COMPRESSIVE MOLECULAR PROBES FOR GENOMIC EDITING AND TRACKING

20240209447

18/287619

April 20, 2022

The present disclosure provides systems, methods, nucleic acids, and kits for barcoding and tracking cells based on CRISPR editing technologies.

1. A system comprising: a Cas12a protein or a vector encoding thereof; a polynucleotide barcode flanked by two PAM sequences of inverse orientation, or a vector encoding thereof, wherein the polynucleotide barcode comprises a first target nucleic acid sequence and a second target nucleic acid sequence; and a pair of guide RNAs (gRNAs) configured to hybridize to the two PAM sequences.

2. The system of claim 1, wherein the system comprises two or more polynucleotide barcodes.

3. The system of claim 1 or 2, wherein the polynucleotide barcode is on the vector encoding the pair of gRNAs.

4. The system of any of claims 1-3, wherein the vector encoding the Cas12a protein is the same vector as the vector encoding the pair of gRNAs.

5. The system of any of claims 1-4, wherein the polynucleotide barcode is on same vector as the pair of gRNAs and the Cas12a protein.

6. The system of any of claims 1-5, wherein the polynucleotide barcode further comprises a linker between the first target nucleic acid sequence and the second target nucleic acid sequence.

7. The system of claim 6, wherein the linker comprises 1-20 nucleotides.

8. The system of claim 6 or claim 7, wherein the linker comprises 10 nucleotides.

9. The system of any of claims 1-8, wherein the polynucleotide barcode comprises less than 200 nucleotides.

10. The system of any of claims 1-9, wherein the polynucleotide barcode comprises less than 150 nucleotides.

11. The system of any of claims 1-10, wherein the polynucleotide barcode comprises 50-60 nucleotides.

12. The system of any of claims 1-11, wherein the polynucleotide barcode comprises 54 nucleotides.

13. The system of any of claims 1-12, wherein the polynucleotide barcode sequence is configured to promote insertions and deletions over time.

14. The system of any of claims 1-13, wherein the polynucleotide barcode comprises GC directly upstream of PAM sequence at the 3′ end of the polynucleotide barcode.

15. The system of any of claims 1-14, wherein the polynucleotide barcode comprises a cytidine at position 39.

16. The system of claim 15, wherein the polynucleotide barcode comprises a guanosine at position 40.

17. The system of any of claims 1-16, wherein the polynucleotide barcode comprises an adenosine at positions 45 and 46.

18. The system of any of claims 1-17, wherein the polynucleotide barcode comprises an adenosine at position 31 and a guanosine at position 32.

19. The system of any of claims 1-18, wherein the polynucleotide barcode does not comprise a thymidine at position 54, position 49, or both.

20. The system of any of claims 1-19, wherein the polynucleotide barcode does not comprise a guanosine at position 50 and a cytidine at position 51.

21. The system of any of claims 1-20, wherein the polynucleotide barcode comprises CACTTG (SEQ ID NO: 1054) at positions 32-37.

22. The system of any of claims 1-21, wherein the polynucleotide barcode comprises CCTAGTAATAG (SEQ ID NO: 1055) at positions 39-49.

23. The system of any of claims 1-22, wherein the polynucleotide barcode comprises CCGG (SEQ ID NO: 1056) directly downstream of PAM sequence at the 5′ end of the polynucleotide barcode.

24. The system of any of claims 1-23, wherein the polynucleotide barcode comprises a sequence with at least 70% similarity to sequences selected from the group consisting of SEQ ID NOs: 1-1053.

25. The system of any of claims 1-24, wherein the polynucleotide barcode sequence is selected from the group consisting of SEQ ID NOs: 1-10.

26. The system of any of claims 1-25, wherein the pair of gRNAs are within a crRNA array.

27. The system of any of claims 1-26, wherein one or each of the pair of gRNAs comprise a guide sequence of less than 25 nucleotides.

28. The system of any of claims 1-27, further comprising at least one gRNA configured to hybridize to a recipient nucleic acid.

29. The system of claim 28, wherein the recipient nucleic acid is a nucleic acid endogenous to a target cell.

30. The system of claim 28 or 29, wherein the recipient nucleic acid is a gene within a target cell.

31. The system of any of claims 28-30, wherein the at least one gRNA configured to hybridize to a recipient nucleic acid is on the vector encoding the pair of gRNAs.

32. The system of any of claims 28-31, wherein the at least one gRNA is within the crRNA array comprising the pair of gRNAs.

33. The system of any of claims 1-32, wherein at least one or all of the gRNAs are non-naturally occurring gRNAs.

34. The system of any of claims 1-33, wherein the vector encoding Cas12a comprises an inducible promoter for Cas12a expression.

35. The system of any of claims 1-34, wherein the system further comprises a recipient nucleic acid.

36. The system of any of claims 1-35, further comprising a sequence tag configured to remain static over time.

37. The system of any of claims 1-36, further comprising a gene editing system.

38. The system of claim 37, wherein the gene editing system comprises a CRISPR/Cas gene editing system.

39. The system of claim 37 or 38, further comprising one or more gene editing gRNAs.

40. The system of claim 39, wherein the one or more gene editing gRNAs are provided in a crRNA array with the pair of guide RNAs.

41. The system of any of claims 1-40, wherein the system is a cell-free system.

42. A cell comprising the system of any one of claims 1-40.

43. The cell of claim 42, wherein the cell is a eukaryotic cell.

44. The cell of claim 42 or 43, wherein the cell is in vitro.

45. The cell of any of claims 42-44, wherein the cell is a cancer cell.

46. A system comprising: a CRISPR associated (Cas) endonuclease or a vector encoding thereof; a polynucleotide barcode comprising less than 100 nucleotides flanked by two PAM sequences; and a pair of guide RNAs (gRNAs) configured to hybridize to the two PAM sequences.

47. The system of claim 46, wherein the system comprises two or more polynucleotide barcodes.

48. The system of claim 46 or 47, wherein the polynucleotide barcode is on the vector encoding the pair of gRNAs.

49. The system of any of claims 46-48, wherein the vector encoding the Cas endonuclease is the same vector as the vector encoding the pair of gRNAs.

50. The system of any of claims 46-49, wherein the polynucleotide barcode is on same vector as the pair of gRNAs and the Cas endonuclease.

51. The system of any of claims 46-50, wherein the polynucleotide barcode further comprises first target nucleic acid sequence and the second target nucleic acid sequence.

52. The system of any of claims 46-51, wherein the PAM sequences are of inverse orientation.

53. The system of any of claims 46-52, wherein the polynucleotide barcode comprises 50-60 nucleotides.

54. The system of any of claims 46-53, wherein the polynucleotide barcode comprises 54 nucleotides.

55. The system of any of claims 46-54, wherein the polynucleotide barcode sequence is configured to promote insertions and deletions over time.

56. The system of any of claims 46-55, wherein the polynucleotide barcode comprises GC directly upstream of PAM sequence at the 3′ end of the polynucleotide barcode.

57. The system of any of claims 46-56, wherein the polynucleotide barcode comprises a cytidine at position 39.

58. The system of claim 57, wherein the polynucleotide barcode comprises a guanosine at position 40.

59. The system of any of claims 46-58, wherein the polynucleotide barcode comprises an adenosine at positions 45 and 46.

60. The system of any of claims 46-59, wherein the polynucleotide barcode comprises an adenosine at position 31 and a guanosine at position 32.

61. The system of any of claims 46-60, wherein the polynucleotide barcode does not comprise a thymidine at position 54, position 49, or both.

62. The system of any of claims 46-61, wherein the polynucleotide barcode does not comprise a guanosine at position 50 and a cytidine at position 51.

63. The system of any of claims 46-62, wherein the polynucleotide barcode comprises CACTTG (SEQ ID NO: 1054) at positions 32-37.

64. The system of any of claims 46-63, wherein the polynucleotide barcode comprises CCTAGTAATAG (SEQ ID NO: 1055) at positions 39-49.

65. The system of any of claims 46-64, wherein the polynucleotide barcode comprises CCGG (SEQ ID NO: 1056) directly downstream of PAM sequence at the 5′ end of the polynucleotide barcode.

66. The system of any of claims 46-65, wherein the polynucleotide barcode comprises a sequence with at least 70% similarity to sequences selected from the group consisting of SEQ ID NOs: 1-1053.

67. The system of any of claims 46-66, wherein the polynucleotide barcode sequence is selected from the group consisting of SEQ ID NOs: 1-10.

68. The system of any of claims 46-67, wherein the pair of gRNAs are within a crRNA array.

69. The system of any of claims 46-68, wherein one or each of the pair of gRNAs comprise a guide sequence of less than 25 nucleotides.

70. The system of any of claims 46-69, further comprising at least one gRNA configured to hybridize to a recipient nucleic acid.

71. The system of claim 70, wherein the recipient nucleic acid is a nucleic acid endogenous to a target cell.

72. The system of claim 70 or 71, wherein the recipient nucleic acid is a gene within a target cell.

73. The system of any of claims 70-72, wherein the at least one gRNA configured to hybridize to a recipient nucleic acid is on the vector encoding the pair of gRNAs.

74. The system of any of claims 70-73, wherein the at least one gRNA is within the crRNA array comprising the pair of gRNAs.

75. The system of any of claims 46-74, wherein at least one or all of the gRNAs are non-naturally occurring gRNAs.

76. The system of any of claims 46-75, wherein the vector encoding Cas12a comprises an inducible promoter for Cas12a expression.

77. The system of any of claims 46-76, wherein the system further comprises a recipient nucleic acid.

78. The system of any of claims 46-77, further comprising a sequence tag configured to remain static over time.

79. The system of any of claims 46-78, further comprising a gene editing system.

80. The system of claim 79, wherein the gene editing system comprises a CRISPR/Cas gene editing system.

81. The system of claim 79 or 80, further comprising one or more gene editing gRNAs.

82. The system of claim 81, wherein the one or more gene editing gRNAs are provided in a crRNA array with the pair of guide RNAs.

83. The system of any of claims 46-82, wherein the system is a cell-free system.

84. A population of cells comprising the system of any one of claims 46-82.

85. The population of cells of claim 84, wherein each of the cells comprises a distinct version of the barcode representing a particular cell generation.

86. The population of cells of claim 85, wherein the population of cells represents up to 1000 cell generations.

87. The population of cells of claim 85 or 86, wherein the population of cells represents about 700 cell generations.

88. The population of cells of any of claims 84-87, wherein the population comprises eukaryotic cells.

89. The population of cells of any of claims 84-88, wherein the population of cells is in vitro.

90. The population of cells of any of claims 84-89, wherein the population of cells comprises cancer cells.

91. A nucleic acid comprising a polynucleotide barcode flanked by a pair of PAM sequences of inverse orientation, wherein the polynucleotide barcode comprises a first target nucleic acid sequence and a second target nucleic acid sequence.

92. The nucleic acid of claim 91, wherein the polynucleotide barcode further comprises a linker between the first target nucleic acid sequence and the second target nucleic acid sequence.

93. The nucleic acid of claim 92, wherein the linker comprises 1-20 nucleotides.

94. The nucleic acid of claim 92 or claim 93, wherein the linker comprises 10 nucleotides.

95. The nucleic acid of any of claims 91-94, wherein the polynucleotide barcode comprises less than 200 nucleotides.

96. The nucleic acid of any of claims 91-95, wherein the polynucleotide barcode comprises less than 150 nucleotides.

97. The nucleic acid of any of claims 91-96, wherein the polynucleotide barcode comprises less than 100 nucleotides.

98. The nucleic acid of any of claims 91-97, wherein the polynucleotide barcode comprises 50-60 nucleotides.

99. The nucleic acid of any of claims 91-98, wherein the polynucleotide barcode sequence is configured to promote insertions and deletions over time.

100. The nucleic acid of any of claims 91-99, wherein the polynucleotide barcode comprises GC at the 3′ end of the polynucleotide barcode.

101. The nucleic acid of any of claims 91-100, wherein the polynucleotide barcode comprises a cytidine at position 39.

102. The nucleic acid of claim 101, wherein the polynucleotide barcode comprises a guanosine at position 40.

103. The nucleic acid of any of claims 91-102, wherein the polynucleotide barcode comprises an adenosine at positions 45 and 46.

104. The nucleic acid of any of claims 91-103, wherein the polynucleotide barcode comprises an adenosine at position 31 and a guanosine at position 32.

105. The nucleic acid of any of claims 91-104, wherein the polynucleotide barcode does not comprise a thymidine at position 54, position 49, or both.

106. The nucleic acid of any of claims 91-105, wherein the polynucleotide barcode does not comprise a guanosine at position 50 and a cytidine at position 51.

107. The nucleic acid of any of claims 91-106, wherein the polynucleotide barcode comprises CACTTG (SEQ ID NO: 1054) at positions 32-37.

108. The nucleic acid of any of claims 91-107, wherein the polynucleotide barcode comprises CCTAGTAATAG (SEQ ID NO: 1055) at positions 39-49.

109. The nucleic acid of any of claims 91-108, wherein the polynucleotide barcode comprises CCGG (SEQ ID NO: 1056) at the 5′ end of the polynucleotide barcode.

110. The nucleic acid of any of claims 91-109, wherein the polynucleotide barcode comprises a sequence with at least 70% similarity to sequences selected from the group consisting of SEQ ID NOs: 1-1053.

111. The nucleic acid of any of claims 91-110, wherein the polynucleotide barcode sequence is selected from the group consisting of SEQ ID NO: 1-10.

112. The nucleic acid of any of claims 91-111, wherein the nucleic acid encodes a pair of gRNAs configured to hybridize to the two PAM sequences flanking the polynucleotide barcode.

113. The nucleic acid of claim 112, wherein one or both of the pair of gRNAs comprises a guide sequence of less than 25 nucleotides.

114. The nucleic acid of any of claims 91-113, wherein the pair of gRNAs are within a crRNA array.

115. The nucleic acid of claim 114, wherein the crRNA array further comprises a termination signal for crRNA expression.

116. The nucleic acid of claim 114 or 115, wherein the crRNA array further comprises at least one gRNA configured to hybridize to a recipient nucleic acid.

117. The nucleic acid of any of claims 91-116, wherein the nucleic acid further comprises a sequence tag configured to remain static over time.

118. A method for introducing a polynucleotide barcode into a cell comprising: introducing into the cell a system of any of claims 1-40, a system of any of claims 46-82, or the nucleic acid of any of claims 91-117 and a CRISPR associated (Cas) endonuclease or a nucleic acid encoding a Cas endonuclease.

119. The method of claim 118, wherein the Cas endonuclease is a Class 2 Cas endonuclease.

120. The method of claim 118 or 119, wherein the Cas endonuclease is a Type V Cas endonuclease.

121. The method of any of claims 118-120, wherein the Cas endonuclease is selected from Cas9, Cas12a, and Cas14.

122. The method of any of claims 118-121, wherein the polynucleotide barcode integrates into genomic DNA.

123. The method of any of claims 118-122, wherein the polynucleotide barcode is passed to daughter cells.

124. The method of any of claims 118-123, wherein the cell is eukaryotic cell.

125. The method of any of claims 118-124, wherein the cell is in vitro.

126. The method of any of claims 118-125, wherein the cell is ex vivo.

127. The method of any of claims 118-126, wherein the cell is in a subject.

128. A method for cell tracking comprising: introducing in the cell: a system of any of claims 1-40, a system of any of claims 46-82, or the nucleic acid of any of claims 91-117 and a Cas endonuclease; isolating cellular nucleic acids at one or more time points; sequencing the polynucleotide barcode at the one or more time points; and tracking changes to original sequence of barcode in the cell at each time point.

129. The method of claim 128, wherein the cell is eukaryotic cell.

130. The method of claim 128 or 129, wherein the cell is in vitro.

131. The method of any of claims 128-130, wherein the cell is ex vivo.

132. The method of claim 128 or 129, wherein the cell is in a subject.

133. The method of any of claims 128-132, wherein the one or more time points are over multiple cell generations.

134. The method of any of claims 128-133, further comprising establishing lineage connections or a sequence of changes in barcode sequence between cells from different generations.

135. The method of any of claims 128-134, wherein expression of Cas12a or the Cas endonuclease is controlled by an inducible promoter.

136. The method of claim 135, further comprising adding varying concentrations of an inducing agent to the cells to vary the change rate of the original barcode sequence.

137. The method of claim 136, wherein increasing concentrations of the inducing agent increases the change rate of the original barcode sequence.

138. The method of any of claims 135-137, wherein the cell is a tumor cell, a neuron, or an adipocyte.

139. The method of claim 138, wherein the inducing agent concentration is low.

140. The method of any of claims 135-137, wherein the cell is a cancerous cell or intestinal epithelial cell.

141. The method of claim 140, wherein the inducing agent concentration is high.

142. The method of any of claims 128-141, further comprising determining single-cell transcriptomic profiles.

143. The method of claim 142, further comprising characterizing heritability of gene expression patterns.

144. The method of claim 143, further comprising determining gene products which have heritable expression patterns.

145. The method of any of claims 128-144, further comprising introducing one or more mutations, insertions, or deletions in one or more target genes of interest in the cell.

146. The method of claim 145, further comprising monitoring the effect of the one or more mutations, insertions, or deletions on cell function, cell viability, or effectiveness of a pharmacological treatment.

147. A computer implemented method for designing a polynucleotide barcode sequence configured to promote insertions and deletions over time comprising: designing a seed barcode sequence based on sequence elements which promote insertions and deletions, sequence elements which suppress insertions and deletions, or both; iteratively mutating the seed barcode sequence; and predicting sequence entropy as measure of insertions and deletions accumulated in the polynucleotide barcode sequence over time.

148. The computer implemented method of claim 147, wherein the seed barcode comprises 50-60 nucleotides.

149. The computer implemented method of claim 147 or 148, wherein the sequence elements which promote insertions and deletions are selected from the group consisting of: a GC dinucleotide at the 3′ end of the barcode; a CG dinucleotide starting at position 39; an AA dinucleotide starting at position 45; an AG dinucleotide starting at position 31; CACTTG (SEQ ID NO: 1054) at positions 32-37; CCTAGTAATAG (SEQ ID NO: 1055) at positions 39-49; CCGG (SEQ ID NO: 1056) at the 5′ end of the polynucleotide barcode; or a combination thereof.

150. The computer implemented method of any of claims 147-149, wherein the sequence elements which suppress insertions and deletions are selected from the group consisting of: a thymidine at position 54; a thymidine at position 49; a GC dinucleotide starting at position 50; or a combination thereof.

12; 12; 12; 12; 12; 16; 16

edspap.20240209447