Compositions and Methods for DNA Modification Detection
AssigneeAltius Institute for Biomedical Sciences
InventorsJohn Stamatoyannopoulos, Pavel Zrazhevskiy
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Abstract
Provided herein are methods and compositions for use in detecting on-target and/or off-target cleavage of genomic DNA in a cell by a nuclease. The on-target and off-target cleavage is detected by using oligonucleotides that bind to the single-stranded 3′-overhang created at the cleavage site. The nuclease may be an engineered nuclease comprising a nucleic acid binding domain that binds to a target nucleic acid of interest.
Claims53
A method for detecting induction of cleavage in genomic DNA by a nuclease, the method comprising:
- contacting a live cell comprising genomic DNA comprising a cleavage site for the nuclease with the nuclease under conditions sufficient for induction of cleavage at the cleavage site;
- fixing the cell and contacting the fixed cell with a plurality of oligonucleotides complementary to a single stranded (ss) 3′-overhang created at the cleavage site, wherein the genomic DNA is not denatured;
- and
- detecting hybridization of the plurality of oligonucleotides to the ss 3′-overhang,
- wherein hybridization of the oligonucleotides indicates induction of the cleavage by the nuclease.
A kit for detecting induction of cleavage at a cleavage site for a recombinant nuclease in genomic DNA in a cell, comprising:
- a plurality of labeled oligonucleotides complementary to a single stranded (ss) 3′-overhang created at the cleavage site for the recombinant nuclease;
- and
- the recombinant nuclease.
Dependencies are inferred from the claim text.