# US 10,501,794 B2 · Genomewide unbiased identification of DSBs evaluated by sequencing (GUIDE-seq)

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- Number: US 10,501,794 B2
- Document ID: US10501794B2
- Status: Granted
- Kind: B2
- Application number: 15/782,037
- Filed: 2017-10-12
- Published: 2018-03-29
- Granted: 2019-12-10
- Earliest claimed: 2014-06-23
- Assignees: The General Hospital Corporation
- Applicants: The General Hospital Corporation
- Inventors: J. Keith Joung; Shengdar Tsai
- Web page: https://patentgrep.com/patent/US10501794B2

## CPC

- C12Q 1/6869: Methods for sequencing
- C12Q 1/6855: Ligating adaptors
- 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

## Abstract

Unbiased, genomewide and highly sensitive methods for detecting mutations, e.g., off-target mutations, induced by engineered nucleases.

## Claims

### Claim 1

A method for detecting double stranded breaks (DSBs) in genomic DNA (gDNA) of a cell, the method comprising:
contacting the cell with a blunt-ended double-stranded oligodeoxynucleotide (dsODN), wherein both strands of the dsODN are orthogonal to the genome of the cell, and further wherein (a) the 5′ ends of the dsODN are phosphorylated, and (b) phosphorothioate linkages are present on both 3′ ends, or phosphorothioate linkages are present on both 3′ ends and both 5′ ends;
maintaining the cell under conditions sufficient for the cell to repair the DSBs, integrating a dsODN at one or more DSBs;
amplifying a portion of gDNA comprising an integrated dsODN; and
sequencing the amplified portion of the gDNA,
thereby detecting a DSB in the gDNA of the cell.

### Claim 2 (depends on 1)

The method of claim 1, wherein amplifying a portion of the gDNA comprises:
fragmenting the gDNA;
ligating ends of the fragmented gDNA from the cell with a universal adapter; and performing polymerase chain reaction (PCR) on the ligated DNA.

### Claim 3 (depends on 1)

The method of claim 1, wherein the cell is a mammalian cell.

### Claim 4 (depends on 1)

The method of claim 1, wherein the dsODN is 30-35 nts long.

### Claim 5 (depends on 1)

The method of claim 1, wherein the dsODN is phosphorylated on the 5′ ends, and phosphorothioated on the 3′ ends.

### Claim 6 (depends on 1)

The method of claim 1, wherein the dsODN contains a randomized DNA barcode.

### Claim 7 (depends on 1)

The method claim 1, comprising:
shearing the gDNA into fragments; and
preparing the fragments for sequencing by end-repair, a-tailing, and ligation of a single-tailed sequencing adapter.

### Claim 8 (depends on 1)

The method of claim 1, wherein the dsODN is 15-50 nts long.

### Claim 9

A method for detecting double stranded breaks (DSBs) in gDNA of a cell, the method comprising:
contacting the cell with a blunt-ended double-stranded oligodeoxynucleotide (dsODN), wherein both strands of the dsODN are orthogonal to the genome of the cell, and further wherein (a) the 5′ ends of the dsODN are phosphorylated, and (b) phosphorothioate linkages are present on both 3′ ends, or phosphorothioate linkages are present on both 3′ ends and both 5′ ends;
maintaining the cell under conditions sufficient for the DSBs to occur in the genomic DNA of the cell, and for the cell to repair the DSBs, integrating a dsODN at one or more DSBs;
amplifying a portion of gDNA comprising an integrated dsODN; and
sequencing the amplified portion of the genomic DNA,
thereby detecting a DSB in the gDNA of the cell.

### Claim 10 (depends on 9)

The method of claim 9, wherein amplifying a portion of the gDNA comprises:
fragmenting the gDNA;
ligating ends of the fragmented gDNA from the cell with a universal adapter; and performing PCR on the ligated DNA.

### Claim 11 (depends on 9)

The method of claim 9, wherein the cell is a mammalian cell.

### Claim 12 (depends on 9)

The method of claim 9, wherein the dsODN is 30-35 nts long.

### Claim 13 (depends on 9)

The method of claim 9, wherein the dsODN is 15-50 nts long.

### Claim 14 (depends on 9)

The method of claim 9, wherein the dsODN is phosphorylated on the 5′ ends, and phosphorothioated on the 3′ ends.

### Claim 15 (depends on 9)

The method of claim 9, wherein the dsODN contains a randomized DNA barcode.

### Claim 16 (depends on 9)

The method claim 9, comprising:
shearing the gDNA into fragments; and
preparing the fragments for sequencing by end-repair, a-tailing, and ligation of a single-tailed sequencing adapter.