METHODS FOR USING DNA REPAIR FOR CELL ENGINEERING (as amended)
AssigneeCaribou Biosciences, Inc.
InventorsDaniel Capurso, Andrew P. May, Megan Van Overbeek
Life of this patent
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Abstract
Methods for gene editing and predicting non-random editing events are described. The methods use Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems to characterize and manipulate DNA repair outcomes at Cas-initiated double-strand breaks (DSBs) to anticipate functional outcomes.
Claims21
1-20. (canceled)
A method of designing a guide polynucleotide for predictably inserting an adenine (A) or a thymine (T) nucleotide at a cut site produced by Cas9-mediated cleavage of DNA in a cell, the method comprising:
- selecting a target region in the DNA in the cell to be modified;
- and
- designing a guide polynucleotide complementary to a protospacer in the target region such that the nucleotide at position 17 of the protospacer corresponds to an A or a T;
- wherein (i) for insertion of an A at the cut site, position 17 of the protospacer is A, or (ii) for insertion of a Tat the cut site, position 17 of the protospacer is T.
A method of predictably inserting an adenine (A) or a thymine (T) nucleotide at a cut site produced by Cas9-mediated cleavage of DNA in a cell, the method comprising:
- selecting a target region in the DNA of the cell to be modified;
- and
- introducing into the cell a complex comprising a Cas9 protein and a guide polynucleotide complementary to a protospacer in the target region such that the nucleotide at position 17 of the protospacer corresponds to an A or a T;
- wherein (i) for insertion of an A at the cut site, position 17 of the protospacer is A, or (ii) for insertion of a T at the cut site, position 17 of the protospacer is T;
- and further wherein the complex cleaves the DNA and produces the cut site, thus facilitating insertion of the A or the T, respectively, at the cut site.
Dependencies are inferred from the claim text.