Full interrogation of nuclease DSBs and sequencing (find-seq)

1. A method of preparing a library of covalently closed DNA fragments, the method comprising:

providing DNA;

randomly shearing the DNA to a defined average length to provide a population of DNA fragments;

preparing the population of DNA fragments for end-ligation;

ligating to the ends of the fragments a first hairpin adapter comprising a single stranded hairpin loop region comprising at least a single deoxyuridine and a first primer site, to prepare a population of ligated fragments;

and

purifying the ligated fragments using an exonuclease, thereby preparing a library of covalently closed DNA fragments.

2. The method of claim 1 , wherein the first primer site is compatible for use in PCR priming and/or sequencing.

3. The method of claim 1 , further comprising:

contacting the library of covalently closed DNA fragments with a nuclease to induce double stranded breaks including site-specific breaks;

ligating a second hairpin adapter comprising a single-stranded hairpin loop comprising at least a single deoxyuridine and a second primer site compatible for use with the first primer site in PCR priming and/or sequencing,

contacting the library with an enzyme to nick the DNA at the deoxyuridine; and

sequencing those fragments having a first and second hairpin adapter.

4. The method of claim 3 , wherein the enzyme that nicks the DNA at the deoxyuridine is a uracil DNA glycosylase (UDG) or endonuclease VIII.

5. The method of claim 3 , wherein treating the sample with a nuclease to induce double stranded breaks comprises contacting the sample with a Cas9 nuclease complexed with a specific guide RNA (gRNA).

6. The method of claim 3 , wherein the nuclease that induces double stranded breaks is selected from the group consisting of meganucleases, MegaTALs, zinc-finger nucleases, transcription activator effector-like nucleases (TALEN), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas RNA-guided nucleases (CRISPR/Cas RGNs).

7. The method of claim 6 , wherein the nuclease is a Cas9 nuclease, and the method also includes expressing in the cells a guide RNA that directs the Cas9 nuclease to a target sequence in the genome.

8. The method of claim 1 , wherein the DNA is randomly sheared to an average length of 200-1000 bps.

9. The method of claim 1 , wherein the DNA is genomic DNA isolated from a mammalian, plant, bacterial, or fungal cell.

10. The method of claim 1 , wherein the DNA is synthetic.

11. The method of claim 1 , wherein the first primer site in the first hairpin adapter comprises a next generation sequencing primer site, a randomized DNA barcode or unique molecular identifier (UMI).

12. A method of preparing a library of fragments comprising nuclease-induced double stranded breaks in DNA, the method comprising:

providing DNA;

randomly shearing the DNA to a defined average length;

optionally end-repairing and then A-tailing the sheared DNA;

ligating to the DNA a first hairpin adapter comprising:

a first region of 10-20 nucleotides;

a second region of 45-65 nucleotides that forms one or more single-stranded hairpin loops and comprises a first primer site compatible for use in PCR priming and/or sequencing; and

a third region of 10-20 nucleotides that is complementary to the first region, with a single deoxyuridine nucleotide between the first and second regions;

contacting the sample with one or more exonucleases, sufficient to degrade any DNA molecules that lack the first hairpin adapter ligated to both of their ends;

treating the sample with a nuclease to induce double stranded breaks, including site-specific breaks, of the DNA;

optionally end-repairing and then A-tailing the resulting ends;

ligating a second hairpin adapter comprising a first region of 10-20 nucleotides; a second region of 40-60 nucleotides that forms one or more single-stranded hairpin loops and comprises a second primer compatible for use with the first primer site in PCR priming and/or sequencing; and a third region of 10-20 nucleotides that is complementary to the first region and that also contains a single deoxyuridine nucleotide between the second and third regions, to create a population wherein the DNA fragments that were cleaved by the nuclease have a first and second hairpin adapter ligated to their respective ends; thereby preparing a library of fragments wherein one end was created by a nuclease-induced double stranded break in the DNA.

13. The method of claim 12 , further comprising:

contacting the library with uracil DNA glycosylase (UDG) and/or endonuclease VIII to nick the DNA at the deoxyuridine.

14. The method of claim 12 , wherein treating the sample with a nuclease to induce site-specific cleavage comprises contacting the sample with a Cas9 nuclease complexed with a specific guide RNA (gRNA).

15. A method for detecting nuclease-induced double stranded breaks (DSBs) in DNA, the method comprising:

providing DNA;

randomly shearing the DNA to a defined average length;

optionally end-repairing and then A-tailing the sheared DNA;

ligating a first hairpin adapter comprising:

a first region of 10-20 nucleotides;

a second region of 45-65 nucleotides that forms one or more single-stranded hairpin loops and comprises a first primer site compatible for use in PCR priming and/or sequencing; and

a third region of 10-20 nucleotides that is complementary to the first region, with a single deoxyuridine nucleotide between the first and second regions;

contacting the sample with one or more exonucleases, sufficient to degrade any DNA molecules that lack the first hairpin adapter ligated to both of their ends;

treating the sample with a nuclease to induce double stranded breaks, including site-specific breaks, of the DNA;

optionally end-repairing and then A-tailing the resulting cleaved ends;

ligating a second hairpin adapter comprising a first region of 10-20 nucleotides; a second region of 40-60 nucleotides that forms one or more single-stranded hairpin loops and comprises a second primer compatible for use with the first primer site in PCR priming and/or sequencing; and a third region of 10-20 nucleotides that is complementary to the first region, and that also contains a single deoxyuridine nucleotide between the second and third regions, to create a population wherein the DNA fragments that were cleaved by the nuclease have the first and second hairpin adapters ligated to their respective ends; thereby preparing a library of fragments comprising nuclease-induced double stranded breaks in the DNA, wherein one end was created by a nuclease-induced double stranded break in the DNA;

contacting the library with uracil DNA glycosylase (UDG) and/or endonuclease VIII, a DNA glycosylase-lyase to nick the DNA at the deoxyuridine; and

sequencing those fragments bearing a first and a second hairpin adapter;

thereby detecting DSBs induced by the nuclease.

16. The method of claim 15 , wherein the nuclease is selected from the group consisting of meganucleases, MegaTALs, zinc-finger nucleases, transcription activator effector-like nucleases (TALEN), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas RNA-guided nucleases (CRISPR/Cas RGNs).

17. The method of claim 15 , wherein treating the sample with a nuclease to induce double stranded breaks comprises contacting the sample with a Cas9 nuclease complexed with a specific guide RNA (gRNA).

18. A method of determining which of a plurality of guide RNAs is most specific, the method comprising, for each of the plurality of guide RNAs:

providing DNA;

randomly shearing the DNA to a defined average length;

end-repairing and then A-tailing the sheared DNA;

ligating a first hairpin adapter comprising:

a first region of 10-20 nucleotides;

a second region of 45-65 nucleotides, that forms one or more single-stranded hairpin loops and comprises a first primer site compatible for use in PCR priming and/or sequencing; and

a third region of 10-20 nucleotides that is complementary to the first region, with a single deoxyuridine nucleotide between the first and second regions;

contacting the sample with one or more exonucleases, sufficient to degrade any DNA molecules that lack the first adapter ligated to both of their ends;

treating the sample with a Cas9 nuclease compatible with the guide RNA to induce site specific double stranded breaks of the DNA;

optionally end-repairing and then A-tailing the resulting cleaved ends;

ligating a second hairpin adapter comprising:

a first region of 10-20, nucleotides;

a second region of 40-60 nucleotides that forms one or more single-stranded hairpin loops and comprises a second primer compatible for use with the first primer site in PCR priming and/or sequencing; and

a third region of 10-20 nucleotides that is complementary to the first region, and that also contains a single deoxyuridine nucleotide between the second and third regions, to create a population wherein the DNA fragments that have a first and second hairpin adapter ligated to their ends are those that were cleaved by the nuclease; thereby preparing a library of fragments comprising nuclease-induced double stranded breaks in DNA wherein one end was created by a nuclease-induced double stranded break in the DNA;

contacting the library with uracil DNA glycosylase (UDG) and/or endonuclease VIII to nick the DNA at the deoxyuridine; and

sequencing those fragments bearing a first and a second hairpin adapter, thereby detecting DSBs induced by the nuclease in each sample;

optionally identifying whether each DSB is on-target or off-target;

comparing the DSBs induced by the nuclease in each sample; and

determining which of the plurality of guide RNAs induced the fewest off-target DSBs.