Methods Of Modifying A Sequence Using CRISPR
First Claim
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1. A method of modifying one or more target nucleic acid sequences comprising:
- (a) contacting the one or more target nucleic acid sequences with;
i) one or more ribonucleic acid (RNA) sequences wherein each RNA sequence comprises a portion that is complementary to all or a portion of one or more of the target nucleic acid sequences;
ii) a CRISPR associated (Cas) protein having nuclease activity;
iii) one or more exogenous nucleic acid sequences wherein at least one exogenous nucleic acid sequence comprises a 5′
adapter sequence that hybridizes to a 5′
flanking sequence of the target nucleic acid sequence and at least one exogenous nucleic acid sequence comprises a 3′
adapter sequence that hybridizes to a 3′
flanking sequence of the target nucleic acid sequence; and
iv) a nucleic acid sequence that interacts with Cas protein;
thereby producing a combination; and
(b) maintaining the combination under conditions in which the one or more RNA sequences hybridize to all or the portion of the one or more target nucleic acid sequences to which each RNA sequence forms a complement thereby forming one or more base paired structures, and the one or more base paired structures and the nucleic acid sequence that interacts with Cas protein direct Cas protein to cleave the one or more target nucleic acid sequences;
thereby modifying the one or more target nucleic acid sequences.
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Abstract
Methods of modifying one or more target nucleic acid sequences using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated (Cas) proteins (CRISPR/Cas) system are disclosed. Methods of introducing one or more exogenous nucleic acid sequences into one or more circular nucleic acid sequences using the CRISPR/Cas system are also disclosed.
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Citations
46 Claims
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1. A method of modifying one or more target nucleic acid sequences comprising:
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(a) contacting the one or more target nucleic acid sequences with; i) one or more ribonucleic acid (RNA) sequences wherein each RNA sequence comprises a portion that is complementary to all or a portion of one or more of the target nucleic acid sequences; ii) a CRISPR associated (Cas) protein having nuclease activity; iii) one or more exogenous nucleic acid sequences wherein at least one exogenous nucleic acid sequence comprises a 5′
adapter sequence that hybridizes to a 5′
flanking sequence of the target nucleic acid sequence and at least one exogenous nucleic acid sequence comprises a 3′
adapter sequence that hybridizes to a 3′
flanking sequence of the target nucleic acid sequence; andiv) a nucleic acid sequence that interacts with Cas protein; thereby producing a combination; and (b) maintaining the combination under conditions in which the one or more RNA sequences hybridize to all or the portion of the one or more target nucleic acid sequences to which each RNA sequence forms a complement thereby forming one or more base paired structures, and the one or more base paired structures and the nucleic acid sequence that interacts with Cas protein direct Cas protein to cleave the one or more target nucleic acid sequences; thereby modifying the one or more target nucleic acid sequences. - View Dependent Claims (2, 3, 4, 5, 8, 13, 14, 18, 20, 22)
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6. (canceled)
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7. (canceled)
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9-12. -12. (canceled)
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15-17. -17. (canceled)
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19. (canceled)
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21. (canceled)
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23. (canceled)
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24. (canceled)
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25. A method of introducing one or more exogenous nucleic acid sequences into one or more circular nucleic acid sequences comprising:
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(a) contacting the one or more circular nucleic acid sequences with; i) one or more ribonucleic acid (RNA) sequences wherein each RNA sequence comprises a portion that is complementary to all or a portion of one or more target sequences within the one or more circular nucleic acid sequences; ii) a CRISPR associated (Cas) protein having nuclease activity; iii) one or more exogenous nucleic acid sequences wherein at least one exogenous nucleic acid sequence comprises a 5′
adapter sequence that hybridizes to a 5′
flanking sequence of the target nucleic acid sequence and at least one exogenous nucleic acid sequence comprises a 3′
adapter sequence that hybridizes to a 3′
flanking sequence of the target nucleic acid sequence;
wherein at least one exogenous nucleic acid sequence comprises one or more additional nucleotides; andiv) a nucleic acid sequence that interacts with Cas protein; thereby producing a combination; and (b) maintaining the combination under conditions in which the one or more RNA sequences hybridize to all or the portion of the one or more target nucleic acid sequences to which each RNA sequence forms a complement thereby forming one or more base paired structures, and the one or more base paired structures and the nucleic acid sequence that interacts with Cas protein direct the Cas protein to cleave the target nucleic acid sequence; thereby introducing the one or more exogenous nucleic acid sequence into the one or more circular nucleic acid sequences. - View Dependent Claims (26, 27, 28, 31, 36, 41, 42, 44)
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29. (canceled)
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30. (canceled)
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32-35. -35. (canceled)
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37-40. -40. (canceled)
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43. (canceled)
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45. (canceled)
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46. (canceled)
Specification