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1. US20190040416 - Cas Discrimination Using Tuned Guide RNA

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Claims

1. A method of altering a target nucleic acid in a cell comprising
providing to the cell a guide RNA having a spacer sequence that differs from a target protospacer sequence of same length by one or two or three nucleotides, wherein the target protospacer sequence is adjacent a PAM sequence, and wherein the target protospacer sequence differs from a nontarget sequence of same length by one or two nucleotides,
providing to the cell a Cas9 protein or ortholog thereof,
wherein the guide RNA spacer sequence binds to the target complementary protospacer sequence and the Cas9 protein or ortholog thereof interacts with the guide RNA to form a co-localization complex with the target nucleic acid and the target nucleic acid is cleaved in a site specific manner.
2. The method of claim 1 wherein the target protospacer sequence is a single point mutation of the nontarget sequence.
3. The method of claim 1 wherein the target protospacer sequence is endogenous to the cell or exogenous to the cell.
4. The method of claim 1 wherein the nontarget sequence is endogenous to the cell or exogenous to the cell.
5. The method of claim 1 wherein cleaving of the target nucleic acid results in cell death.
6. The method of claim 1 wherein the guide RNA spacer sequence discriminates between the target protospacer sequence and the nontarget sequence.
7. The method of claim 1 wherein the guide RNA spacer sequence and the nontarget sequence are nonbinding.
8. The method of claim 1 wherein the Cas9 protein or ortholog thereof is a Cas9 enzyme, a Cas9 nickase or a nuclease null Cas9 with a nuclease attached thereto.
9. The method of claim 1 wherein the Cas9 protein is naturally occurring or engineered.
10. The method of claim 1 wherein the cell is in vitro, in vivo or ex vivo.
11. The method of claim 1 wherein the cell is a eukaryotic cell or a prokaryotic cell.
12. The method of claim 1 wherein the cell is a bacteria cell, a yeast cell, a fungal cell, a mammalian cell, a human cell, a stem cell, a progenitor cell, an induced pluripotent stem cell, a human induced pluripotent stem cell, a plant cell or an animal cell.
13. The method of claim 1 wherein the target nucleic acid is genomic DNA, mitochondrial DNA, plastid DNA, viral DNA, or exogenous DNA.
14. The method of claim 1 wherein the one or two nucleotide difference is proximal to the PAM sequence.
15. The method of claim 1 further comprising providing the cell with a donor nucleic acid, wherein the donor nucleic acid is inserted into the target nucleic acid.
16. The method of claim 1 further including
providing to the cell a plurality of guide RNAs each having a corresponding spacer sequence that differs from a target protospacer sequence of same length by one or two or three nucleotides, wherein the target protospacer sequence is adjacent a PAM sequence of a target nucleic acid, and wherein the target protospacer sequence differs from a nontarget sequence of same length by one or two nucleotides,
providing to the cell a Cas9 protein or ortholog thereof,
wherein the plurality of guide RNA spacer sequences bind to the corresponding target protospacer sequences and the Cas9 protein or ortholog thereof interacts with each of the plurality of guide RNAs to form co-localization complexes with target nucleic acids and the target nucleic acids are cleaved in a site specific manner.
17. The method of claim 1 wherein the guide RNA is provided to the cell by introducing into the cell a first foreign nucleic acid encoding the guide RNA.
18. The method of claim 1 wherein the Cas9 is provided to the cell by introducing into the cell a second nucleic acid encoding the Cas9.
19. The method of claim 1 wherein the guide RNA is provided to the cell by introducing into the cell a first foreign nucleic acid encoding the guide RNA within a vector and wherein the Cas9 is provided to the cell by introducing into the cell a second nucleic acid encoding the Cas9 within a vector, and wherein the first foreign nucleic acid and the second foreign nucleic acid are provided on the same or different vectors.
20. The method of claim 1 wherein the guide RNA is provided to the cell as a native guide RNA.
21. The method of claim 1 wherein the Cas9 is provided to the cell as a native species.
22. A method of genetically modifying a cell to include a genome editing system which targets single point mutations in cell progeny for target nucleic acid cleavage comprising
providing to the cell a first nucleic acid encoding a guide RNA having a spacer sequence
that differs from a target protospacer sequence of same length by one or two nucleotides, wherein the target protospacer sequence is a single point mutation of the nontarget sequence,
providing to the cell a second nucleic acid encoding a Cas9 protein,
providing the cell under conditions where first cell progeny is produced including the target protospacer sequence and second cell progeny is produced including the nontarget sequence,
wherein the guide RNA spacer sequence binds to the target protospacer sequence and the Cas9 protein interacts with the guide RNA to form a co-localization complex with the target nucleic acid and the target nucleic acid is cleaved in a site specific manner
23. The method of claim 22 wherein cleaving of the target nucleic acid results in cell death.
24. The method of claim 22 wherein the guide RNA spacer sequence and the nontarget sequence of the second cell progeny are nonbinding.
25. The method of claim 22 wherein the Cas9 protein is a Cas9 enzyme, a Cas9 nickase or a nuclease null Cas9 protein with a nuclease attached thereto.
26. The method of claim 22 wherein the cell is in vitro, in vivo or ex vivo.
27. The method of claim 22 wherein the cell is a eukaryotic cell or prokaryotic cell.
28. The method of claim 1 wherein the cell is a bacteria cell, a yeast cell, a fungal cell, a mammalian cell, a human cell, a stem cell, a progenitor cell, an induced pluripotent stem cell, a human induced pluripotent stem cell, a plant cell or an animal cell.
29. The method of claim 22 wherein the target nucleic acid is genomic DNA, mitochondrial DNA, plastid DNA, viral DNA, or exogenous DNA.
30. The method of claim 22 wherein the one or two nucleotide difference is proximal to the PAM sequence.
31. A cell comprising
a guide RNA having a spacer sequence that differs from a target protospacer sequence of same length by one or two nucleotides, wherein the target protospacer sequence is adjacent a PAM sequence, and wherein the target protospacer sequence differs from a nontarget sequence of same length by one nucleotide, and
a Cas9 protein or ortholog thereof, wherein the guide RNA and the Cas9 protein or ortholog thereof are members of a co-localization complex for a target nucleic acid.
32. The cell of claim 31 wherein the cell is a eukaryotic cell or prokaryotic cell.
33. The method of claim 1 wherein the cell is a bacteria cell, a yeast cell, a fungal cell, a mammalian cell, a human cell, a stem cell, a progenitor cell, an induced pluripotent stem cell, a human induced pluripotent stem cell, a plant cell or an animal cell.
34. A cell comprising
a first nucleic acid encoding a guide RNA having a spacer sequence that differs from a target protospacer sequence of same length by one or two nucleotides, wherein the target protospacer sequence is adjacent a PAM sequence, and wherein the target protospacer sequence differs from a nontarget sequence of same length by one nucleotide, and
a second nucleic acid encoding a Cas9 protein or ortholog thereof, wherein the guide RNA and the Cas9 protein or ortholog thereof are members of a co-localization complex for a target nucleic acid.
35. The cell of claim 34 wherein the cell is a eukaryotic cell or prokaryotic cell.
36. The method of claim 1 wherein the cell is a bacteria cell, a yeast cell, a fungal cell, a mammalian cell, a human cell, a stem cell, a progenitor cell, an induced pluripotent stem cell, a human induced pluripotent stem cell, a plant cell or an animal cell.
37. An organism including a cell of claim 31.
38. An organism including a cell of claim 34.
39. The method of claim 1 wherein the spacer sequence differs from the target protospacer sequence of same length by one or two nucleotides, and wherein the target protospacer sequence differs from the nontarget sequence of same length by one nucleotide.
40. A method of altering a target nucleic acid in a cell comprising
providing to the cell a guide RNA having a spacer sequence entirely complementary to a target protospacer sequence of same length except for a one or two nucleotide mismatch with the target protospacer sequence, wherein the target protospacer sequence is adjacent a PAM sequence, and wherein the target protospacer sequence differs from a nontarget sequence of same length by one nucleotide,
providing to the cell a Cas9 protein or ortholog thereof,
wherein the guide RNA spacer sequence binds to the target protospacer sequence and the Cas9 protein or ortholog thereof interacts with the guide RNA to form a co-localization complex with the target nucleic acid and the target nucleic acid is cleaved in a site specific manner.
41. The method of claim 40 wherein the target protospacer sequence is a single point mutation of the nontarget sequence.
42. The method of claim 40 wherein the target protospacer sequence is endogenous to the cell or exogenous to the cell.
43. The method of claim 40 wherein the nontarget sequence is endogenous to the cell or exogenous to the cell.
44. The method of claim 40 wherein cleaving of the target nucleic acid results in cell death.
45. The method of claim 40 wherein the guide RNA spacer sequence discriminates between the target protospacer sequence and the nontarget sequence.
46. The method of claim 40 wherein the guide RNA spacer sequence and the nontarget sequence are nonbinding.
47. The method of claim 40 wherein the Cas9 protein or ortholog thereof is a Cas9 enzyme, a Cas9 nickase or a nuclease null Cas9 with a nuclease attached thereto.
48. The method of claim 40 wherein the Cas9 protein is naturally occurring or engineered.
49. The method of claim 40 wherein the cell is in vitro, in vivo or ex vivo.
50. The method of claim 40 wherein the cell is a eukaryotic cell or a prokaryotic cell.
51. The method of claim 40 wherein the cell is a bacteria cell, a yeast cell, a fungal cell, a mammalian cell, a human cell, a stem cell, a progenitor cell, an induced pluripotent stem cell, a human induced pluripotent stem cell, a plant cell or an animal cell.
52. The method of claim 40 wherein the target nucleic acid is genomic DNA, mitochondrial DNA, plastid DNA, viral DNA, or exogenous DNA.
53. The method of claim 40 wherein the one or two nucleotide mismatch is proximal to the PAM sequence.
54. The method of claim 40 further comprising providing the cell with a donor nucleic acid, wherein the donor nucleic acid is inserted into the target nucleic acid.
55. The method of claim 40 further including providing to the cell a plurality of guide RNAs each having a corresponding spacer sequence entirely complementary to a corresponding target protospacer sequence of same length except for a one or two nucleotide mismatch with the target protospacer sequence, wherein the target protospacer sequence is adjacent a PAM sequence of a target nucleic acid, and wherein the target protospacer sequence differs from a nontarget sequence of same length by one nucleotide,
providing to the cell a Cas9 protein or ortholog thereof,
wherein the plurality of guide RNA spacer sequences bind to the corresponding target protospacer sequences and the Cas9 protein or ortholog thereof interacts with each of the plurality of guide RNAs to form co-localization complexes with target nucleic acids and the target nucleic acids are cleaved in a site specific manner.
56. The method of claim 40 wherein the guide RNA is provided to the cell by introducing into the cell a first foreign nucleic acid encoding the guide RNA.
57. The method of claim 40 wherein the Cas9 is provided to the cell by introducing into the cell a second nucleic acid encoding the Cas9.
58. The method of claim 40 wherein the guide RNA is provided to the cell by introducing into the cell a first foreign nucleic acid encoding the guide RNA within a vector and wherein the Cas9 is provided to the cell by introducing into the cell a second nucleic acid encoding the Cas9 within a vector, and wherein the first foreign nucleic acid and the second foreign nucleic acid are provided on the same or different vectors.
59. The method of claim 40 wherein the guide RNA is provided to the cell as a native guide RNA.
60. The method of claim 40 wherein the Cas9 is provided to the cell as a native species.
61. A method of genetically modifying a cell to include a genome editing system which targets single point mutations in cell progeny for target nucleic acid cleavage comprising
providing to the cell a first nucleic acid encoding a guide RNA having a spacer sequence entirely complementary to a target protospacer sequence except for a one or two nucleotide mismatch with the target protospacer sequence, wherein the target protospacer sequence is a single point mutation of the nontarget sequence,
providing to the cell a second nucleic acid encoding a Cas9 protein,
providing the cell under conditions where first cell progeny is produced including the target protospacer sequence and second cell progeny is produced including the nontarget sequence,
wherein the guide RNA spacer sequence binds to the target protospacer sequence and the Cas9 protein interacts with the guide RNA to form a co-localization complex with the target nucleic acid and the target nucleic acid is cleaved in a site specific manner
62. The method of claim 61 wherein cleaving of the target nucleic acid results in cell death.
63. The method of claim 61 wherein the guide RNA spacer sequence and the nontarget sequence of the second cell progeny are nonbinding.
64. The method of claim 61 wherein the Cas9 protein is a Cas9 enzyme, a Cas9 nickase or a nuclease null Cas9 protein with a nuclease attached thereto.
65. The method of claim 61 wherein the cell is in vitro, in vivo or ex vivo.
66. The method of claim 61 wherein the cell is a eukaryotic cell or prokaryotic cell.
67. The method of claim 61 wherein the cell is a bacteria cell, a yeast cell, a fungal cell, a mammalian cell, a plant cell or an animal cell.
68. The method of claim 61 wherein the target nucleic acid is genomic DNA, mitochondrial DNA, plastid DNA, viral DNA, or exogenous DNA.
69. The method of claim 61 wherein the one or two nucleotide mismatch is proximal to the PAM sequence.
70. A cell comprising
a guide RNA having a spacer sequence entirely complementary to a target protospacer sequence of same length except for a one or two nucleotide mismatch with the target protospacer sequence, wherein the target protospacer sequence is adjacent a PAM sequence, and wherein the target protospacer sequence differs from a nontarget sequence of same length by one nucleotide, and
a Cas9 protein or ortholog thereof, wherein the guide RNA and the Cas9 protein or ortholog thereof are members of a co-localization complex for a target nucleic acid.
71. The cell of claim 70 wherein the cell is a eukaryotic cell or prokaryotic cell.
72. The cell of claim 70 wherein the cell is a bacteria cell, a yeast cell, a fungal cell, a mammalian cell, a plant cell or an animal cell.
73. A cell comprising
a first nucleic acid encoding a guide RNA having a spacer sequence entirely complementary to a target protospacer sequence of same length except for a one or two nucleotide mismatch with the target protospacer sequence, wherein the target protospacer sequence is adjacent a PAM sequence, and wherein the target protospacer sequence differs from a nontarget sequence of same length by one nucleotide, and
a second nucleic acid encoding a Cas9 protein or ortholog thereof, wherein the guide RNA and the Cas9 protein or ortholog thereof are members of a co-localization complex for a target nucleic acid.
74. The cell of claim 73 wherein the cell is a eukaryotic cell or prokaryotic cell.
75. The cell of claim 73 wherein the cell is a bacteria cell, a yeast cell, a fungal cell, a mammalian cell, a plant cell or an animal cell.
76. An organism including a cell of claim 70.
77. An organism including a cell of claim 73.
78. A method of identifying one or more discriminatory guide RNA from a library of guide RNA that discriminate between a target protospacer sequence that differs from a nontarget sequence of same length by one nucleotide wherein the library of guide RNA have a spacer sequence entirely complementary to the target protospacer sequence of same length except for a one or two nucleotide mismatch with the target protospacer sequence comprising
(a) combining the library of guide RNA with a strain including the nontarget sequence and a Cas9 enzyme and determining cutting rate of candidate guide RNA for the nontarget sequence,
(b) combining the library of guide RNA with a strain including the nontarget sequence, a Cas9 enzyme and a plasmid including the target protospacer sequence and determining cutting rate of candidate guide RNA for the target protospacer sequence,
(c) normalizing the cutting rates of step (a) and step (b) wherein guide RNA having a high cutting rate of the target protospacer sequence and a low cutting rate of the nontarget sequence are discriminatory guide RNA.
79. A method of designing a guide RNA to discriminate between a target protospacer sequence and a nontarget sequence of same length wherein the target protospacer sequence differs from the nontarget sequence of same length by a first nucleotide comprising
designing the guide RNA to be exactly complementary to the target protospacer sequence except for a one or two nucleotide mismatch with the target protospacer sequence.
80. A method of identifying one or more discriminatory guide RNA from a library of guide RNA that discriminate between a target protospacer sequence that differs from a nontarget sequence of same length by one nucleotide wherein the library of guide RNA have a spacer sequence that differs from a target protospacer sequence of same length by one or two nucleotides comprising
(a) combining the library of guide RNA with a strain including the nontarget sequence and a Cas9 enzyme and determining cutting rate of candidate guide RNA for the nontarget sequence,
(b) combining the library of guide RNA with a strain including the nontarget sequence, a Cas9 enzyme and a plasmid including the target protospacer sequence and determining cutting rate of candidate guide RNA for the target protospacer sequence,
(c) normalizing the cutting rates of step (a) and step (b) wherein guide RNA having a high cutting rate of the target protospacer sequence and a low cutting rate of the nontarget sequence are discriminatory guide RNA.
81. A method of identifying one or more discriminatory guide RNA from a library of guide RNA that discriminate between a target protospacer sequence that differs from a nontarget sequence of same length by one nucleotide wherein the library of guide RNA have a spacer sequence entirely complementary to the target protospacer sequence of same length except for a one or two nucleotide mismatch with the target protospacer sequence comprising
(a) combining the library of guide RNA with a strain including a Cas9 enzyme and a plasmid including the nontarget sequence and determining cutting rate of candidate guide RNA for the nontarget sequence,
(b) combining the library of guide RNA with a strain including Cas9 and a plasmid including the target protospacer sequence and determining cutting rate of candidate guide RNA for the target protospacer sequence,
(c) normalizing the cutting rates of step (a) and step (b) wherein guide RNA having a high cutting rate of the target protospacer sequence and a low cutting rate of the nontarget sequence are discriminatory guide RNA.
82. A method of identifying one or more discriminatory guide RNA from a library of guide RNA that discriminate between a target protospacer sequence that differs from a nontarget sequence of same length by one nucleotide wherein the library of guide RNA have a spacer sequence that differs from a target protospacer sequence of same length by one or two nucleotides comprising
(a) combining the library of guide RNA with a strain including a Cas9 enzyme and a plasmid including the nontarget sequence and determining cutting rate of candidate guide RNA for the nontarget sequence,
(b) combining the library of guide RNA with a strain including Cas9 and a plasmid including the target protospacer sequence and determining cutting rate of candidate guide RNA for the target protospacer sequence,
(c) normalizing the cutting rates of step (a) and step (b) wherein guide RNA having a high cutting rate of the target protospacer sequence and a low cutting rate of the nontarget sequence are discriminatory guide RNA.