Methods for the identification of variant recognition sites for rare-cutting engineered double-strand-break-inducing agents and compositions and uses thereof

An in vitro method to identify a Cas endonuclease double strand break site in genomic DNA, said method comprising:

• a. contacting the genomic DNA with a Cas endonuclease-guide RNA complex, wherein the Cas endonuclease-guide RNA complex introduces a double-strand break in the genomic DNA, resulting in a blunt end;
• b. creating a nucleotide overhang from the blunt end of (a);
• c. ligating a first adapter to the nucleotide overhang of (b), resulting in a ligated DNA;
• d. obtaining fragments of the ligated DNA of (c) and ligating a second adapter to the DNA fragments to allow for the amplification and sequencing of the DNA fragments that each comprise the first adapter on one end and the second adapter on the other end;
• e. amplifying and sequencing the DNA fragments of (d);
• f. aligning nucleotide sequences of the DNA fragments of (e) with a reference DNA sequence;
• and,
• g. identifying the double-strand break site.