method according to claim 1, further comprising a blend.

16. Claim 1, wherein the low dNTP concentration includes the dNTP concentration found in non-dividing cells. Method of description.

17. The aforementioned low dNTP concentration is less than the dNTP concentration found in activated peripheral blood mononuclear cells. The method according to claim 1.

18. The low dNTP concentration includes a dNTP concentration of less than 1 micromol, according to claim 1. The method.

19. Increasing the dNTP concentration compared to the baseline dNTP measurement value The dNTP concentration is reduced to at least 5%, at least 10%, at least 15%, and less at least 20%, at least 25%, at least 30%, at least 40%, at least 5 0%, at least 60%, at least 70%, at least 80%, at least 90%, small The method according to claim 1, comprising increasing by at least 100% or more.

20. The dNTP concentration is the deoxyadenosine triphosphate (dATP) concentration, deoxycytidine dCTP concentration, deoxyguanosine triphosphate (dGTP) concentration, or Deoxythymidine triphosphate (dTTP) concentration, or any combination thereof, please The method described in any one of the requests 1 to 19.

21. A composition comprising Cas nickas and reverse transcriptase, wherein the Cas nickas and the At least a portion of the reverse transcriptase is contained in a separate polypeptide chain, and the Cas2 A composition comprising casase and the reverse transcriptase forming a Cas-reverse transcriptase heterodimer.

22. The Cas-reverse transcriptase heterodimer is fused to the Cas nickase, forming the first heterodimer. It comprises a heterodimer domain and a second heterodimer domain fused to the reverse transcriptase. The first heterodimer domain binds to the second heterodimer domain, The composition according to claim 21, which forms a Cas-reverse transcriptase heterodimer.

23. The first heterodimer domain is a leucine zipper, and the second heterodimer The composition according to claim 22, wherein the somatic domain is a leucine zipper.

24. The reverse transcriptase is sequence number 3 to sequence number 22 or sequence number 40 to sequence number 80 A sequence having at least 80% sequence identity with respect to any one of them, or A composition according to any one of claims 21 to 23, comprising a fragment of the

25. The aforementioned reverse transcriptase is fused to a portion of the Cas nickase, forming a non-long-terminated repeat retrotran. Includes domains from sposable (retrotransposable) elements or the composition according to any one of claims 21 to 24.

26. The aforementioned reverse transcriptase is fused to a portion of the Cas nickase, forming a bacterial group II int. A composition according to any one of claims 21 to 24, comprising a sequence from ron.

27. The aforementioned reverse transcriptase is fused to a portion of the Cas nickase in the retrovirus gag-p A set according to any one of claims 21 to 24, comprising a domain from an ol polyprotein. Finished product.

28. A composition comprising Cas nicasse, reverse transcriptase, and guide nucleic acid, wherein the first The polypeptide comprises the Cas nicasse, and the second polypeptide comprises the reverse transcriptase. The composition includes the guide nucleic acid which binds to the Cas nicasse and the reverse transcriptase. thing.

29. The reverse transcriptase comprises the mcp peptide, according to any one of claims 21 to 28. composition.

30. The reverse transcriptase comprises a loop region, according to any one of claims 21 to 29. thing.

31. The composition according to claim 30, wherein the loop region is a 2a loop or a 3a loop.

32. The guide nucleic acid comprises an MS2 hairpin, as described in any one of claims 28 to 31. composition.

33. One of the following sequence numbers: Sequence ID 3 to Sequence ID 22 or Sequence ID 40 to Sequence ID 80 A sequence having at least 80% sequence identity with respect to the one in question, or a sequence that can be fused to Cas nickase. A composition comprising a reverse transcriptase having the combined fragments thereof.

34. From non-long-term repeat retrotransposable elements fused to Cas nickase A composition comprising a reverse transcriptase containing a domain.

35. Reverse transcription yeast containing sequences from bacterial group II introns fused to Cas nickase A composition containing an element.

36. DOM from retroviral gag-pol polyprotein fused to Cas nickase A composition comprising a reverse transcriptase containing yin.

37. A composition comprising Cas nickas and reverse transcriptase, wherein the Cas nickas and the reverse transcriptase comprises a separate polypeptide chain, and the Cas nicasse and reverse transcriptase A composition in which the enzyme has not been manipulated to undergo heterodimerization.

38. It contains a guide nucleic acid that forms a complex with the Cas nicasse, and when the complex is formed, the C The claim states that AS nickase can introduce single-strand breaks at target sites in target nucleic acids. A composition according to any one of items 21 to 37.

39. The target nucleic acid is CFTR nucleic acid, USH2A nucleic acid, ABCA4 nucleic acid, ATP7B nucleic acid, Or a composition according to any one of claims 21 to 38, comprising HTT nucleic acid.

40. A claim comprising a nuclear localization signal fused to the Cas nicasse or the reverse transcriptase. A composition according to any one of items 21 to 39.

41. The reverse transcriptase is a cleaved reverse transcriptase, according to any one of claims 21 to 40. The composition described.

42. Claim 2, the reverse transcriptase has increased processing capacity compared to natural reverse transcriptase. A composition according to any one of items 1 to 41.

43. Claims 21-4, wherein the reverse transcriptase has increased processing capacity compared to mlvRT. The composition according to any one of item 2.

44. The aforementioned reverse transcriptase modifies the target sequence with a longer window length compared to mlvRT. A composition according to any one of claims 21 to 43.

45. Claims 21-4, wherein the reverse transcriptase has reduced immunogenicity compared to mlvRT. The composition according to any one of item 4.

46. Claim 21, wherein the reverse transcriptase has improved delivery to cells compared with mlvRT. The composition described in any one of the items 45 to 45.

47. The aforementioned reverse transcriptase binds to 20 or more, 40 or more, or 45 or more in a single binding event. 50 or more, 60 or more, 81 or more, 100 or more, 500 or more, or 1000 or more A composition according to any one of claims 21 to 46, wherein the nucleotides above are polymerized.

48. It is a guide nucleic acid, A spacer that is inversely complementary to the first region of the target nucleic acid, A scaffold configured to bond to Cas nickase, A reverse transcriptase template encoding a sequence to be inserted into the target nucleic acid, The second region of the target nucleic acid includes a first chain primer binding site which is inversely complementary to the target nucleic acid, Idonic acid.

49. Claim 48 further comprises a second chain primer including an sequence of the region of the reverse transcriptase template. The guide nucleic acid described.

50. The first region of the target nucleic acid is on the first strand of the target nucleic acid, and the target nucleic acid The guide according to claim 48 or 49, wherein the second region is located on the second strand of the target nucleic acid. Nucleic acid.

51. All or part of the first region of the target nucleic acid is the second region of the target nucleic acid The gas according to any one of claims 48 to 50, which is inversely complementary to all or part of the region. Idonic acid.

52. Any one of claims 48 to 51 further comprises a cleavable sequence at the 3' end of the guide nucleic acid. Guide nucleic acids as described in item 1.

53. The guide according to claim 52, wherein the cleavable sequence is a ribozyme-cleavable sequence. Nucleic acid.

54. The guide nucleus according to claim 52, wherein the cleavable sequence is a tRNA-cleavable sequence. acid.

55. The first chain primer binding site hybridizes to the second region of the target nucleic acid. The reverse transcriptase template is configured such that the 3' end of the second region of the target nucleic acid any one of claims 48 to 54, configured to function as a template for reverse transfer from The guide nucleic acid described in item 1.

56. The second chain primer is for the transfer from a template that is inversely complementary to the reverse transcriptase template. A configuration configured to function as a primer, according to any one of claims 48 to 55 Guide nucleic acid.

57. The first synthetic chain is used as a template for the synthesis of the second chain from the second chain primer. A guide nucleic acid according to any one of claims 48 to 56, which is capable of performing the function.

58. Claim 48 further includes a Velcro region that hybridizes to a Velcro binding site. Guide nucleic acids as described in any one of items ~57.

59. The Velcro binding site is 100% inversely complementary to the Velcro region. The Velcro binding site is at least 50% of the Velcro region. At least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, At least 92%, at least 93%, at least 94%, at least 95%, and at least They are inversely complementary in 96%, at least 97%, at least 98%, and at least 99% of cases. and / or the Velcro binding site is 55% or less of the Velcro region, 60% or less, 65% or less, 70% or less, 75% or less, 80% or less, 85% or less, 90% or less Lower, 91% or less, 92% or less, 93% or less, 94% or less, 95% or less, 96% or less, 97 The guide nucleic acid according to claim 58, which is less than % of, less than 98%, less than 99%, and inversely complementary.

60. The reverse transcriptase template region includes the Velcro binding site, according to claim 58 or 59. The guide nucleic acid described.

61. The claim states that the Velcro binding site is 3' of the first chain primer binding site. Guide nucleic acids as described in 58 or 59.

62. The Velcro region is 3' of the reverse transcriptase template, according to any of claims 48 to 61. The guide nucleic acid described in one of the items.

63. The Velcro region is 5' of the scaffold, according to any one of claims 48 to 62. The guide nucleic acid described.

64. The target nucleic acid is CFTR nucleic acid, USH2A nucleic acid, ABCA4 nucleic acid, ATP7B nucleic acid, Or a guide nucleic acid according to any one of claims 48 to 63, comprising HTT nucleic acid.

65. The spacer has at least 8 of any one of sequence numbers 96 to 119. A guide nucleic acid according to any one of claims 48 to 64, comprising 5% identical nucleic acid sequences.

66. A first guide nucleus including the guide described in any one of claims 28 to 32 or 37 to 65 A composition comprising an acid and a second guide nucleic acid.

67. The second guide nucleic acid is any of claims 28, 32, or 37, or 48 to 65. The composition according to claim 66, comprising the guide nucleic acid described in item 1.

68. The reverse transcriptase template of the second guide nucleic acid is the reverse transcriptase of the first guide nucleic acid The claim is that the plain mold is complementary (or at least partially complementary) to at least a portion of it. The composition described in item 67.

69. The first guide nucleic acid binds to the first Cas nickase, and the second guide nucleic acid The composition according to any one of claims 66 to 68, wherein the second Cas nicasse is bonded. 。

70. The first spacer of the first guide nucleic acid binds to the first Cas nickase, The second spacer of the second guide nucleic acid binds to the second Cas nickase, and the first The first scaffold of the guide nucleic acid binds to the second Cas nicasse, and the second guide nucleus Any of claims 66 to 68, wherein the second acidic scaffold is bonded to the first Cas nicasse. The composition described in item 1.

71. The first guide nucleic acid comprises a first linker, and the second guide nucleic acid comprises a second linker. A claim including a linker, wherein the first linker hybridizes with the second linker. The composition according to any one of paragraphs 66 to 68 or 70.

72. The composition according to any one of claims 21 to 47 or 66 to 71, or claim 38 To deliver Orf1p to cells expressing the guide nucleic acid described in any one of items ~45. Methods to increase genome editing efficiency, including those mentioned above.

73. To code for a composition according to any one of claims 21 to 47 or 66 to 71, Or one or more nucleic acids comprising the guide nucleic acid described in any one of claims 48 to 65.

74. A viral vector comprising the nucleic acid described in claim 73.

75. The composition according to any one of claims 21 to 47 or 66 to 71, claims 48 to 6 The guide nucleic acid described in any one of item 5, the nucleic acid described in claim 73, or the guide nucleic acid described in claim 74 Cells containing a viral vector.

76. The method according to claim 72 or the cell according to claim 75, wherein the cell is a prokaryotic cell.

77. The method according to claim 72 or the cell according to claim 75, wherein the cell is a eukaryotic cell.

78. A method to increase genome editing efficiency, including the expression of the Vpx protein in cells. 。

79. The cells are the composition according to any one of claims 21 to 47 or 66 to 71, The following is the case of claim 78, which expresses the guide nucleic acid described in any one of claims 48 to 65. method.

80. A method for increasing genome editing efficiency by increasing intracellular dNTP concentration, example. For example, a method for increasing genome editing efficiency, including inhibiting SAMHD1 in cells.

81. The aforementioned cells produce Cas9 programmable nuclease, reverse transcriptase, and guide nucleic acid. The method according to claim 80.

82. Inhibiting SAMHD1 includes causing the expression of Vpx protein within the cells. The method according to claim 80 or 81.

83. Inhibiting SAMHD1 leads to the development of microRNAs targeting SAMHD1 within the cells. This includes expressing the cell, or treating the cell with a small molecule SAMHD1 inhibitor. The method according to claim 80 or 81.

84. Includes one or more point mutations or insertion mutations that enable or improve intein catalysis. A composition comprising Cas9 programmable nuclease.

85. The Cas9 programmable nuclease is the Cas9 programmable nuclease It includes a point mutation or insertion mutation located in the C-terminal half of ze, or the said point mutation or insertion mutation. The input mutation occurs at any amino acid position after position 574 of the Cas9 programmable nuclease. The composition according to claim 84, located at a specific location.

86. The aforementioned point mutation is a cysteine ​​point mutation, serine point mutation, threonine point mutation, or alani. It includes a point mutation, or the insertion mutation is a cysteine ​​insertion mutation, a serine insertion mutation, a threone insertion mutation. The composition according to claim 85, comprising a nin insertion mutation or an alanine insertion mutation.

87. The point mutation includes a cysteine ​​point mutation, or the insertion mutation is a cysteine ​​insertion mutation The composition according to claim 85, comprising:

88. Claim 84, wherein the Cas9 programmable nuclease is Cas9 nicasse. The composition described in any one of the items ~87.

89. The Cas9 programmable nuclease is S. Pyogenes Cas9. or the composition according to any one of claims 84 to 88.

90. The aforementioned point mutations are D1079, D1125, D1 of S. Pyogenes Cas9. 130, G1133, A1140, I1168, S1173, D1180, G1186, The insertion mutation is located at L1203 or R1212, or the insertion mutation is located at S. Pyog ENES Cas9 D1079, D1125, D1130, G1133, A1140, I1168, S1173, D1180, G1186, L1203, or R1212 The composition according to claim 89, located immediately upstream.

91. The Cas9 programmable nuclease is sequence number 85 to sequence number 87 or sequence number Any of claims 84 to 90, comprising one sequence from sequence number 90 to sequence number 92. The composition described in item one.

92. The Cas9 programmable nuclease is expressed as two or more segments. or the composition according to any one of claims 84 to 91.

93. The first segment of the two or more segments is the Cas9 programmable The nuclease comprises the N-terminal portion and the first intein, and the two or more segments The second segment of the Cas9 programmable nuclease is the C-terminal portion and The composition according to claim 92, comprising two inteins.

94. The cysteine ​​point mutation is the C-terminal portion of the Cas9 programmable nuclease. The composition according to claim 93, located at the N-terminus.

95. The first intein is the N-terminal portion of the Cas9 programmable nuclease. The second intein is fused to the C-terminus of the Cas9 programmable nucleus. The composition according to claim 93 or 94, wherein the C-terminal portion of Ze is fused to the N-terminal portion.

96. The first segment includes the sequence of sequence number 90, and the second segment includes the sequence A composition according to any one of claims 93 to 95, comprising the sequence number 91.

97. The second segment of the two or more segments is the Cas9 programmer Claims 93 to 96 include a reverse transcriptase fused to the C-terminal portion of brunuclease. Any of the compositions described in item one.

98. The reverse transcriptase is the C-terminal portion of the Cas9 programmable nuclease. The composition according to claim 97, comprising an N-terminus fused to the end.

99. The reverse transcriptase comprises mlvRT or a variant thereof, as described in claim 97 or 98. The composition of the listed items.

100. A method for optimizing genome editing efficiency, which increases catalytic efficiency at low dNTP concentrations. Modified to reduce (for example, modified to reduce its Km relative to dNTPs) () Using Moloney leukemia virus reverse transcriptase (mlvRT) to perform genome editing A method, including the act of administering something.

101. A method for optimizing genome editing efficiency under limited dNTP conditions, comprising reverse transcriptase Moloney leukemia virus reverse transcriptase (m) containing a point mutation at position 221 or 223. A method comprising performing genome editing using lvRT, or a variant thereof.

102. Claim 100 or the mlvRT or its variant includes a point variation at position 221 The method described in 101.

103. The method according to claim 102, wherein the point variation at position 221 includes Q221R.

104. Claim 100 or the mlvRT or its variant includes a point variation at position 223 The method described in 101.

105. The method according to claim 104, wherein the point variation at position 223 includes V223A.

106. The method according to claim 104, wherein the point variation at position 223 includes V223M.

107. The reverse transcriptase is located at positions P51, S67, Q84, L139, Q221, V223, T Point mutations in 197, D653, T664, L671, L435, H204, or D524 A composition comprising any one of claims 21 to 47 or 66 to 71.

108. The aforementioned reverse transcriptases are P51L, S67R, Q84A, L139P, Q221R, and V22 3A, V223M, T197A, D653N, T664N, L671P, L435G, H Any of claims 21 to 47 or 66 to 71, which include a point mutation including 204R or D524A The composition described in any one of the items.

109. The aforementioned reverse transcriptase has amino acid positions Q84, L139, Q221, V223, T664, Or, according to any one of claims 21 to 47 or 66 to 71, L671 includes a point variation. composition.

110. The aforementioned reverse transcriptases are S67R, Q84A, L139P, Q221R, V223A, V2 Claims 21 to 4 include point mutations including 23M, T664N, L671P, or D524A. The composition according to any one of paragraphs 7 or 66 to 71.

111. Claim 2, wherein the Cas nicasse and RT are encoded by polynucleotides. A composition according to any one of items 1 to 47.

112. AAV comprising the polynucleotide described in claim 111.

113. At least a portion of the Cas nickase and RT is included by a separate AAV. The AAV described in claim 112.

114. It contains a first polynucleotide that encodes at least a portion of Cas niccas. A first AAV and a second AAV containing a second polynucleotide encoding reverse transcriptase Adeno-associated viruses (AAVs), including ,

115. The aforementioned AAVs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AA V7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-DJ, AA V-DJ / 8, AAV-Rh10, AAV-Rh74, AAV-retro, AAV-P HP.B, AAV8-PHP.eB, or AAV-PHP.S, or a combination thereof AAV according to any one of claims 112 to 114, including a blended version.

116. The Cas nicasse and the reverse transcriptase form a heterodimer with each other, according to the claim. AAV as described in 114 or 115.

117. The first or second polynucleotide is used in the Cas nicasse and the reverse transcriptase. Further encoding a guide nucleic acid that binds to form a complex, and the Cas nickel of the complex Any of claims 114 to 116, wherein the enzyme introduces a single-strand break at a target site in the target nucleic acid. AAV as described in item 1.

118. The aforementioned Cas nickase is Cas9 such as S. Pyogenes Cas9 nickase. The reverse transcriptase comprises nickase, and the reverse transcriptase comprises mlvRT or a variant thereof. The transcriptases are P51, S67, Q84, L139, Q221, V223, T197, and D6. Claims including point variations in 53, T664, L671, L435, H204, or D524. The AAV described in any one of items 114 to 117.

119. The aforementioned point mutations are P51L, S67R, Q84A, L139P, Q221R, V223A , V223M, T197A, D653N, T664N, L671P, L435G, H20 The AAV according to claim 118, comprising 4R or D524A.

120. The Cas9 nickas is said to include S. Pyogenes Cas9 nickas, and the The reverse transcriptase comprises mlvRT or a variant thereof, and the reverse transcriptase contains P51, S 67, Q84, L139, Q221, V223, T197, D653, T664, L67 1. Claims 114-1, which include an insertion mutation immediately upstream of L435, H204, or D524. The AAV specified in any one of item 19.

121. A composition comprising the first or second AAV described in any one of claims 114 to 120, A method for editing a genome, including administration to a target or cells.

122. A composition containing AAV according to any one of claims 112 to 120 is applied to a target or cell. A method for editing a genome, including administration.

123. Claim 121 or 122 further includes measuring genome editing within the subject or cells. Methods used.

124. Gene editing efficiency in cells with low deoxynucleoside triphosphate (dNTP) concentrations A method for increasing, The cells are genetically modified for efficient catalytic activity at the low dNTP concentration. Contacting the gene editing enzyme, or expressing the gene editing enzyme within the cell, Methods that include...

125. The gene editing enzyme is located at positions Q84, L139, Q221, V223, T664, or Claim 124 comprises a reverse transcriptase modified by introducing a point mutation at L671. Method of description.

126. A method for screening or identifying improved reverse transcriptase (RT), Overexpress SAMHD1 in cells, or phosphate groups of residues in mutant SAMHD1. Expressing a mutant SAMHD1 that has been mutated to prevent the transformation, Identifying the RT activity within the aforementioned cells, This includes identifying the RT as an improved RT based on the RT activity, method.

127. A spacer, a reverse transcriptase template including the desired edits, and a primer binding site, RN A system comprising A or polynucleotide, wherein the primer binding site is the same Nucleic acids that do not contain any portion of the nucleic acid region targeted or bound by the ser, This is a nucleic acid that is inversely complementary to the nucleic acid targeted or bound by the spacer. A system that combines.

128. It is a system, The first guide nucleic acid, A spacer that is inversely complementary to the first region of the target nucleic acid, A scaffold configured to bind to Cas nuclease, A reverse transcriptase template encoding a sequence to be inserted into the target nucleic acid, Not including any part of the first region, and not including any of the inverse complements of the first region. A first chain primer binding site that binds to the region of the target nucleic acid, excluding the portion thereof, A GPS region that hybridizes to a GPS binding site on a second guide nucleic acid, a system including a first guide nucleic acid.

129. The second guide nucleic acid comprising the GPS binding site, according to claim 128. Stem.

130. The second guide nucleic acid includes a second spacer that is inversely complementary to another region of the target nucleic acid. The system described in claim 129.

131. The second guide nucleic acid brings the primer binding site close to the genome flap. The system described in item 129 or 130.

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