# US 12,601,009 B2 · Highly sensitive in vitro assays to define substrate preferences and sites of nucleic-acid binding, modifying, and cleaving agents

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- Number: US 12,601,009 B2
- Document ID: US12601009B2
- Status: Granted
- Kind: B2
- Application number: 18/502,817
- Filed: 2023-11-06
- Published: 2024-09-19
- Granted: 2026-04-14
- Earliest claimed: 2018-04-17
- Assignees: The General Hospital Corporation
- Applicants: The General Hospital Corporation
- Inventors: J. Keith Joung; Vikram Pattanayak; Karl Petri; Jason Michael Gehrke; Kanae Esther Sasaki
- Web page: https://patentgrep.com/patent/US12601009B2

## CPC

- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 15/1058: {Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms}
- C12Q 1/6827: for detection of mutation or polymorphism
- C12Q 1/6853: using modified primers or templates
- C12Y 305/04005: Cytidine deaminase (3.5.4.5)
- 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)
- C12Y: ENZYMES

## 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

### Claim 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.

### Claim 2 (depends on 1)

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.

### Claim 3 (depends on 1)

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.

### Claim 4 (depends on 1)

The method of claim 1, wherein the library members comprise 1,000 to 1011 different sequences.

### Claim 5 (depends on 1)

The method of claim 1, wherein the library members comprise sequences that are 50 to 500 bp long.

### Claim 6 (depends on 1)

The method of claim 1, wherein the nuclease is a Cas9 nuclease.