Genomewide unbiased identification of DSBs evaluated by sequencing (GUIDE-Seq)

• US 9,822,407 B2
• Filed: 06/24/2016
• Issued: 11/21/2017
• Est. Priority Date: 06/23/2014
• Status: Active Grant

• Current Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Original Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Inventors: Joung, J. Keith, Tsai, Shengdar
• Primary Examiner(s): DUNSTON, JENNIFER ANN
• Application Number:
  US15/192,753
• Publication Number:
  US 20160304950A1
• Time in Patent Office: 515 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2525/113 incorporating modified backbone
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2525/204 specific length of the oligonucleotides
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing

Abstract

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

Claims

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;

expressing or activating an exogenous engineered nuclease in the cell, for a time sufficient for the nuclease to induce DSBs 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 genomic DNA comprising an integrated dsODN; and

sequencing the amplified portion of the genomic DNA,

thereby detecting a DSB in the genomic DNA of the cell.

2. The method of claim 1, wherein amplifying a portion of the genomic DNA comprises:

fragmenting the DNA;

ligating ends of the fragmented genomic DNA from the cell with a universal adapter; and

performing polymerase chain reaction (PCR) on the ligated DNA.

3. The method of claim 1, wherein the engineered nuclease is selected from the group consisting of meganucleases, zinc-finger nucleases, transcription activator effector-like nucleases (TALEN), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas RNA-guided nucleases (CRISPR/Cas RGNs).

4. The method of claim 1, wherein the DSBs are off-target DSBs.

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

6. The method of claim 1, wherein the engineered 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.

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

8. The method of claim 1, wherein the dsODN is phosphorylated on the 5′

ends, and phosphorothioated on the 3′

ends.

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

10. 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.

11. The method of claim 1, wherein the dsODN is between 15 and 50 nts long.

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

contacting a first population of cells with a first guide RNA and 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;

expressing or activating an exogenous Cas9 engineered nuclease in the first population of cells, for a time sufficient for the nuclease to induce DSBs in the genomic DNA of the cells, and for the cells to repair the DSBs, integrating a dsODN at one or more DSBs;

amplifying a portion of genomic DNA from the first population of cells comprising an integrated dsODN;

sequencing the amplified portion of the genomic DNA from the first population of cells;

determining a number of sites at which the dsODN integrated into the genomic DNA of the first population of cells;

contacting a second population of cells with a second guide RNA and 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;

expressing or activating an exogenous Cas9 engineered nuclease in the second population of cells, for a time sufficient for the nuclease to induce DSBs in the genomic DNA of the second population of cells, and for the cells to repair the DSBs, integrating a dsODN at one or more DSBs;

amplifying a portion of genomic DNA comprising an integrated dsODN from the second population of cells;

sequencing the amplified portion of the genomic DNA from the second population of cells;

determining a number of sites at which the dsODN integrated into the genomic DNA of the second population of cells; and

comparing the number of sites at which the dsODN integrated into the genomic DNA of the first population of cells to the number of sites at which the dsODN integrated into the genomic DNA of the second population of cells to determine if the first or second guide RNA is more specific.