# US 11,028,429 B2 · Full interrogation of nuclease DSBs and sequencing (FIND-seq)

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- Number: US 11,028,429 B2
- Document ID: US11028429B2
- Status: Granted
- Kind: B2
- Application number: 15/994,939
- Filed: 2018-05-31
- Published: 2018-09-20
- Granted: 2021-06-08
- Earliest claimed: 2015-09-11
- Assignees: The General Hospital Corporation
- Applicants: The General Hospital Corporation
- Inventors: J. Keith Joung; Shengdar Tsai
- Web page: https://patentgrep.com/patent/US11028429B2

## CPC

- C12Q 1/6855: Ligating adaptors
- C12N 15/1093: {General methods of preparing gene libraries, not provided for in other subgroups}
- C40B 40/06: Libraries containing nucleotides or polynucleotides, or derivatives thereof
- C40B 50/06: Biochemical methods, e.g. using enzymes or whole viable microorganisms
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60)

## Abstract

Sensitive, unbiased methods for genome-wide detection of potential off-target nuclease cleavage sites in DNA, e.g., in cell type-specific genomic DNA samples.

## Claims

### Claim 1

A library of covalently closed DNA fragments, the library obtained by a 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 first population of ligated fragments;
purifying the ligated fragments using an exonuclease, to prepare a population of purified ligated fragments;
treating the purified ligated fragments 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; and
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 producing a library of covalently closed DNA fragments,
wherein one end was created by a nuclease-induced double stranded break in the DNA,
wherein the first hairpin adapter comprises:
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,
wherein purifying the ligated fragments using an exonuclease comprises 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, and
wherein preparing the population of DNA fragments for end-ligation comprises end-repairing and then A-tailing the sheared DNA.

### Claim 2 (depends on 1)

The library of claim 1, wherein treating the purified ligated fragments with a nuclease to induce double stranded breaks comprises contacting the sample with a Cas9 nuclease complexed with a specific guide RNA (gRNA).

### Claim 3 (depends on 1)

The library of claim 1, 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).

### Claim 4 (depends on 1)

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

### Claim 5 (depends on 1)

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

### Claim 6 (depends on 1)

The library of claim 1, wherein the DNA is synthetic.

### Claim 7 (depends on 1)

The library 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).

### Claim 8 (depends on 1)

A library of DNA fragments, the library obtained by a method comprising:
contacting the library of covalently closed DNA fragments of claim 1 with uracil DNA glycosylase (UDG) and/or endonuclease VIII to nick the DNA at the deoxyuridine, thereby producing a library of DNA fragments.