Comprehensive in vitro reporting of cleavage events by sequencing (CIRCLE-seq)

• US 10,738,303 B2
• Filed: 12/22/2017
• Issued: 08/11/2020
• Est. Priority Date: 09/30/2015
• 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): Zhang, Kaijiang
• Application Number:
  US15/853,275
• Publication Number:
  US 20180245071A1
• Time in Patent Office: 963 Days
• Field of Search: None
• CPC Class Codes: C12N 15/1031 mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing

Abstract

Sensitive, unbiased methods for genome-wide detection of potential CRISPR-Cas9 off-target cleavage sites from cell type-specific genomic DNA samples.

Claims

1. A method of preparing a library of covalently closed circular double-stranded DNA (dsDNA) fragments from a subject'"'"'s genomic DNA, the method comprising:

providing a sample comprising the subject'"'"'s genomic DNA (gDNA);

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

preparing the fragments for end-ligation;

ligating to the ends of the fragments a stem-loop adapter comprising a single deoxyuridine adjacent to or within a single-stranded loop sequence comprising a palindromic sequence for intramolecular ligation, to prepare a population of ligated linear dsDNA fragments;

contacting the population of ligated linear dsDNA fragments with an exonuclease to degrade any remaining linear fragments with unligated ends, to produce a purified population of ligated linear dsDNA fragments,

contacting the purified population of ligated linear dsDNA fragments with enzymes that nick the ligated dsDNA fragments at the deoxyuridine and remove a 3′

terminal phosphate;

incubating the nicked linear dsDNA fragments under conditions sufficient to promote intramolecular ligation and formation of circular dsDNA fragments;

purifying the ligated circular dsDNA fragments using an exonuclease to degrade unligated noncircular fragments, thereby preparing a library of covalently closed fully circular dsDNA fragments from the subject'"'"'s gDNA.

2. The method of claim 1, further comprising:

contacting the library of covalently closed fully circular dsDNA fragments with an engineered nuclease to induce site-specific cleavage;

preparing the cleaved fragments for end-ligation;

ligating a sequencing adapter comprising at least a single deoxyuridine and a primer site compatible for use in PCR priming or sequencing, at the cleavage site;

contacting the library with enzymes that nick at the deoxyuridine; and

sequencing resulting fragments using primers that bind to the sequencing adapter.

3. The method of claim 2, wherein the engineered nuclease cleaves at one or both of on- and off-target sites.

4. The method of claim 2, wherein the engineered nuclease induces blunt or staggered/overhanging ends.

5. The method of claim 2, wherein the engineered nuclease is selected from the group consisting of meganucleases, MegaTALs, zinc-finger nucleases, transcription activator effector-like nucleases (TALEN), Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas RNA-guided nucleases (CRISPR/Cas RGNs), and Fokl-dCas9 fusion proteins.

6. The method of claim 5, wherein the CRISPR/Cas RGN is Cas9 or Cpf1.

7. The method of claim 2, wherein treating the sample with an engineered nuclease to induce site-specific cleavage comprises contacting the sample with a Cas9 nuclease complexed with a specific guide RNA (gRNA).

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

9. The method of claim 2, wherein the primer site comprises a next generation sequencing primer binding sequence, a randomized DNA barcode or unique molecular identifier (UMI).

10. The method of claim 2, wherein the sequencing adapter comprises:

a first region;

a second region that forms one or more hairpin loops and comprises a primer site compatible for use in PCR priming and/or sequencing;

a third region that is complementary to the first region with one additional nucleotide; and

wherein the single deoxyuridine is between the second and third regions.

11. The method of claim 10, further comprising:

contacting the library with enzymes to nick at the deoxyuridine in the sequencing adapter;

using PCR amplification to enrich for adapter-ligated fragments and to add a full sequencing adapter, and

sequencing those fragments bearing a sequencing adapter.

12. The method of claim 11, wherein the enzymes to nick at the deoxyuridine comprise uracil DNA glycosylase (UDG) and/or endonuclease VIII.

13. The method of claim 2, further comprising comparing the sequences of the resulting fragments to sequence of an intended target site of the engineered nuclease.

14. The method of claim 13, further comprising identifying fragments with mismatches as compared to the intended target site as comprising off-target sites.

15. The method of claim 1, wherein randomly shearing the gDNA comprises randomly shearing the gDNA to an average length of 200-500 bps.

16. The method of claim 1, wherein the exonuclease used to degrade any remaining linear fragments with unligated ends is a cocktail of nucleases comprising one or more of bacteriophage Lambda exonuclease, E. coli Exonuclease I, and an ATP-dependent exonuclease.

17. The method of claim 1, wherein the method comprises one or both of end-repairing and A-tailing the sheared DNA.

18. The method of claim 1, wherein the enzyme that nicks at the deoxyuridine comprises one or both of uracil DNA glycosylase (UDG) and endonuclease VIII, and the enzyme that removes the terminal 3′

phosphate comprises T4 Polynucleotide Kinase.

19. A method of preparing a library of fragments comprising sites of engineered nuclease-induced double stranded breaks in double-stranded DNA (dsDNA) molecules from a subject'"'"'s genomic DNA (gDNA), the method comprising:

providing dsDNA, wherein the dsDNA comprises a subject'"'"'s gDNA;

randomly shearing the dsDNA to a defined average length;

end-repairing and/or A-tailing the sheared dsDNA;

ligating a stem-loop adapter comprising;

a first region;

a second region that forms one or more loops and comprises a single deoxyuridine nucleotide adjacent to a palindromic sequence for intramolecular ligation; and

a third region that is complementary to the first region with one additional nucleotide;

contacting the library enzymes that nick the dsDNA at the deoxyuridine and remove a terminal 3′

phosphate

incubating the nicked dsDNA under conditions sufficient to promote intramolecular ligation and formation of a sample comprising circular dsDNA molecules;

contacting the sample with one or more exonucleases sufficient to degrade any DNA molecules that are not circular;

treating the sample with an engineered nuclease to induce site-specific cleavage;

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

ligating a sequencing adapter comprising;

a first region;

a second region that forms one or more hairpin loops and comprises a primer site compatible for use in PCR priming and/or sequencing;

a third region that is complementary to the first region with one additional nucleotide; and

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 sequencing adapter ligated to each end;

thereby preparing a library enriched for nuclease-cleaved adapter-ligated fragments.

20. The method of claim 19, wherein the method comprises, prior to ligating a stem-loop adapter or prior to ligating a sequencing adapter, end-repairing and then A-tailing the resulting ends.