Highly sensitive in vitro assays to define substrate preferences and sites of nucleic-acid binding, modifying, and cleaving agents

An in vitro method of identifying double stranded DNA sequences that are cleaved by a nuclease, the method comprising:

• (i) providing a library of defined linear dsDNA oligonucleotides of known sequences, each library member having a known sequence comprising, in order:
• a first sequence common to each of the oligonucleotides in the library;
• a first barcode sequence unique to the library member;
• a potential DNA substrate sequence for the nuclease;
• a second copy of the barcode sequence;
• and
• a second sequence common to each of the oligonucleotides in the library;
• (ii) incubating the library in the presence of a site-specific nuclease under conditions sufficient for cleavage of one or more of the linear dsDNA oligonucleotides to occur;
• (iii) ligating DNA adapters comprising primer sequences to the cleaved ends of the dsDNA oligonucleotides;
• (iv) amplifying at least one of the cleaved dsDNA oligonucleotides using a primer specific to a primer sequence of the DNA adapter and a primer specific to at least one of the common sequences to generate amplified fragments;
• and
• (v) determining the barcode sequence(s) of the amplified fragments, thereby identifying double stranded DNA sequences that are cleaved by a nuclease.