Using Truncated Guide RNAs (tru-gRNAs) to Increase Specificity for RNA-Guided Genome Editing
First Claim
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1. A method of increasing specificity of RNA-guided genome editing in a cell, the method comprising contacting the cell with a guide RNA that includes a complementarity region consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a selected target genomic sequence, thereby increasing specificity of RNA-guided genome editing in a cell.
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Abstract
Methods for increasing specificity of RNA-guided genome editing, e.g., editing using CRISPR/Cas9 systems, using truncated guide RNAs (tru-gRNAs).
233 Citations
39 Claims
- 1. A method of increasing specificity of RNA-guided genome editing in a cell, the method comprising contacting the cell with a guide RNA that includes a complementarity region consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a selected target genomic sequence, thereby increasing specificity of RNA-guided genome editing in a cell.
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2. A method of inducing a break in a target region of a double-stranded DNA molecule in a cell, the method comprising expressing in or introducing into the cell:
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a Cas9 nuclease or nickase; and a guide RNA that includes a complementarity region consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of double-stranded DNA molecule, thereby inducing a break in a target region of a double-stranded DNA molecule in a cell. - View Dependent Claims (4, 5, 13, 14, 18, 19, 20, 26, 27, 29)
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3. A method of modifying a target region of a double-stranded DNA molecule in a cell, the method comprising expressing in or introducing into the cell:
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a dCas9-heterologous functional domain fusion protein (dCas9-HFD); and a guide RNA that includes a complementarity region consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a selected target genomic sequence, thereby modifying a target region of a double-stranded DNA molecule in a cell.
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- 6. A guide RNA molecule having a target complementarity region of 17-18 nucleotides.
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10-12. -12. (canceled)
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21-25. -25. (canceled)
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28. (canceled)
Specification