Granted Patent
B2
US 12,601,009 · App. 18/502,817 · Granted Apr 14, 2026
Highly sensitive in vitro assays to define substrate preferences and sites of nucleic-acid binding, modifying, and cleaving agents
Inventors:
J. Keith Joung (Winchester, MA); Vikram Pattanayak (Wellesley, MA); Karl Petri (Cambridge, MA); Jason Michael Gehrke (Cambridge, MA); Kanae Esther Sasaki (Somerville, MA)
Assignee:
The General Hospital Corporation
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Abstract
Described herein are, among other things, in vitro methods of identifying double stranded DNA sequences that are cleaved by a nuclease. For example, by providing a library of defined linear dsDNA oligonucleotides of known sequences, incubating the library in the presence of a nuclease, ligating DNA adapters, amplifying cleaved oligonucleotides, and determining the sequence(s) of the amplified fragments.
Claims (21)
1 . An in vitro method of identifying double stranded DNA sequences that are cleaved by a nuclease, the method comprising:
(i) providing a library of defined linear dsDNA oligonucleotides of known sequences, each library member having a known sequence comprising, in order:
a first sequence common to each of the oligonucleotides in the library;
a first barcode sequence unique to the library member;
a potential DNA substrate sequence for the nuclease;
a second copy of the barcode sequence; and
a second sequence common to each of the oligonucleotides in the library;
(ii) incubating the library in the presence of a site-specific nuclease under conditions sufficient for cleavage of one or more of the linear dsDNA oligonucleotides to occur;
(iii) ligating DNA adapters comprising primer sequences to the cleaved ends of the dsDNA oligonucleotides;
(iv) amplifying at least one of the cleaved dsDNA oligonucleotides using a primer specific to a primer sequence of the DNA adapter and a primer specific to at least one of the common sequences to generate amplified fragments; and
(v) determining the barcode sequence(s) of the amplified fragments, thereby identifying double stranded DNA sequences that are cleaved by a nuclease.
2 . The method of claim 1 , wherein the linear dsDNA oligonucleotides comprise:
(i) a set of all potential off-target sequences in a reference genome bearing mismatches relative to an identified on-target site;
(ii) a comprehensive set of potential off-target sites bearing mismatches;
(iii) a library of potential off-target sequences present in a set of variant genomes from defined populations; or
(iv) another relevant defined set of potential off-target sites.
3 . The method of claim 1 , wherein:
the library members are first synthesized as individual single-stranded DNA sequences; and the single-stranded DNA sequences are converted into double-stranded DNA molecules by priming against the first and/or second common sequence.
4 . The method of claim 1 , wherein the library members comprise 1,000 to 10 11 different sequences.
5 . The method of claim 1 , wherein the library members comprise sequences that are 50 to 500 bp long.
6 . The method of claim 1 , wherein the nuclease is a Cas9 nuclease.
Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Jan 2, 2024
From: JOUNG, J. KEITH; PATTANAYAK, VIKRAM; PETRI, KARL; SASAKI, KANAE ESTHER; GEHRKE, JASON MICHAEL
To: THE GENERAL HOSPITAL CORPORATION
Reel/Frame 065994/0967 →
Continuity (5)
Provisional Application
62767633
· Nov 15, 2018
Provisional Application
62659073
· Apr 17, 2018
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