DELIVERY, ENGINEERING AND OPTIMIZATION OF TANDEM GUIDE SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION

• US 20160186213A1
• Filed: 12/17/2015
• Published: 06/30/2016
• 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: ZHANG, Feng, LIN, Chie-yu, RAN, Fei
• Granted Patent: US 11,008,588 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• 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 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• 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 2310/3519 Fusion with another nucleic acid
• 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 a genomic locus of interest in a cell comprising

delivering a non-naturally occurring or engineered composition comprising a tandem guideRNA construct comprising:

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

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

(b) a first tracr mate sequence,

(c) a first tracr sequence,

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

(e) a second tracr mate sequence, and

(f) a second tracr sequence, and

II. a polynucleotide sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences (NLS)s,

wherein the polynucleotide sequence comprises a linker sequence between the first tracr sequence and the second guide sequence, whereby the first guide sequence and the second guide sequence are in tandem,

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 hybridized 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 hybridized 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 genomic locus.

2. The method of claim 1, wherein the modifying of the genomic locus comprises inducing an indel and/or a microdeletion.

3. The method of claim 2, wherein the microdeletion comprises deletion of a sequence between the first and second target sequences.

4. The method of claim 1, wherein the linker sequence comprises at least 5 nucleotides.

5. The method of claim 1, wherein the linker sequence comprises at least 10 nucleotides.

6. The method of claim 1, wherein the linker sequence comprises at least 20 nucleotides.

7. The method of claim 1, wherein the linker sequence has 8 or 12 nucleotides.

8. The method of claim 1, wherein the first or second guide sequence, the first or second tracr sequence and/or the first or second tracr mate sequences are modified.

9. The method of claim 8, wherein the modification comprises optimized first or second tracr sequence and/or optimized first or second guide sequence and/or, co-fold structure of first or second tracr sequence and/or first or second tracr mate sequence(s) respectively and/or stabilizing secondary structures (hair pins) of first or second tracr sequence and/or first or second tracr sequence with a reduced region of base-pairing and/or first or second tracr sequence fused RNA elements.

10. 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.

11. The method of claim 1, wherein the polynucleotides encoding 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.

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

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

14. The method of claim 1, wherein the CRISPR enzyme is a Cas9 enzyme functioning as a nuclease or a Cas9 enzyme functioning as a nickase.

15. The method of claim 14, wherein the CRISPR enzyme is SpCas9.

16. A method of modifying an organism or a non-human organism 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,

(b) a first tracr mate sequence,

(c) a first tracr sequence,

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

(e) a second tracr mate sequence, and

(f) a second tracr sequence, and

wherein a linker sequence is present between the first tracr sequence and the second guide sequence, whereby the first guide sequence and the second guide sequence are in tandem,

and

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

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

when transcribed, a first tracr mate sequence hybridizes to a first 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 hybridized 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 hybridized 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.

17. The method of claim 16, wherein the modifying of the organism or the non-human organism comprises inducing an indel and/or a microdeletion.

18. The method of claim 17, wherein the microdeletion comprises deletion of a sequence between the first and second target sequences.

19. The method of claim 16, wherein the linker sequence comprises at least 5 nucleotides.

20. The method of claim 16, wherein the linker sequence comprises at least 10 nucleotides.

21. The method of claim 16, wherein the linker sequence comprises at least 20 nucleotides.

22. The method of claim 16, wherein the linker sequence has 8 or 12 nucleotides.

23. The method of claim 16, wherein the first or second guide sequence, the first or second tracr sequence and/or the first or second tracr mate sequences are modified.

24. The method of claim 23, wherein the modification comprises optimized first or second tracr sequence and/or optimized first or second guide sequence and/or, co-fold structure of first or second tracr sequence and/or first or second tracr mate sequence(s) respectively and/or stabilizing secondary structures (hair pins) of first or second tracr sequence and/or first or second tracr sequence with a reduced region of base-pairing and/or first or second tracr sequence fused RNA elements.

25. The method of claim 16, 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.

26. The method of claim 16, wherein the polynucleotides encoding 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.

27. The method of claim 16, wherein the first and second tracr mate sequence share 100% identity.

28. The method of claim 16, wherein the first and second tracr sequence share 100% identity.

29. The method of claim 16, wherein the CRISPR enzyme is a Cas9 enzyme functioning as a nuclease or a Cas9 enzyme functioning as a nickase.

30. The method of claim 29, wherein the CRISPR enzyme is SpCas9.

31. A non-naturally occurring or engineered composition for modifying an organism or a non-human organism 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:

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

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

(b) a first tracr mate sequence,

(c) a first tracr sequence,

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

(e) a second tracr mate sequence, and

(f) a second tracr sequence, and

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

wherein the polynucleotide sequence comprises a linker sequence between the first tracr sequence and the second guide sequence, whereby the first guide sequence and the second guide sequence are in tandem,

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 hybridized 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 hybridized 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 to modify the organism or the non-human organism.

32. The composition of claim 31, wherein the modifying of the organism or the non-human organism with a reduction in likelihood of off-target modifications comprises inducing an indel and/or a microdeletion.

33. The composition of claim 32, wherein the microdeletion comprises deletion of a sequence between the first and second target sequences.

34. The composition of claim 31, wherein the linker sequence comprises at least 5 nucleotides.

35. The composition of claim 31, wherein the linker sequence comprises at least 10 nucleotides.

36. The composition of claim 31, wherein the linker sequence comprises at least 20 nucleotides.

37. The composition of claim 31, wherein the linker sequence has 8 or 12 nucleotides.

38. The composition of claim 31, wherein the first or second guide sequence, the first or second tracr sequence and/or the first or second tracr mate sequences are modified.

39. The composition of claim 38, wherein the modification comprises optimized first or second tracr sequence and/or optimized first or second guide sequence and/or, co-fold structure of first or second tracr sequence and/or first or second tracr mate sequence(s) respectively and/or stabilizing secondary structures (hair pins) of first or second tracr sequence and/or first or second tracr sequence with a reduced region of base-pairing and/or first or second tracr sequence fused RNA elements.

40. The composition of claim 31, 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.

41. The composition of claim 31, wherein the polynucleotides encoding 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.

42. The composition of claim 31, wherein the first and second tracr mate sequence share 100% identity.

43. The composition of claim 31, wherein the first and second tracr sequence share 100% identity.

44. The composition of claim 31, wherein the CRISPR enzyme is a Cas9 enzyme functioning as a nuclease or a Cas9 enzyme functioning as a nickase.

45. The composition of claim 44, wherein the CRISPR enzyme is SpCas9.

46. A method of modifying an organism or a non-human organism 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 CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, wherein the polynucleotide sequence comprises;

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

(b) a first tracr mate sequence,

(c) a first tracr sequence,

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

(e) a second tracr mate sequence, and

(f) a second tracr sequence, and

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

wherein the polynucleotide sequence comprises a linker sequence between the first tracr sequence and the second guide sequence, whereby the first guide sequence and the second guide sequence are in tandem,

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 hybridized 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 hybridized 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.

47. The method of claim 46, 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.

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

overhang is at most 200 base pairs.

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

overhand is at most 100 base pairs.

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

overhang is at most 50 base pairs.

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

overhang is at least 26 base pairs.

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

overhang is at least 30 base pairs.

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

overhang is 34-50 base pairs.

54. The method of claim 46, 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.

55. The method of claim 46, wherein the first and second tracr mate sequence share 100% identity.

56. The method of claim 46, wherein the first and second tracr sequence share 100% identity.

57. The method of claim 46, wherein the CRISPR enzyme is a Cas9 enzyme.

58. The method of claim 57, wherein the CRISPR enzyme is SpCas9.

59. The method of claim 58, wherein the CRISPR enzyme comprises one or more mutations in a catalytic domain.

60. The method of claim 59, wherein the CRISPR enzyme comprises one or more mutations selected from the group consisting of D10A, E762A, H840A, N854A, N863A and D986A.

61. The method of claim 60, wherein the CRISPR enzyme has the D10A mutation.

62. The method of claim 46, wherein the modification comprises optimized first or second tracr sequence and/or optimized first or second guide sequence and/or, co-fold structure of first or second tracr sequence and/or first or second tracr mate sequence(s) respectively and/or stabilizing secondary structures (hair pins) of first or second tracr sequence and/or first or second tracr sequence with a reduced region of base-pairing and/or first or second tracr sequence fused RNA elements.

97. The method of claims 46 or 63, wherein the linker sequence comprises at least 5 nucleotides.

98. The method of claims 46 or 63, wherein the linker sequence comprises at least 10 nucleotides.

99. The method of claims 46 or 63, wherein the linker sequence comprises at least 20 nucleotides.

100. The method of claims 46 or 63, wherein the linker sequence has 8 nucleotides.

63. A method of modifying an organism or a non-human organism 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,

(b) a first tracr mate sequence,

(c) a first tracr sequence,

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

(e) a second tracr mate sequence, and

(f) a second tracr sequence, and

wherein a linker sequence is present between the first tracr sequence and the second guide sequence, whereby the first guide sequence and the second guide sequence are in tandem,

and

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

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

when transcribed, a first tracr mate sequence hybridizes to a first 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 hybridized 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 hybridized 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.

64. The method of claim 63, 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.

65. The method of claim 64, wherein the 5′

overhang is at most 200 base pairs.

66. The method of claim 64, wherein the 5′

overhand is at most 100 base pairs.

67. The method of claim 64, wherein the 5′

overhang is at most 50 base pairs.

68. The method of claim 64, wherein the 5′

overhang is at least 26 base pairs.

69. The method of claim 64, wherein the 5′

overhang is at least 30 base pairs.

70. The method of claim 64, wherein the 5′

overhang is 34-50 base pairs.

71. The method of claim 64, 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.

72. The method of claim 63, wherein the first and second tracr mate sequence share 100% identity.

73. The method of claim 63, wherein the first and second tracr sequence share 100% identity.

74. The method of claim 63, wherein the CRISPR enzyme is a Cas9 enzyme.

75. The method of claim 74, wherein the CRISPR enzyme is SpCas9.

76. The method of claim 75, wherein the CRISPR enzyme comprises one or more mutations in a catalytic domain.

77. The method of claim 76, wherein the CRISPR enzyme comprises one or more mutations selected from the group consisting of D10A, E762A, H840A, N854A, N863A and D986A.

78. The method of claim 77, wherein the CRISPR enzyme has the D10A mutation.

79. The method of claim 63, wherein one or more of the viral vectors are delivered via nanoparticles, exosomes, microvesicles, or a gene-gun.

80. A non-naturally occurring or engineered composition modifying an organism or a non-human organism 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:

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

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

(b) a first tracr mate sequence,

(c) a first tracr sequence,

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

(e) a second tracr mate sequence, and

(f) a second tracr sequence, and

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

wherein the polynucleotide sequence comprises a linker sequence between the first tracr sequence and the second guide sequence, whereby the first guide sequence and the second guide sequence are in tandem,

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 hybridized 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 hybridized 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 to modify the organism or the non-human organism.

81. The composition of claim 80, 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.

82. The composition of claim 81, wherein the 5′

overhang is at most 200 base pairs.

83. The composition of claim 81, wherein the 5′

overhand is at most 100 base pairs.

84. The composition of claim 81, wherein the 5′

overhang is at most 50 base pairs.

85. The composition of claim 81, wherein the 5′

overhang is at least 26 base pairs.

86. The composition of claim 81, wherein the 5′

overhang is at least 30 base pairs.

87. The composition of claim 81, wherein the 5′

overhang is 34-50 base pairs.

88. The composition of claim 80, 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.

89. The composition of claim 80, wherein the first and second tracr mate sequence share 100% identity.

90. The composition of claim 80, wherein the first and second tracr sequence share 100% identity.

91. The composition of claim 80, wherein the CRISPR enzyme is a Cas9 enzyme.

92. The composition of claim 91, wherein the CRISPR enzyme is SpCas9.

93. The composition of claim 92, wherein the CRISPR enzyme comprises one or more mutations in a catalytic domain.

94. The composition of claim 93, wherein the CRISPR enzyme comprises one or more mutations selected from the group consisting of D10A, E762A, H840A, N854A, N863A and D986A.

95. The composition of claim 94, wherein the CRISPR enzyme has the D10A mutation.

96. The composition of claim 80, wherein the modification comprises optimized first or second tracr sequence and/or optimized first or second guide sequence and/or, co-fold structure of first or second tracr sequence and/or first or second tract mate sequence(s) respectively and/or stabilizing secondary structures (hair pins) of first or second tracr sequence and/or first or second tracr sequence with a reduced region of base-pairing and/or first or second tracr sequence fused RNA elements.

101. The composition of claim 80, wherein the linker sequence comprises at least 5 nucleotides.

102. The composition of claim 80, wherein the linker sequence comprises at least 10 nucleotides.

103. The composition of claim 80, wherein the linker sequence comprises at least 20 nucleotides.

104. The composition of claim 80, wherein the linker sequence has 8 or 12 nucleotides.

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

I. a polynucleotide comprising;

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

(b) a first tracr mate sequence,

(c) a first tracr sequence,

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

(e) a second tracr mate sequence, and

(f) a second tracr sequence,

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

to 3′

orientation and a linker sequence is present between (c) and (d), whereby the first guide sequence and the second guide sequence are in tandem,

and

II. a polynucleotide comprising a sequence encoding a CRISPR enzyme and one or more nuclear localization sequences,

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 guide sequence and the second guide sequence 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 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 hybridized 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 hybridized 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.

107. The method of claim 105 or 106, wherein said CRISPR enzyme is a nickase enzyme, optionally a Cas9 enzyme comprising at least one mutation in a catalytic domain.

108. The method of claim 107 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.

109. The method according to any one of claims 105 to 108, wherein said offset between the 5′

ends of the first polynucleotide and the second polynucleotide is 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.

110. The method according to any one of claims 105 to 109, 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.

111. The method according to any one of claims 105 to 110, wherein said overhang is at most 200 bases, at most 100 bases, or at most 50 bases.

112. The method according to claim 111, 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.

113. The method according to claim 111, wherein the overhang is between 34 and 50 bases or between 1 and 34 bases.

114. The method of any one of claims 105 to 113 wherein said double strand break induces a microdeletion in the locus of interest, preferably between the first and second target sequences.

115. The method of any one of claims 105 to 109, wherein the linker sequence comprises at least 5 nucleotides, at least 10 nucleotides, or at least 20 nucleotides, or wherein the linker consists of 8 nucleotides.

116. The method according to any one of claims 105 to 110, 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.

117. The method according to any one of claims 105 to 111, wherein the first and second tracr mate sequence share 100% identity and/or the first and second tracr sequence share 100% identity.

118. The method according to claim 105, wherein each of I and II is provided in a vector, optionally wherein each is provided in the same or a different vector.

119. The method according to any one of claims 105 to 113, wherein the first or second guide sequence, the first or second tracr sequence and/or the first or second tracr mate sequences are modified, optionally wherein the modification comprises optimized first or second tracr sequence and/or optimized first or second guide sequence and/or, co-fold structure of first or second tracr sequence and/or first or second tracr mate sequence(s) respectively and/or stabilizing secondary structures (hair pins) of first or second tracr sequence and/or first or second tracr sequence with a reduced region of base-pairing and/or first or second tracr sequence fused RNA elements.

120. The method according to any one of claims 105 to 119, 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.

121. The method according to claim 120, 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.

122. The method according to any one of claims 105 to 121, 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.

123. The method according to any one of claims 105 to 122, 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.

124. The method according to claim 123, 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.

126. Use of a kit or composition according to claim 125 in a method according to any one of claims 105 to 124.

106. 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 comprising a regulatory element operably linked to

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

(b) a first tracr mate sequence,

(c) a first tracr sequence,

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

(e) a second tracr mate sequence, and

(f) a second tracr sequence, and

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

to 3′

orientation and a linker sequence is present between (c) and (d), whereby the first guide sequence and the second guide sequence are in tandem,

and

II. a second polynucleotide comprising a second regulatory element operably linked to a sequence encoding a CRISPR enzyme;

wherein components I and II 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 guide sequence and the second guide sequence 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 hybridized 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 hybridized 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.

125. A kit or composition comprising:

I. a polynucleotide comprising;

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

(b) a first tracr mate sequence,

(c) a first tracr sequence,

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

(e) a second tracr mate sequence, and

(f) a second tracr sequence,

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

to 3′

orientation and a linker sequence is present between (c) and (d), whereby the first guide sequence and the second guide sequence are in tandem,

and

II. a polynucleotide comprising a sequence encoding a CRISPR enzyme and one or more nuclear localization sequences,

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 guide sequence and the second guide sequence are offset relative to each other by at least one base pair of the duplex;

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

127. Use of a kit or composition according to claim 125 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.