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

1. 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 first strand comprising, from 5′ to 3′:

a first known common sequence common to each of the oligonucleotides in the plurality;

a first known barcode sequence unique to the library member;

one and only one copy of a known potential DNA substrate sequence for the nuclease comprising a cognate protospacer adjacent motif (PAM);

a second known barcode sequence identical to the first barcode sequence; and

a second known common sequence common to each of the oligonucleotides in the library; and

a second strand complementary to the known sequences of the first strand;

(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, thereby creating first and second dsDNA oligonucleotide fragments;

(iii) ligating double stranded DNA adapters comprising primer sequences to the cleaved ends of the first and second dsDNA oligonucleotide fragments;

(iv) amplifying the first dsDNA oligonucleotide fragments using one primer specific to the primer sequence of the DNA adapter and one primer specific to the first common sequence and amplifying the second dsDNA oligonucleotide fragments using one primer specific to the primer sequence of the DNA adapter and one primer specific to the second common sequence; and

(v) determining the sequence of a barcode sequence of one or more of the amplified dsDNA oligonucleotide fragments, thereby identifying double stranded DNA sequences that are cleaved by a nuclease.

2. The method of claim 1 , wherein the linear dsDNA oligonucleotides comprise potential DNA substrate sequences comprising:

(i) a set of all potential off-target sequences for the site-specific nuclease in a reference genome bearing up to a certain number of substitutions, single base pair deletions, and/or single base pair insertions relative to an identified on-target site for the site-specific nuclease;

(ii) a comprehensive set of all potential off-target sequences for the site-specific nuclease bearing up to a certain number of substitutions, single base pair deletions, and/or single base pair insertions relative to an identified on-target site for the site-specific nuclease;

(iii) a set of potential off-target sequences for the site-specific nuclease present in a set of variant genomes from defined populations, optionally genomic DNA libraries designed to reflect DNA sequence variants present in a population of individuals bearing up to a certain number of substitutions, single base pair deletions, and/or single base pair insertions relative to an identified on-target site for the site-specific nuclease;

(iv) a set of all potential off-target sequences for the site-specific nuclease in the coding sequence of a reference genome bearing up to a certain number of substitutions, single base pair deletions, and/or single base pair insertions relative to an identified on-target site for the site-specific nuclease; or

(v) a set of all potential off-target sequences for the site-specific nuclease of an oncogene hotspots and/or tumor suppressor gene of a reference genome bearing up to a certain number of substitutions, single base pair deletions and/or single base pair insertions relative to an identified on-target site for the site-specific nuclease.

3. The method of claim 1 , wherein

the linear dsDNA oligonucleotides are first synthesized as individual single-stranded DNA sequences, optionally on high-density oligonucleotide arrays, and wherein the single-stranded DNA sequences are converted into double-stranded DNA molecules.

4. The method of claim 1 , wherein the linear dsDNA oligonucleotides comprise 1,000 to 10 11 different sequences.

5. The method of claim 1 , wherein the linear dsDNA oligonucleotides comprise sequences that are 50 to 500 bp long.

6. The method of claim 1 , wherein the site-specific nuclease is Cas9.