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1. WO2020142754 - PROGRAMMABLE NUCLEASE IMPROVEMENTS AND COMPOSITIONS AND METHODS FOR NUCLEIC ACID AMPLIFICATION AND DETECTION

Note: Text based on automatic Optical Character Recognition processes. Please use the PDF version for legal matters

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CLAIMS

WHAT IS CLAIMED IS:

1. A composition comprising a programmable nuclease having at least 60% sequence identity to SEQ ID NO: 11 and a non-naturally occurring guide nucleic acid

2. The composition of claim 1, wherein the programmable nuclease comprises a turnover rate of at least about 0.1 cleaved detector nucleic acid molecules per minute.

3. The composition of claim 1 or 2, wherein the programmable nuclease recognizes a protospacer adjacent motif of YYN.

4. A composition comprising programmable nuclease having a turnover rate of at least about 0.1 cleaved detector nucleic acid molecules per minute and a non-naturally occurring guide nucleic acid.

5. The composition of claim 6, wherein the programmable nuclease recognizes a protospacer adjacent motif of YYN.

6. A composition comprising a non-naturally occurring guide nucleic acid and a programmable nuclease, wherein the programmable nuclease comprises a turnover rate of at least about 0.1 cleaved detector nucleic acid molecules per minute and recognizes a protospacer adjacent motif of YYN.

7. The composition of any one of claims 1-6, wherein the programmable nuclease is a Type V programmable nuclease.

8. The composition any one of claims 1-7, wherein the programmable nuclease is a

Casl2 nuclease.

9. The composition of any one of claims 1-8, wherein the programmable nuclease comprises three partial RuvC domains.

10. The composition of any one of claims 1-9, wherein the programmable nuclease comprises a RuvC-I subdomain, a RuvC-II subdomain, and a RuvC-III subdomain.

11. The composition any one of claims 4-10, wherein the programmable nuclease has at least 60% sequence identity to SEQ ID NO: 11.

12. The composition of any one of claims 1-11, wherein the programmable nuclease has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to SEQ ID NO: 11.

13. The method of any one of claims 1-12, wherein the programmable nuclease is SEQ ID NO: 11.

14. The composition of any one of claims 3 or 5-13, wherein the Y is a C or T nucleotide.

15. The composition of any one of claims 3 or 5-14, wherein the N is any nucleotide.

16. The composition of any one of claims 1-15, wherein the composition further comprises a buffer.

17. The composition of claim 16, wherein the buffer comprises a buffering agent, a salt, a crowding agent, a detergent, or any combination thereof.

18. The composition of claim 17, wherein the buffering agent is at a concentration of from 5 mM to 100 mM.

19. The composition of any one of claims 17-18, wherein the buffering agent is at a concentration of from 10 mM to 40 mM.

20. The composition of any one of claims 17-19, wherein the buffering agent is at a concentration of about 20 mM.

21. The composition of any one of claims 17-20, wherein the salt is from 5 mM to 100 mM.

22. The composition of any one of claims 17-21, wherein the salt is from 5 mM to 10 mM.

23. The composition of any one of claims 17-22, wherein the crowding agent is from 0.5% (v/v) to 2% (v/v).

24. The composition of any one of claims 17-23, wherein the crowding agent is about

1%.

25. The composition of any one of claims 17-24, wherein the detergent is about 2% (v/v) or less

26. The composition of any one of claims 17-25, wherein the detergent is about 0.00016% (v/v).

27. The composition of any one of claims 17-26, wherein the buffering agent is HEPES.

28. The composition of any one of claims 17-27, wherein the salt is potassium acetate, magnesium acetate, sodium chloride, magnesium chloride, or any combination thereof.

29. The composition of any one of claims 17-28, wherein the crowding agent is glycerol.

30. The composition of any one of claims 17-29, wherein the detergent is Tween, Triton-X, or any combination thereof.

31. The composition of any one of claims 1 -30, wherein a pH of the composition is from 7 to 8.

32. The composition of any one of claims 1-31, wherein a pH of the composition is 7.5.

33. The composition of any one of claims 1-32, wherein the composition is at a temperature of from 25°C to 45°C.

34. The composition of any one of claims 1-33, wherein the programmable nuclease exhibits catalytic activity at a temperature of from 25°C to 45°C.

35. The composition of any one of claims 1-34, wherein the programmable nuclease exhibits catalytic activity after heating the composition to a temperature of greater than 45 °C and restoring the temperature to from 25°C to 45°C.

36. A method of assaying for a segment of a target nucleic acid in a sample, the method comprising:

contacting the sample to:

a detector nucleic acid; and

the composition of any one of claims 1-35, wherein the guide nucleic acid hybridizes to a segment of the target nucleic acid; and

assaying for a signal produced by cleavage of the detector nucleic acid.

37. A method of assaying for a segment of a target nucleic acid in a sample from a subject comprising:

contacting the sample comprising a population of nucleic acids to:

a guide nucleic acid that hybridizes to the segment of the target nucleic acid;

a detector nucleic acid; and

a Casl2 nuclease that cleaves the detector nucleic acid upon hybridization of the guide nucleic acid to the segment of the target nucleic acid; and

assaying for a signal produced by cleavage of the detector nucleic acid, wherein the signal is at least two-fold greater when the segment of the target nucleic acid is present in the sample than the signal when the sample lacks the segment of the target nucleic acid and wherein the subject has a disease when the segment of the target nucleic acid is present.

38. The method of claim 37, the method further comprising administering a treatment for the disease.

39. A method of assaying for a segment of a target nucleic acid comprising:

contacting a sample comprising a population of nucleic acids, wherein the population comprises at least one nucleic acid comprising a segment having less than 100% sequence identity to the segment of the target nucleic acid and having no less than 50% sequence identity to the segment of the target nucleic acid to:

a guide nucleic acid that hybridizes to the segment of the target nucleic acid;

a detector nucleic acid; and

a Casl2 nuclease that cleaves the detector nucleic acid upon

hybridization of the guide nucleic acid to the segment of the target nucleic acid; and

assaying for a signal produced by cleavage of the detector nucleic acid, wherein the signal is at least two-fold greater when the segment of the target nucleic acid is present in the sample than the signal when the sample lacks the segment of the target nucleic acid.

40. The method of any one of claims 37-39, wherein the segment of the at least one nucleic acid comprises at least two base mutations compared to the segment of the target nucleic acid.

41. The method of any one of claims 39-40, wherein the segment of the at least one nucleic acid comprises from one to ten base mutations compared to the segment of the target nucleic acid.

42. The method of any one of claims 39-40, wherein the segment of the at least one nucleic acid comprises one base mutation compared to the segment of the target nucleic acid.

43. The method of any one of claims 39-42, wherein the signal is from two-fold to 20-fold greater when the segment of the target nucleic acid is present in the sample than the signal when the sample lacks the segment of the target nucleic acid.

44. The method of any one of claims 39-43, wherein the signal is from two-fold to 10-fold greater when the segment of the target nucleic acid is present in the sample than the signal when the sample lacks the segment of the target nucleic acid.

45. The method of any one of claims 39-44, wherein the signal is from five-fold to 10-fold greater when the segment of the target nucleic acid is present in the sample than the signal when the sample lacks the segment of the target nucleic acid.

46. The method of claim 45, wherein the guide nucleic acid is reverse complementary to the segment of the target nucleic acid.

47. The method of claim 46, wherein the guide nucleic acid and the second guide nucleic acid lack synthetic mismatches.

48. The method of any one of claims 39-47, wherein the guide nucleic acid is at least

10 bases.

49. The method of any one of claims 39-48, wherein the guide nucleic acid is from 10 to 50 bases.

50. The method of any one of claims 39-49, wherein the guide nucleic acid is at least 25 bases.

51. The method of any one of claims 39-50, wherein the target nucleic acid is in the population of nucleic acids at a minor allele frequency of 10% or less.

52. The method of any one of claims 39-51 , wherein the target nucleic acid is in the population of nucleic acids at a minor allele frequency of from 0.1% to 10%.

53. The method of any one of claims 39-52, wherein the target nucleic acid is in the population of nucleic acids at a minor allele frequency of from 0.1% to 5%.

54. The method of any one of claims 39-53, wherein the target nucleic acid is in the population of nucleic acids at a minor allele frequency of from 0.1% to 1%.

55. The method of any one of claims 39-54, wherein the Casl2 nuclease is Casl2a, Casl2b, Casl2c, CasY, or Casl2e.

56. The method of any one of claims 39-55, wherein the Casl2 nuclease is Casl2a.

57. The method of any one of claims 39-56, wherein the Casl2 nuclease has at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 1 - SEQ ID NO: 11, SEQ ID NO: 282, or SEQ ID NO: 571 - SEQ ID NO: 602.

58. The method of any one of claims 39-57, wherein the Casl2 nuclease has at least 60% sequence identity to SEQ ID NO: 11.

59. The method of any one of claims 39-58, wherein the contacting is carried out in a buffer comprising a buffering agent, a salt, a crowding agent, a detergent, a reducing agent, a competitor, or any combination thereof.

60. The method of claim 59, wherein the buffering agent is at a concentration of from 5 mM to 100 mM.

61. The method of any one of claims 59-60, wherein the buffering agent is at a concentration of from 10 mM to 30 mM.

62. The method of any one of claims 59-61, wherein the salt is from 5 mM to 100 mM.

63. The method of any one of claims 59-62, wherein the salt is from 5 mM to 10 mM.

64. The method of any one of claims 59-63, wherein the crowding agent is from 0.5% (v/v) to 10% (v/v).

65. The method of any one of claims 59-64, wherein the crowding agent is from 1% (v/v) to 5% (v/v).

66. The method of any one of claims 59-65, wherein the detergent is at 2% (v/v) or less

67. The method of any one of claims 59-66, wherein the reducing agent is from 0.01 mM to 100 mM.

68. The method of any one of claims 59-67, wherein the reducing agent is from 0.1 mM to 10 mM.

69. The method of any one of claims 59-68, wherein the reducing agent is from 0.5 mM to 2 mM.

70. The method of any one of claims 59-69, wherein the competitor is from 1 ug/ml to 100 ug/ml.

71. The method of any one of claims 59-70, wherein the competitor is from 40 ug/ml to 60 ug/ml.

72. The method of any one of claims 59-71, wherein the buffering agent is HEPES, Tris, or any combination thereof.

73. The method of any one of claims 59-72, wherein the salt is potassium acetate, magnesium acetate, sodium chloride, magnesium chloride, or any combination thereof.

74. The method of any one of claims 59-72, wherein the crowding agent is glycerol.

75. The method of any one of claims 59-74, wherein the detergent is Tween, Triton-X, or any combination thereof.

76. The method of any one of claims 59-75, wherein the reducing agent is DTT.

77. The method of any one of claims 59-76, wherein the competitor is heparin.

78. The method of any one of claims 59-77, wherein a pH of the composition is from

7 to 8.

79. The method of any one of claims 37-78, further comprising amplifying the target nucleic acid before the contacting.

80. The method of claim 79, wherein the amplifying the target nucleic acid before the contacting comprises using a blocking primer.

81. The method of claim 80, wherein the target nucleic acid segment comprises a single nucleotide mutation.

82. The method of claim 81, wherein the blocking primer binds to a nucleic acid comprising encoding the wild type sequence of the target nucleic acid segment.

83. The method of claim 79, wherein the amplifying comprises COLD-PCR.

84. The method of claim 83, wherein the COLD-PCR comprises full COLD-PCR.

85. The method of claim 83, wherein the COLD-PCR comprises fast COLD-PCR.

86. The method of claim 79, wherein the amplifying comprises fast COLD-PCR.

87. The method of claim 79, wherein the amplifying comprises allele-specific PCR.

88. The method of claim 87, wherein the amplifying further comprises COLD-PCR.

89. A composition comprising a programmable nuclease and a buffer, wherein the buffer comprises a salt at less than about 110 mM and wherein the buffer comprises a pH of from 7 to 8.

90. The composition of claim 89, wherein the salt is from 1 mM to 110 mM.

91. The composition of claim 89 or 90, wherein the salt is from 1 mM to 60 mM.

92. The composition of any one of claims 89-91, wherein the salt is from 1 mM to 10 mM.

93. The composition of any one of claims 89-92, wherein the salt is at about 105 mM.

94. The composition of any one of claims 89-93, wherein the salt is at about 55 mM.

95. The composition of any one of claims 89-94, wherein the salt is at about 7 mM.

96. The composition of any one of claims 89-95, wherein the salt comprises potassium acetate, magnesium acetate, sodium chloride, magnesium chloride, potassium chloride, or any combination thereof.

97. The composition of any one of claims 89-96, wherein the salt comprises potassium acetate and magnesium acetate.

98. The composition of any one of claims 89-96, wherein the salt comprises sodium chloride and magnesium chloride.

99. The composition of any one of claims 89-96, wherein the salt comprises potassium chloride and magnesium chloride.

100. The composition of any one of claims 89-99, wherein the pH comprises about 7.5.

101. The composition of any one of claims 89-99, wherein the pH comprises about 8.

102. The composition of any one of claims 89-101, wherein the buffer comprises a crowding agent or a competitor.

103. The composition of claim 102, wherein the crowding agent is present from 1% (v/v) to 10% (v/v).

104. The composition of any one of claims 102-103, wherein the crowding agent or the competitor is present from 1% (v/v) to 5% (v/v).

105. The composition of any one of claims 102-103, wherein the crowding agent or the competitor is present at about 5% (v/v).

106. The composition of any one of claims 102-103, wherein the crowding agent or the competitor is present at about 1% (v/v).

107. The composition of any one of claims 102-106, wherein the crowding agent or the competitor is present from 1 ug/mL to 100 ug/ml.

108. The composition of any one of claims 102-107, wherein the crowding agent or the competitor is present from 30 ug/ml to 70 ug/ml.

109. The composition of any one of claims 102-108, wherein the crowding agent or the competitor is present at about 50 ug/ml.

110. The composition of any one of claims 102-109, wherein the crowding agent or the competitor is present from 1 mM to 50 mM.

111. The composition of any one of claims 102-110, wherein the crowding agent or the competitor is present from 10 mM to 30 mM.

112. The composition of any one of claims 102-111, wherein the crowding agent or the competitor is present at about 20 mM.

113. The composition of any one of claims 102-112, wherein the crowding agent or the competitor is selected from the group consisting of: glycerol, heparin, bovine serum albumin, imidazole, and any combination thereof.

114. The composition of any one of claims 102-113, wherein the crowding agent or the competitor comprises glycerol.

115. The composition of any one of claims 102-114, wherein the crowding agent or the competitor comprises glycerol and heparin.

116. The composition of any one of claims 102-115, wherein the crowding agent or the competitor comprises glycerol, bovine serum albumin, and imidazole.

117. The composition of any one of claims 88-116, wherein the buffer comprises a buffering agent.

118. The composition of claim 117, wherein the buffering agent is present from 1 mM to 50 mM.

119. The composition of any one of claims 116-118, wherein the buffering agent is present from 1 mM to 30 mM.

120. The composition of any one of claims 116-119, wherein the buffering agent is present at about 20 mM.

121. The composition of any one of claims 116-120, wherein the buffering agent is HEPES.

122. The composition of any one of claims 116-120, wherein the buffering agent is

Tris.

123. The composition of any one of claims 89-122, wherein the buffer comprises a detergent.

124. The composition of claim 123, wherein the detergent is present from 0.00001% (v/v) to 0.1% (v/v).

125. The composition of any one of claims 123-124, wherein the detergent is present from 0.00001% (v/v) to 0.01% (v/v).

126. The composition of any one of claims 123-125, wherein the detergent is at about 0.00016% (v/v).

127. The composition of any one of claims 123-125, wherein the detergent is at about 0.01% (v/v).

128. The composition of any one of claims 123-127, wherein the detergent is Triton-X.

129. The composition of any one of claims 123-127, wherein the detergent is IGEPAL CA-630.

130. The composition of any one of claims 89-129, wherein the buffer comprises a reducing agent.

131. The composition of claim 130, wherein the reducing agent is present from 0.01 mM to 100 mM.

132. The composition of any one of claims 130-131 , wherein the reducing agent is present from 0.1 mM to 10 mM.

133. The composition of any one of claims 130-132, wherein the reducing agent is present at about 1 mM.

134. The composition of any one of claims 130-133, wherein the reducing agent is

DTT.

135. The composition of any one of claims 89-134, wherein the programmable nuclease comprises a RuvC domain.

136. The composition of any one of claims 89-135, wherein the programmable nuclease comprises a Type V Cas protein.

137. The composition of any one of claims 89-136, wherein the programmable nuclease is a Cas 12 protein.

138. The composition of claim 137, wherein the Cas 12 protein is Cas 12a, Cas 12b, Cas 12c, CasY, or Casl2e.

139. The composition of any one of claims 89-138, wherein the programmable nuclease has at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 1 -SEQ ID NO: 11, SEQ ID NO: 282, or SEQ ID NO: 571 - SEQ ID NO: 602.

140. The composition of any one of claims 89-139, wherein the programmable nuclease has at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 1.

141. The composition of any one of claims 89-139, wherein the programmable nuclease has at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 11.

142. The composition of any one of claims 89-134, wherein the programmable nuclease comprises at least two HEPN domains.

143. The composition of any one of claims 89-134, wherein the programmable nuclease is a Type VI Cas protein.

144. The composition of any one of claims 89-134, wherein the programmable nuclease is a Cas 13 protein.

145. The composition of claim 144, wherein the Casl3 protein is Casl3a, Casl3b, Casl3c, Casl3d, or Casl3e.

146. The composition of any one of claims 89-145, wherein the programmable nuclease has at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 103 - SEQ ID NO: 137.

147. The composition of any one of claims 89-146, wherein the programmable nuclease has at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 104.

148. The composition of any one of claims 89-92, 95-97, 100, 102-105, 113-114, 117- 121. 123-126, 128, 135-139, or 141, wherein the programmable nuclease has at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 11 and the buffer comprises about 20 mM HEPES, about 2 mM potassium acetate, about 5 mM magnesium acetate, about 1% (v/v) glycerol, about 0.00016% (v/v) Triton-X, and a pH of about 7.5.

149. The composition of any one of claims 89-90, 93, 96, 98, 91-105, 107-109, 113,

115, 117-120, 122, or 130-140, wherein the programmable nuclease has at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 and the buffer comprises about 20 mM Tris, about 100 mM sodium chloride, about 5 mM magnesium chloride, about 5% (v/v) glycerol, about 50 ug/mL heparin, about 1 mM DTT, and a pH of about 8.

150. The composition of any one of claims 89-92, 94, 96, 99-100, 102-103, 110-113,

116. 123-125, 127, 129, or 142-147, wherein the programmable nuclease has at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 104 and the buffer comprises about 50 mM potassium chloride, about 5 mM magnesium chloride, about 10 ug/ml bovine serum albumin, about 5% glycerol, about 20 mM imidazole, about 0.01% (v/v) IGEPAL CA-630, and a pH of about 7.5.

151. The composition of any one of claims 89-150, further comprising a guide nucleic acid.

152. The composition of any one of claims 89-151, further comprising a detector nucleic acid.

153. A composition comprising:

a nucleic acid from a sample, wherein the nucleic acid comprises a PAM and a segment that hybridizes to a guide nucleic acid, wherein the PAM has a sequence of dUdUdUN;

a guide nucleic acid that hybridizes to the segment of the nucleic acid; and

a programmable nuclease that exhibits sequence independent cleavage of a detector nucleic acid upon hybridization of the guide nucleic acid to the segment of the target nucleic acid.

154. The composition of claim 153, further comprising a primer, wherein the primer comprises a first region that is reverse complementary to the PAM and a second region that is reverse complementary to a first segment of the nucleic acid.

155. A method of assaying for a target nucleic acid in a sample, wherein the target nucleic acid lacks a PAM, the method comprising:

amplifying the target nucleic acid from a sample using a primer comprising a first region that is reverse complementary to a PAM and a second region that is reverse complementary to a first segment of the target nucleic acid, wherein the PAM is dUdUdUN, thereby producing a PAM target nucleic acid;

contacting the PAM target nucleic acid to:

a guide nucleic acid that hybridizes to a segment of the PAM target nucleic acid;

a programmable nuclease that exhibits sequence independent cleavage of a detector nucleic acid upon hybridization of the guide nucleic acid to a segment of the PAM target nucleic acid; and

a detector nucleic acid; and

assaying for a signal produced by cleavage of the detector nucleic acid.

156. The composition or method of any one of claims 153-155, wherein the second region comprises from 4 to 12 bases.

157. The composition or method of any one of claims 152-156, wherein the second region comprises from 4 to 10 bases.

158. The composition or method of any one of claims 152-157, wherein the second region comprises from 4 to 7 bases.

159. The method of any one of claims 152-158, wherein the amplifying comprises thermal cycling amplification.

160. The method of any one of claims 152-159, wherein the amplifying comprises isothermal amplification.

161. The method of claim 160, wherein the isothermal amplification comprises isothermal recombinase polymerase amplification (RPA), transcription mediated amplification (TMA), strand displacement amplification (SDA), helicase dependent amplification (HD A), loop mediated amplification (LAMP), rolling circle amplification (RCA), single primer isothermal amplification (SPIA), ligase chain reaction (LCR), simple method amplifying RNA targets (SMART), improved multiple displacement amplification (IMDA), or nucleic acid sequence-based amplification (NASBA).

162. The method of any one of claims 160-161, wherein the isothermal amplification comprises loop mediated amplification (LAMP).

163. The composition or method of any one of claims 152-162, wherein a sequence of the primer and a sequence of the guide nucleic acid overlap by 50% or less.

164. The composition or method of any one of claims 152-162, wherein a sequence of the primer and a sequence of the guide nucleic acid do not overlap.

165. The composition or method of any one of claims 152-164, wherein the primer is a forward primer, a reverse primer, a forward inner primer, or a reverse inner primer.

166. The composition or method of any one of claims 152-165, wherein the segment of the nucleic acid or the segment of the target nucleic acid comprises at least one base mutation compared to at least one other segment of a nucleic acid in the sample.

167. The composition or method of claim 166, wherein the at least one base mutation is no more than 13 nucleotides 3’ of the PAM in the nucleic acid or the PAM target nucleic acid.

168. The method of any one of claims 166- 167, wherein the at least one base mutation is no more than 10 nucleotides 3’ of the PAM in the nucleic acid or the PAM target nucleic acid.

169. The method of any one of claims 166- 168, wherein the at least one base mutation is no more than 9 nucleotides 3’ of the PAM in the nucleic acid or in the PAM target nucleic acid.

170. The method of any one of claims 166-169, wherein the at least one base mutation is no more than 8 nucleotides 3’ of the PAM in the nucleic acid or in the PAM target nucleic acid.

171. The composition or method of any one of claims 166-170, wherein the at least one base mutation is a single nucleotide polymorphism.

172. The composition or method of any one of claims 152-171, wherein the

programmable nuclease comprises a RuvC domain.

173. The composition or method of any one of claims 152-172, wherein the

programmable nuclease comprises three partial RuvC domains.

174. The composition or method of any one of claims 152-173, wherein the

programmable nuclease comprises a RuvC-I subdomain, a RuvC-II subdomain, and a RuvC-III subdomain.

175. The composition or method of any one of claims 152-174, wherein the

programmable nuclease comprises a Type V Cas protein.

176. The composition or method of any one of claims 152-175, wherein the

programmable nuclease is a Cas 12 protein.

177. The composition or method of claim 176, wherein the Casl2 protein is Casl2a, Cas 12b, Cas 12c, CasY, or Casl2e.

178. The composition or method of any one of claims 152-177, wherein the

programmable nuclease has at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 1 - SEQ ID NO: 11, SEQ ID NO: 282, SEQ ID NO: 571 - SEQ ID NO: 602.

179. A Casl2 nuclease for use in diagnosis, wherein the Casl2 nuclease detects the segment of the target nucleic acid according to the method of any one of claims 37 to 88.

180. The composition according to any one of claims 1 to 35 for use in diagnosis.

181. The composition according to any one of claims 89 to 152 for use in diagnosis.

182. The composition according to any one of claims 153, 154, 156-158, 163-167 or 171-178 for use in diagnosis.

183. A programmable nuclease for use in diagnosis, wherein the programmable nuclease detects the target nucleic acid according to any one of claims 155 or 156-178.