OPTIMIZED CRISPR-CAS DOUBLE NICKASE SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION

• US 20160153006A1
• Filed: 12/17/2015
• Published: 06/02/2016
• Est. Priority Date: 06/17/2013
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation), Massachusetts Institute of Technology, The Broad Institute, Inc.
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation), Massachusetts Institute of Technology, The Broad Institute, Inc.
• Inventors: LIN, Chie-yu, ZHANG, Feng, HSU, Patrick, RAN, Fei
• Granted Patent: US 10,711,285 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61K 48/005 characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/1082 Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/90 Stable introduction of foreign DNA into chromosome
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2310/20 involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12N 2800/80 Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

The invention provides for delivery, engineering and optimization of systems, methods, and compositions for manipulation of sequences and/or activities of target sequences. Provided are vectors and vector systems, some of which encode one or more components of a CRISPR complex, as well as methods for the design and use of such vectors. Also provided are methods of directing CRISPR complex formation in prokaryotic and eukaryotic cells to ensure enhanced specificity for target recognition and avoidance of toxicity.

Claims

1. A method of modifying an organism or a non-human organism by minimizing off-target modifications by manipulation of a first and a second target sequence on opposite strands of a DNA duplex in a genomic locus of interest in a cell comprising

delivering a non-naturally occurring or engineered composition comprising:

I. a first CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, wherein the first polynucleotide sequence comprises;

(a) a first guide sequence capable of hybridizing to the first target sequence,

(b) a first tracr mate sequence, and

(c) a first tracr sequence,

II. a second CRISPR-Cas system chiRNA polynucleotide sequence, wherein the second polynucleotide sequence comprises;

(a) a second guide sequence capable of hybridizing to the second target sequence,

(b) a second tracr mate sequence, and

(c) a second tracr sequence, and

III. a polynucleotide sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences and comprising one or more mutations,

wherein (a), (b) and (c) are arranged in a 5′

to 3′

orientation,

wherein when transcribed, the first and the second tracr mate sequence hybridize to the first and second tracr sequence respectively and the first and the second guide sequence directs sequence-specific binding of a first and a second CRISPR complex to the first and second target sequences respectively,

wherein the first CRISPR complex comprises the CRISPR enzyme complexed with (1) the first guide sequence that is hybridizable to the first target sequence, and (2) the first tracr mate sequence that is hybridized to the first tracr sequence,

wherein the second CRISPR complex comprises the CRISPR enzyme complexed with (1) the second guide sequence that is hybridizable to the second target sequence, and (2) the second tracr mate sequence that is hybridized to the second tracr sequence,

wherein the polynucleotide sequence encoding a CRISPR enzyme is DNA or RNA, and

wherein the first guide sequence directs cleavage of one strand of the DNA duplex near the first target sequence and the second guide sequence directs cleavage of other strand near the second target sequence inducing a double strand break, thereby modifying the organism or the non-human organism by minimizing off-target modifications.

2. The method of claim 1, wherein the first guide sequence directing cleavage of one strand of the DNA duplex near the first target sequence and the second guide sequence directing cleavage of other strand near the second target sequence results in a 5′

overhang.

3. The method of claim 2, wherein the 5′

overhang is at most 200 base pairs.

4. The method of claim 2, wherein the 5′

overhang is at most 100 base pairs.

5. The method of claim 2, wherein the 5′

overhang is at most 50 base pairs.

6. The method of claim 2, wherein the 5′

overhang is at least 26 base pairs.

7. The method of claim 2, wherein the 5′

overhang is at least 30 base pairs.

8. The method of claim 2, wherein the 5′

overhang is 34-50 base pairs.

9. The method of claim 2, wherein the 5′

overhang is at least 15 base pairs.

10. The method of claim 2, wherein the 5′

overhang is at least 10 base pairs.

11. The method of claim 2, wherein the 5′

overhang is at least 1 base pair.

12. The method of claim 2, wherein the 5′

overhang is 1-34 base pairs.

13. The method of claim 1, wherein the first guide sequence directing cleavage of one strand of the DNA duplex near the first target sequence and the second guide sequence directing cleavage of other strand near the second target sequence results in a blunt cut.

14. The method of claim 1, wherein the first guide sequence directing cleavage of one strand of the DNA duplex near the first target sequence and the second guide sequence directing cleavage of other strand near the second target sequence results in a 3′

overhang.

15. The method of claim 14, wherein the 3′

overhang is at most 150 base pairs.

16. The method of claim 14, wherein the 3′

overhang is at most 100 base pairs.

17. The method of claim 14, wherein the 3′

overhang is at most 50 base pairs.

18. The method of claim 14, wherein the 3′

overhang is at most 25 base pairs.

19. The method of claim 14, wherein the 3′

overhang is at least 15 base pairs.

20. The method of claim 14, wherein the 3′

overhang is at least 10 base pairs.

21. The method of claim 14, wherein the 3′

overhang is at least 1 base pair.

22. The method of claim 14, wherein the 3′

overhang is 1-100 base pairs.

23. The method of claim 1, wherein any or all of the polynucleotide sequence encoding the CRISPR enzyme, the first and the second guide sequence, the first and the second tracr mate sequence or the first and the second tracr sequence, is/are RNA.

24. The method of claim 1, wherein the polynucleotides comprising the sequence encoding the CRISPR enzyme, the first and the second guide sequence, the first and the second tracr mate sequence or the first and the second tracr sequence, is/are RNA and are delivered via nanoparticles, exosomes, microvesicles, or a gene-gun.

25. The method of claim 1, wherein the first and second tracr mate sequence share 100% identity.

26. The method of claim 1, wherein the first and second tracr sequence share 100% identity.

27. The method of claim 1, wherein the CRISPR enzyme is a Cas9 enzyme.

28. The method of claim 27, wherein the CRISPR enzyme is SpCas9.

29. The method of claim 28, wherein the CRISPR enzyme comprises one or more mutations in a catalytic domain.

30. The method of claim 29, wherein the one or more mutations is in a RuvC domain.

31. The method of claim 30, wherein the one or more mutations is selected from the group consisting of D10A, E762A and D986A.

32. The method of claim 31, wherein the CRISPR enzyme has the D10A mutation.

33. The method of claim 29, wherein the one or more mutations is in a HNH domain.

34. The method of claim 33, wherein the one or more mutations is selected from the group consisting of H840A, N854A and N863A.

35. The method of claim 34, wherein the CRISPR enzyme has the H840A mutation.

36. A method of modifying an organism or a non-human organism by minimizing off-target modifications by manipulation of a first and a second target sequence on opposite strands of a DNA duplex in a genomic locus of interest in a cell comprising delivering a non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising

I. a first regulatory element operably linked to

(a) a first guide sequence capable of hybridizing to the first target sequence, and

(b) at least one or more tracr mate sequences,

II. a second regulatory element operably linked to

(a) a second guide sequence capable of hybridizing to the second target sequence, and

(b) at least one or more tracr mate sequences,

III. a third regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, and

IV. a fourth regulatory element operably linked to a tracr sequence,

wherein components I, II, III and IV are located on the same or different vectors of the system,

when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the first and the second guide sequence directs sequence-specific binding of a first and a second CRISPR complex to the first and second target sequences respectively,

wherein the first CRISPR complex comprises the CRISPR enzyme complexed with (1) the first guide sequence that is hybridizable to the first target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence,

wherein the second CRISPR complex comprises the CRISPR enzyme complexed with (1) the second guide sequence that is hybridizable to the second target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence,

wherein the polynucleotide sequence encoding a CRISPR enzyme is DNA or RNA, and

wherein the first guide sequence directs cleavage of one strand of the DNA duplex near the first target sequence and the second guide sequence directs cleavage of other strand near the second target sequence inducing a double strand break, thereby modifying the organism or the non-human organism by minimizing off-target modifications.

37. The method of claim 36, wherein the first guide sequence directing cleavage of one strand of the DNA duplex near the first target sequence and the second guide sequence directing cleavage of other strand near the second target sequence results in a 5′

overhang.

38. The method of claim 37, wherein the 5′

overhang is at most 200 base pairs.

39. The method of claim 37, wherein the 5′

overhang is at most 100 base pairs.

40. The method of claim 37, wherein the 5′

overhang is at most 50 base pairs.

41. The method of claim 37, wherein the 5′

overhang is at least 26 base pairs.

42. The method of claim 37, wherein the 5′

overhang is at least 30 base pairs.

43. The method of claim 37, wherein the 5′

overhang is 34-50 base pairs.

44. The method of claim 37, wherein the 5′

overhang is at least 15 base pairs.

45. The method of claim 37, wherein the 5′

overhang is at least 10 base pairs.

46. The method of claim 37, wherein the 5′

overhang is at least 1 base pair.

47. The method of claim 37, wherein the 5′

overhang is 1-34 base pairs.

48. The method of claim 36, wherein the first guide sequence directing cleavage of one strand of the DNA duplex near the first target sequence and the second guide sequence directing cleavage of other strand near the second target sequence results in a blunt cut.

49. The method of claim 36, wherein the first guide sequence directing cleavage of one strand of the DNA duplex near the first target sequence and the second guide sequence directing cleavage of other strand near the second target sequence results in a 3′

overhang.

50. The method of claim 49, wherein the 3′

overhang is at most 150 base pairs.

51. The method of claim 49, wherein the 3′

overhang is at most 100 base pairs.

52. The method of claim 49, wherein the 3′

overhang is at most 50 base pairs.

53. The method of claim 49, wherein the 3′

overhang is at most 25 base pairs.

54. The method of claim 49, wherein the 3′

overhang is at least 15 base pairs.

55. The method of claim 49, wherein the 3′

overhang is at least 10 base pairs.

56. The method of claim 49, wherein the 3′

overhang is at least 1 base pair.

57. The method of claim 49, wherein the 3′

overhang is 1-100 base pairs.

58. The method of claim 36, wherein any or all of the polynucleotide sequence encoding the CRISPR enzyme, the first and the second guide sequence, the first and the second tracr mate sequence or the first and the second tracr sequence, is/are RNA.

59. The method of claim 36, wherein the first and second tracr mate sequence share 100% identity.

60. The method of claim 36, wherein the first and second tracr sequence share 100% identity.

61. The method of claim 36, wherein the CRISPR enzyme is a Cas9 enzyme.

62. The method of claim 61, wherein the CRISPR enzyme is SpCas9.

63. The method of claim 62, wherein the CRISPR enzyme comprises one or more mutations in a catalytic domain.

64. The method of claim 63, wherein the one or more mutations is in a RuvC domain.

65. The method of claim 64, wherein the one or more mutations is selected from the group consisting of D10A, E762A and D986A.

66. The method of claim 65, wherein the CRISPR enzyme has the D10A mutation.

67. The method of claim 63, wherein the one or more mutations is in a HNH domain.

68. The method of claim 67, wherein the one or more mutations is selected from the group consisting of H840A, N854A and N863A.

69. The method of claim 68, wherein the CRISPR enzyme has the H840A mutation.

70. The method of claim 36, wherein one or more of the viral vectors are delivered via nanoparticles, exosomes, microvesicles, or a gene-gun.

71. A method of modifying a genomic locus of interest by minimizing off-target modifications by introducing into a cell containing and expressing a double stranded DNA molecule encoding the gene product an engineered, non-naturally occurring CRISPR-Cas system comprising a Cas protein having one or more mutations and two guide RNAs that target a first strand and a second strand of the DNA molecule respectively, whereby the guide RNAs target the DNA molecule encoding the gene product and the Cas protein nicks each of the first strand and the second strand of the DNA molecule encoding the gene product, whereby expression of the gene product is altered;

and, wherein the Cas protein and the two guide RNAs do not naturally occur together.

72. The method of claim 71, wherein the Cas protein nicking each of the first strand and the second strand of the DNA molecule encoding the gene product results in a 5′

overhang.

73. The method of claim 71, wherein the 5′

overhang is at most 200 base pairs.

74. The method of claim 71, wherein the 5′

overhang is at most 100 base pairs.

75. The method of claim 71, wherein the 5′

overhang is at most 50 base pairs.

76. The method of claim 71, wherein the 5′

overhang is at least 26 base pairs.

77. The method of claim 71, wherein the 5′

overhang is at least 30 base pairs.

78. The method of claim 71, wherein the 5′

overhang is 34-50 base pairs.

79. The method of claim 71, wherein the 5′

overhang is at least 15 base pairs.

80. The method of claim 71, wherein the 5′

overhang is at least 10 base pairs.

81. The method of claim 71, wherein the 5′

overhang is at least 1 base pair.

82. The method of claim 71, wherein the 5′

overhang is 1-34 base pairs.

83. The method of claim 71, wherein the Cas protein nicking each of the first strand and the second strand of the DNA molecule encoding the gene product results in a blunt cut.

84. The method of claim 71, wherein the Cas protein nicking each of the first strand and the second strand of the DNA molecule encoding the gene product results in a 3′

overhang.

85. The method of claim 84, wherein the 3′

overhang is at most 150 base pairs.

86. The method of claim 84, wherein the 3′

overhang is at most 100 base pairs.

87. The method of claim 84, wherein the 3′

overhang is at most 50 base pairs.

88. The method of claim 84, wherein the 3′

overhang is at most 25 base pairs.

89. The method of claim 84, wherein the 3′

overhang is at least 15 base pairs.

90. The method of claim 84, wherein the 3′

overhang is at least 10 base pairs.

91. The method of claim 84, wherein the 3′

overhang is at least 1 base pair.

92. The method of claim 84, wherein the 3′

overhang is 1-100 base pairs.

93. The method of claim 71, wherein the guide RNAs comprise a guide sequence fused to a tracr mate sequence and a tracr sequence.

94. The method of claim 71, wherein the Cas protein is codon optimized for expression in a eukaryotic cell.

95. The method of claim 94, wherein the eukaryotic cell is a mammalian cell.

96. The method of claim 95, wherein the mammalian cell is a human cell.

97. The method of claim 71, wherein the Cas protein is a type II CRISPR-Cas protein.

98. The method of claim 97, wherein the Cas protein is a Cas9 protein.

99. The method of claim 98, wherein the Cas protein is SpCas9.

100. The method of claim 99, wherein the Cas protein comprises one or more mutations in a catalytic domain.

101. The method of claim 100, wherein the one or more mutations is in a RuvC domain.

102. The method of claim 101, wherein the one or more mutations is selected from the group consisting of D10A, E762A and D986A.

103. The method of claim 102, wherein the Cas protein has the D10A mutation.

104. The method of claim 100, wherein the one or more mutations is in a HNH domain.

105. The method of claim 104, wherein the one or more mutations is selected from the group consisting of H840A, N854A and N863A.

106. The method of claim 105, wherein the Cas protein has the H840A mutation.

107. The method of claim 71, wherein the expression of the gene product is decreased.

108. The method of claim 71, wherein a template polynucleotide is further introduced into the DNA molecule encoding the gene product or an intervening sequence is excised precisely allowing 5′

overhangs to reanneal and ligate.

109. The method of claim 71, wherein the expression of the gene product is increased or the activity or function of the gene product is altered.

110. The method of claim 71, wherein the gene product is a protein.

111. An engineered, non-naturally occurring CRISPR-Cas system comprising a Cas protein having one or more mutations and two guide RNAs that target a first strand and a second strand respectively of a double stranded DNA molecule encoding a gene product in a cell, whereby the guide RNAs target the DNA molecule encoding the gene product and the Cas protein nicks each of the first strand and the second strand of the DNA molecule encoding the gene product, whereby expression of the gene product is altered;

and, wherein the Cas protein and the two guide RNAs do not naturally occur together.

112. The CRISPR-Cas system of claim 111, wherein the guide RNAs comprise a guide sequence fused to a tracr mate sequence and a tracr sequence.

113. The CRISPR-Cas system of claim 111, wherein the Cas protein is a type II CRISPR-Cas protein.

114. The CRISPR-Cas system of claim 113, wherein the Cas protein is a Cas9 protein.

115. The CRISPR-Cas system of claim 114, wherein the Cas protein is SpCas9.

116. The CRISPR-Cas system of claim 115, wherein the Cas protein comprises one or more mutations in a catalytic domain.

117. The CRISPR-Cas system of claim 116, wherein the one or more mutations is in a RuvC domain.

118. The CRISPR-Cas system of claim 117, wherein the one or more mutations is selected from the group consisting of D10A, E762A and D986A.

119. The CRISPR-Cas system of claim 118, wherein the Cas protein has the D10A mutation.

120. The CRISPR-Cas system of claim 116, wherein the one or more mutations is in a HNH domain.

121. The CRISPR-Cas system of claim 120, wherein the one or more mutations is selected from the group consisting of H840A, N854A and N863A.

122. The CRISPR-Cas system of claim 121, wherein the Cas protein has the H840A mutation.

123. The CRISPR-Cas system of claim 111, wherein the Cas protein is codon optimized for expression in a eukaryotic cell.

124. The CRISPR-Cas system of claim 123, wherein the eukaryotic cell is a mammalian cell.

125. The CRISPR-Cas system of claim 124, wherein the mammalian cell is a human cell.

126. The CRISPR-Cas system of claim 111, wherein the expression of the gene product is decreased.

127. The CRISPR-Cas system of claim 111, wherein a template polynucleotide is further introduced into the DNA molecule encoding the gene product or an intervening sequence is excised precisely allowing 5′

or 3′

overhangs to reanneal and ligate.

128. The CRISPR-Cas system of claim 111, wherein the expression of the gene product is increased or the activity or function of the gene product is altered.

129. The CRISPR-Cas system of claim 111, wherein the gene product is a protein.

130. An engineered, non-naturally occurring vector system comprising one or more vectors comprising:

a) a first regulatory element operably linked to each of two CRISPR-Cas system guide RNAs that target a first strand and a second strand respectively of a double stranded DNA molecule encoding a gene product,

b) a second regulatory element operably linked to a Cas protein,

wherein components (a) and (b) are located on same or different vectors of the system,

whereby the guide RNAs target the DNA molecule encoding the gene product and the Cas protein nicks each of the first strand and the second strand of the DNA molecule encoding the gene product, whereby expression of the gene product is altered; and

, wherein the Cas protein and the two guide RNAs do not naturally occur together.

131. The vector system of claim 130, wherein the guide RNAs comprise a guide sequence fused to a tracr mate sequence and a tracr sequence.

132. The vector system of claim 130, wherein the Cas protein is a type II CRISPR-Cas protein.

133. The vector system of claim 132, wherein the Cas protein is a Cas9 protein.

134. The vector system of claim 133, wherein the Cas protein is SpCas9.

135. The vector system of claim 134, wherein the Cas protein comprises one or more mutations in a catalytic domain.

136. The vector system of claim 135, wherein the one or more mutations is in a RuvC domain.

137. The vector system of claim 136, wherein the one or more mutations is selected from the group consisting of D10A, E762A and D986A.

138. The vector system of claim 137, wherein the Cas protein has the D10A mutation.

139. The vector system of claim 135, wherein the one or more mutations is in a HNH domain.

140. The vector system of claim 139, wherein the one or more mutations is selected from the group consisting of H840A, N854A and N863A.

141. The vector system of claim 140, wherein the Cas protein has the H840A mutation.

142. The vector system of claim 130, wherein the Cas protein is codon optimized for expression in a eukaryotic cell.

143. The vector system of claim 142, wherein the eukaryotic cell is a mammalian cell.

144. The vector system of claim 143, wherein the mammalian cell is a human cell.

145. The vector system of claim 130, wherein the gene product is a protein.

146. The vector system of claim 130, wherein the expression of the gene product is decreased.

147. The vector system of claim 130, wherein a template polynucleotide is further introduced into the DNA molecule encoding the gene product or an intervening sequence is excised precisely allowing 5′

or 3′

overhangs to reanneal and ligate.

148. The vector system of claim 130, wherein the expression of the gene product is increased or the activity or function of the gene product is altered.

149. The vector system of claim 130, wherein the vectors of the system are viral vectors.

150. The vector system of claim 130, wherein the vectors of the system are delivered via nanoparticles, exosomes, microvesicles, or a gene-gun.

151. A method of modifying an organism comprising a first and a second target sequence on opposite strands of a DNA duplex in a genomic locus of interest in a cell by promoting homology directed repair comprising delivering a non-naturally occurring or engineered composition comprising:

I. a first CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, wherein the first polynucleotide sequence comprises;

(a) a first guide sequence capable of hybridizing to the first target sequence,

(b) a first tracr mate sequence, and

(c) a first tracr sequence,

II. a second CRISPR-Cas system chiRNA polynucleotide sequence, wherein the second polynucleotide sequence comprises;

(a) a second guide sequence capable of hybridizing to the second target sequence,

(b) a second tracr mate sequence, and

(c) a second tracr sequence, and

III. a polynucleotide sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences and comprising one or more mutations,

IV. a repair template comprising a synthesized or engineered single-stranded oligonucleotide,

wherein (a), (b) and (c) are arranged in a 5′

to 3′

orientation,

wherein when transcribed, the first and the second tracr mate sequence hybridize to the first and second tracr sequence respectively and the first and the second guide sequence directs sequence-specific binding of a first and a second CRISPR complex to the first and second target sequences respectively,

wherein the first CRISPR complex comprises the CRISPR enzyme complexed with (1) the first guide sequence that is hybridizable to the first target sequence, and (2) the first tracr mate sequence that is hybridized to the first tracr sequence,

wherein the second CRISPR complex comprises the CRISPR enzyme complexed with (1) the second guide sequence that is hybridizable to the second target sequence, and (2) the second tracr mate sequence that is hybridized to the second tracr sequence,

wherein the polynucleotide sequence encoding a CRISPR enzyme is DNA or RNA,

wherein the first guide sequence directs cleavage of one strand of the DNA duplex near the first target sequence and the second guide sequence directs cleavage of other strand near the second target sequence inducing a double strand break, and

wherein the repair template is introduced into the DNA duplex by homologous recombination, whereby the organism is modified.

152. The method of claim 151 wherein the repair template further comprises a restriction endonuclease restriction site.

153. The method of claim 151, wherein the first guide sequence directing cleavage of one strand of the DNA duplex near the first target sequence and the second guide sequence directing cleavage of other strand near the second target sequence results in a 5′

or 3′

overhang.

154. The method of claim 153, wherein the 5′

overhang is 1-200 base pairs.

155. The method of claim 153, wherein the 3′

overhang is 1-100 base pairs.

156. The method of claim 151, wherein any or all of the polynucleotide sequence encoding the CRISPR enzyme, the first and the second guide sequence, the first and the second tracr mate sequence or the first and the second tracr sequence, is/are RNA.

157. The method of claim 151, wherein the polynucleotides comprising the sequence encoding the CRISPR enzyme, the first and the second guide sequence, the first and the second tracr mate sequence or the first and the second tracr sequence, is/are RNA and are delivered via nanoparticles, exosomes, microvesicles, or a gene-gun.

158. The method of claim 151, wherein the first and second tracr mate sequence share 100% identity.

159. The method of claim 151, wherein the first and second tracr sequence share 100% identity.

160. The method of claim 151, wherein the CRISPR enzyme is a Cas9 enzyme.

161. The method of claim 160, wherein the CRISPR enzyme is SpCas9.

162. The method of claim 161, wherein the CRISPR enzyme comprises one or more mutations in a catalytic domain.

163. The method of claim 162, wherein the one or more mutations is in a RuvC domain.

164. The method of claim 163, wherein the one or more mutations is selected from the group consisting of D10A, E762A and D986A.

165. The method of claim 164, wherein the CRISPR enzyme has the D10A mutation.

166. The method of claim 162, wherein the one or more mutations is in a HNH domain.

167. The method of claim 166, wherein the one or more mutations is selected from the group consisting of H840A, N854A and N863A.

168. The method of claim 167, wherein the CRISPR enzyme has the H840A mutation.

169. A method of modifying an organism comprising a first and a second target sequence on opposite strands of a DNA duplex in a genomic locus of interest in a cell by facilitating non homologous end joining (NHEJ) mediated ligation comprising

delivering a non-naturally occurring or engineered composition comprising:

I. a first CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, wherein the first polynucleotide sequence comprises;

(a) a first guide sequence capable of hybridizing to the first target sequence,

(b) a first tracr mate sequence, and

(c) a first tracr sequence,

II. a second CRISPR-Cas system chiRNA polynucleotide sequence, wherein the second polynucleotide sequence comprises;

(a) a second guide sequence capable of hybridizing to the second target sequence,

(b) a second tracr mate sequence, and

(c) a second tracr sequence, and

III. a polynucleotide sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences and comprising one or more mutations,

IV. a repair template comprising a first set of overhangs,

wherein (a), (b) and (c) are arranged in a 5′

to 3′

orientation,

wherein when transcribed, the first and the second tracr mate sequence hybridize to the first and second tracr sequence respectively and the first and the second guide sequence directs sequence-specific binding of a first and a second CRISPR complex to the first and second target sequences respectively,

wherein the first CRISPR complex comprises the CRISPR enzyme complexed with (1) the first guide sequence that is hybridizable to the first target sequence, and (2) the first tracr mate sequence that is hybridized to the first tracr sequence,

wherein the second CRISPR complex comprises the CRISPR enzyme complexed with (1) the second guide sequence that is hybridizable to the second target sequence, and (2) the second tracr mate sequence that is hybridized to the second tracr sequence,

wherein the polynucleotide sequence encoding a CRISPR enzyme is DNA or RNA,

wherein the first guide sequence directs cleavage of one strand of the DNA duplex near the first target sequence and the second guide sequence directs cleavage of other strand near the second target sequence inducing a double strand break with a second set of overhangs,

wherein the first set of overhangs is compatible with and matches the second set of overhangs, and

wherein the repair template is introduced into the DNA duplex by ligation, whereby the organism is modified.

170. The method according to claim 169 wherein the repair template is a synthesized or engineered double-stranded oligonucleotide duplex.

171. The method according to claim 169 wherein the repair template is generated from a piece of DNA that is introduced into the cell and is enzymatically processed.

172. The method of claim 169, wherein the first guide sequence directing cleavage of one strand of the DNA duplex near the first target sequence and the second guide sequence directing cleavage of other strand near the second target sequence results in a 5′

or 3′

overhang.

173. The method of claim 172, wherein the 5′

overhang is 1-200 base pairs.

174. The method of claim 172, wherein the 3′

overhang is 1-100 base pairs.

175. The method of claim 169, wherein any or all of the polynucleotide sequence encoding the CRISPR enzyme, the first and the second guide sequence, the first and the second tracr mate sequence or the first and the second tracr sequence, is/are RNA.

176. The method of claim 169, wherein the polynucleotides comprising the sequence encoding the CRISPR enzyme, the first and the second guide sequence, the first and the second tracr mate sequence or the first and the second tracr sequence, is/are RNA and are delivered via nanoparticles, exosomes, microvesicles, or a gene-gun.

177. The method of claim 169, wherein the first and second tracr mate sequence share 100% identity.

178. The method of claim 169, wherein the first and second tracr sequence share 100% identity.

179. The method of claim 169, wherein the CRISPR enzyme is a Cas9 enzyme.

180. The method of claim 179, wherein the CRISPR enzyme is SpCas9.

181. The method of claim 180, wherein the CRISPR enzyme comprises one or more mutations in a catalytic domain.

182. The method of claim 181, wherein the one or more mutations is in a RuvC domain.

183. The method of claim 182, wherein the one or more mutations is selected from the group consisting of D10A, E762A and D986A.

184. The method of claim 183, wherein the CRISPR enzyme has the D10A mutation.

185. The method of claim 181, wherein the one or more mutations is in a HNH domain.

186. The method of claim 185, wherein the one or more mutations is selected from the group consisting of H840A, N854A and N863A.

187. The method of claim 186, wherein the CRISPR enzyme has the H840A mutation.

188. A method of modifying a DNA duplex at a locus of interest in a cell, the method comprising delivering to the cell:

I. a first polynucleotide comprising;

(a) a first guide sequence capable of hybridizing to a first target sequence,

(b) a first tracr mate sequence, and

(c) a first tracr sequence;

II. a second polynucleotide comprising;

(a) a second guide sequence capable of hybridizing to a second target sequence,

(b) a second tracr mate sequence, and

(c) a second tracr sequence;

and

III. a third polynucleotide comprising a sequence encoding a CRISPR enzyme and one or more nuclear localization sequences

wherein (a), (b) and (c) in said first and second polynucleotides are arranged in a 5′

to 3′

orientation;

wherein the first target sequence is on a first strand of the DNA duplex and the second target sequence is on the opposite strand of the DNA duplex, and when the first and second guide sequences are hybridized to said target sequences in the duplex, the 5′

ends of the first polynucleotide and the second polynucleotide are offset relative to each other by at least one base pair of the duplex;

wherein when transcribed, the first and the second tracr mate sequences hybridize to the first and second tracr sequences, respectively, and the first and the second guide sequences direct sequence-specific binding of a first and a second CRISPR complex to the first and second target sequences respectively,

wherein the first CRISPR complex comprises the CRISPR enzyme complexed with (1) the first guide sequence that is hybridizable to the first target sequence, and (2) the first tracr mate sequence that is hybridized to the first tracr sequence,

wherein the second CRISPR complex comprises the CRISPR enzyme complexed with (1) the second guide sequence that is hybridizable to the second target sequence, and (2) the second tracr mate sequence that is hybridized to the second tracr sequence,

and wherein said first strand of the DNA duplex is cleaved near said first target sequence, and said opposite strand of the DNA duplex is cleaved near said second target sequence, resulting in a double strand break with a 5′

overhang or a 3′

overhang.

190. The method of claim 188 or 189, wherein said CRISPR enzyme is a nickase enzyme, optionally a Cas9 enzyme comprising at least one mutation in a catalytic domain.

191. The method of claim 190 wherein said at least one mutation is in the RuvC domain and optionally is selected from the group consisting of D10A, E762A and D986A, or is in the HNH domain and optionally is selected from the group consisting of H840A, N854A and N863A.

192. The method according to any one of claims 190 to 191, wherein said offset between the 5′

ends of the first polynucleotide and the second polynucleotide is greater than −

8 bp or −

278 to +58 bp or −

200 to +200 bp or up to or over 100 bp or −

4 to 20 bp or +23 bp or +16 or +20 or +16 to +20 bp or −

3 to +18 bp.

193. The method according to any one of claims 190 to 192, wherein said cleavage of said first strand and of said opposite strand of the DNA duplex occurs 3′

to a PAM (Protospacer adjacent motif) on each strand, and wherein said PAM on said first strand is separated from said PAM on said opposite strand by from 30 to 150 base pairs.

194. The method according to any one of claims 190 to 193, wherein said overhang is at most 200 bases, at most 100 bases, or at most 50 bases.

195. The method according to claim 194, wherein the overhang is at least 1 base, at least 10 bases, at least 15 bases, at least 26 bases or at least 30 bases.

196. The method according to claim 194, wherein the overhang is between 34 and 50 bases or between 1 and 34 bases.

197. The method according to any one of claims 188 to 197, wherein any or all of the polynucleotide sequence encoding the CRISPR enzyme, the first and the second guide sequence, the first and the second tracr mate sequence or the first and the second tracr sequence, is/are RNA, and optionally wherein any or all of I, II and III are delivered via nanoparticles, exosomes, microvesicles, or a gene-gun.

198. The method according to any one of claims 188 to 198, wherein the first and second tracr mate sequence share 100% identity and/or the first and second tracr sequence share 100% identity.

199. The method according to claim 188, wherein each of I, II and III is provided in a vector, optionally wherein each is provided in the same or a different vector.

200. The method according to any one of claims 188 to 200, wherein said locus of interest comprises a gene and wherein said method results in a change in the expression of said gene, or in a change in the activity or function of the gene product.

202. The method according to any one of claims 188 to 201, further comprising:

delivering to the cell a double-stranded oligodeoxynucleotide (dsODN) comprising overhangs complimentary to the overhangs created by said double strand break, wherein said dsODN is integrated into the locus of interest;

or

delivering to the cell a single-stranded oligodeoxynucleotide (ssODN), wherein said ssODN acts as a template for homology directed repair of said double strand break.

203. The method according to any one of claims 188 to 201, which is for the prevention or treatment of disease in an individual, optionally wherein said disease is caused by a defect in said locus of interest.

204. The method according to claim 203, wherein the method is conducted in vivo in the individual or ex vivo on a cell taken from the individual, optionally wherein said cell is returned to the individual.

206. Use of a kit or composition according to claim 205 in a method according to any one of claims 188 to 204.

189. A method of modifying a DNA duplex at a locus of interest in a cell, the method comprising delivering to the cell a vector system comprising one or more vectors comprising:

I. a first polynucleotide sequence comprising a regulatory element operably linked to

(a) a first guide sequence capable of hybridizing to a first target sequence, and

(b) at least one or more tracr mate sequences,

II. a second polynucleotide sequence comprising a second regulatory element operably linked to

(a) a second guide sequence capable of hybridizing to a second target sequence, and

(b) at least one or more tracr mate sequences,

III. a third polynucleotide sequence comprising a third regulatory element operably linked to a sequence encoding a CRISPR enzyme, and

IV. a fourth polynucleotide sequence comprising a fourth regulatory element operably linked to a tracr sequence,

wherein components I, II, III and IV are located on the same or different vectors of the system

wherein the first target sequence is on a first strand of the DNA duplex and the second target sequence is on the opposite strand of the DNA duplex, and when the first and second guide sequences are hybridized to said target sequences in the duplex, the 5′

ends of the first polynucleotide and the second polynucleotide are offset relative to each other by at least one base pair of the duplex;

wherein when transcribed, the first and the second tracr mate sequences hybridize to a tracr sequence, and the first and the second guide sequences direct sequence-specific binding of a first and a second CRISPR complex to the first and second target sequences respectively,

wherein the first CRISPR complex comprises the CRISPR enzyme complexed with (1) the first guide sequence that is hybridizable to the first target sequence, and (2) the first tracr mate sequence that is hybridized to a tracr sequence,

wherein the second CRISPR complex comprises the CRISPR enzyme complexed with (1) the second guide sequence that is hybridizable to the second target sequence, and (2) the second tracr mate sequence that is hybridized to a tracr sequence,

and wherein said first strand of the DNA duplex is cleaved near said first target sequence, and said opposite strand of the DNA duplex is cleaved near said second target sequence, resulting in a double strand break with a 5′

overhang or a 3′

overhang.

201. The method according to claim 201, wherein said gene product is a protein, and/or wherein said change in expression, activity or function is a reduction in said expression, activity or function.

205. A kit or composition comprising:

I. a first polynucleotide comprising;

(a) a first guide sequence capable of hybridizing to a first target sequence,

(b) a first tracr mate sequence, and

(c) a first tracr sequence;

II. a second polynucleotide comprising;

(a) a second guide sequence capable of hybridizing to a second target sequence,

(b) a second tracr mate sequence, and

(c) a second tracr sequence;

and

III. a third polynucleotide comprising a sequence encoding a CRISPR enzyme and one or more nuclear localization sequences

wherein (a), (b) and (c) in said first and second polynucleotides are arranged in a 5′

to 3′

orientation;

wherein the first target sequence is on a first strand of a DNA duplex and the second target sequence is on the opposite strand of the DNA duplex, and when the first and second guide sequences are hybridized to said target sequences in the duplex, the 5′

ends of the first polynucleotide and the second polynucleotide are offset relative to each other by at least one base pair of the duplex,

and optionally wherein each of I, II and III is provided in the same or a different vector.

207. Use of a kit or composition according to claim 205 in the manufacture of a medicament, optionally wherein said medicament is for the prevention or treatment of a disease caused by a defect in said locus of interest.