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

1. A method of selecting for double stranded DNA sequence(s) that are bound by a DNA-binding domain, the method comprising:

(i) providing a plurality of linear dsDNA oligonucleotides of known sequences;

(ii) incubating the plurality of linear dsDNA oligonucleotides in the presence of an affinity-tagged DNA binding domain that can be bound to a substrate molecule under conditions sufficient for binding of the affinity-tagged DNA-binding domain and one or more of the plurality of linear dsDNA oligonucleotide(s) to occur, thereby creating bound linear dsDNA oligonucleotide(s); and

(iii) selecting for bound linear dsDNA oligonucleotide(s) to create selected linear dsDNA oligonucleotide(s), thereby selecting for double stranded DNA sequence(s) that are bound by a DNA-binding domain.

2. A method of identifying double stranded DNA sequence(s) that are bound by a DNA-binding domain, the method comprising determining the sequence(s) of the selected linear dsDNA oligonucleotide(s) produced by the method of claim 1 , thereby identifying double stranded DNA sequence(s) that are bound by the DNA-binding domain.

3. A method of enriching for double stranded DNA sequence(s) that are bound by a DNA-binding domain, the method comprising:

(i) incubating the selected linear dsDNA oligonucleotides produced by the method of claim 1 in the presence of an affinity-tagged DNA binding domain that binds to a substrate molecule under conditions sufficient for binding of the affinity-tagged DNA-binding domain and one or more of the plurality of selected linear dsDNA oligonucleotide(s) to occur, thereby creating bound selected linear dsDNA oligonucleotide(s); and

(ii) selecting for bound selected linear dsDNA oligonucleotide(s) to create enriched linear dsDNA oligonucleotide(s), thereby enriching for double stranded DNA sequence(s) that are bound by a DNA-binding domain.

4. A method of identifying double stranded DNA sequence(s) that are bound by a DNA-binding domain, the method comprising determining the sequence(s) of the enriched linear dsDNA oligonucleotide(s) produced by the method of claim 3 thereby identifying double stranded DNA sequence(s) that are bound by the DNA-binding domain.

5. The method of claim 3 , wherein selecting for bound selected linear dsDNA oligonucleotide(s) comprises:

(i) incubating the bound selected linear dsDNA oligonucleotide(s) under conditions sufficient for binding of the affinity tag to the substrate molecule, thereby creating substrate bound selected linear dsDNA oligonucleotide(s);

(ii) separating the substrate bound selected linear dsDNA oligonucleotide(s) from unbound linear dsDNA oligonucleotide(s); and

(ii) eluting the substrate bound selected linear dsDNA oligonucleotide(s) in either (a) a buffer that promotes dissociation of the substrate bound linear dsDNA oligonucleotide(s) or (b) a buffer containing a protease under conditions effective to degrade bead-bound protein and release substrate bound linear dsDNA oligonucleotide(s) to create enriched linear dsDNA oligonucleotides, thereby enriching for double stranded DNA sequence(s) that are bound by a DNA-binding domain.

6. The method of claim 1 , wherein the affinity-tagged DNA-binding domain is a Cas9 protein complexed with a sgRNA, a variant of a Cas9 protein complexed with a sgRNA, a Cas9 fusion protein complexed with a sgRNA, or a variant of a Cas9 fusion protein complexed with a sgRNA.

7. The method of claim 6 , wherein the sgRNA targets a site selected from the group consisting of EMX1, FANCF, HBB, HEK2, HEK3, HEK4, RNF2, ABE14, ABE16, ABE 18, and VEGFA3.

8. The method of claim 1 , wherein the affinity-tagged DNA-binding domain is inactivated Cas9 (dCas9) complexed with a sgRNA.

9. The method of claim 1 , wherein the affinity-tagged DNA-binding domain is an engineered zinc finger array.

10. The method of claim 1 , wherein the affinity-tagged DNA-binding domain is an engineered TALE repeat array.

11. The method of claim 1 , wherein the substrate molecule is a magnetic bead carrying a molecule that binds to the affinity tag.

12. The method of claim 1 , wherein the affinity tag is a molecule that can be covalently bound to benzylguanine and the substrate molecule is a benzylguanine-carrying substrate molecule.

13. The method of claim 1 , wherein said selecting for bound linear dsDNA oligonucleotide(s) in step (iii) comprises:

(i) incubating the bound linear dsDNA oligonucleotide(s) under conditions sufficient for binding of the affinity tag to the substrate molecule, thereby creating substrate bound linear dsDNA oligonucleotide(s);

(ii) separating the substrate bound linear dsDNA oligonucleotide(s) from unbound linear dsDNA oligonucleotide(s); and

(iii) eluting the substrate bound linear dsDNA oligonucleotide(s) in either (a) a buffer that promotes dissociation of the substrate bound linear dsDNA oligonucleotide(s) or (b) a buffer containing a protease under conditions effective to degrade bead-bound protein and release substrate bound linear dsDNA oligonucleotide(s) to create selected linear dsDNA oligonucleotides, thereby selecting for double stranded DNA sequence(s) that are bound by a DNA-binding domain.

14. The method of claim 13 , wherein the protease is proteinase K.

15. The method of claim 1 , wherein the linear dsDNA oligonucleotides comprise 16 to 10 8 different sequences.

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

17. 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 DNA-binding domain in a reference genome bearing substitutions, single base pair deletions, and/or single base pair insertions relative to an identified on-target site for the DNA-binding domain;

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

(iii) a set of potential off-target sequences for the DNA-binding domain bearing substitutions, single base pair deletions, and/or single base pair insertions relative to an identified on-target site for the DNA-binding domain;

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

(v) a set of all potential off-target sequences for the DNA-binding domain in the sequence of an oncogene hotspot and/or tumor suppressor gene of a reference genome bearing substitutions, single base pair deletions, and/or single base pair insertions relative to an identified on-target site for the DNA-binding domain.