# US 11,898,203 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 11,898,203 B2
- Document ID: US11898203B2
- Status: Granted
- Kind: B2
- Application number: 16/386,472
- Filed: 2019-04-17
- Published: 2020-01-09
- Granted: 2024-02-13
- Earliest claimed: 2018-04-17
- Assignees: The General Hospital Corporation
- Applicants: The General Hospital Corporation
- Inventors: J. Keith Joung; Vikram Pattanayak; Karl Petri
- Web page: https://patentgrep.com/patent/US11898203B2

## 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

Provided herein are methods and compositions for performing highly sensitive in vitro assays to define substrate preferences and off-target sites of nucleic-acid binding, modifying, and cleaving agents.

## Claims

### Claim 1

An in vitro method of identifying double stranded DNA sequences that are modified by a cytidine deaminase base editing enzyme that converts deoxycytidine to deoxyuridine and generates a nick on the opposite strand, the method comprising:
(i) providing a library of defined linear dsDNA oligonucleotides of known sequences, each library member having a first strand comprising, from 5′ to 3′:
a first known common sequence common to each of the oligonucleotides in the library;
a first known barcode sequence unique to the library member;
one and only one copy of a known potential DNA substrate sequence for the cytidine deaminase base editing enzyme comprising a cognate protospacer adjacent motif (PAM);
a second known barcode sequence identical to the first barcode sequence; and
a second known common sequence common to each of the oligonucleotides in the library; and
a second strand complementary to the known sequences of the first strand;
(ii) incubating the library in the presence of the cytidine deaminase base editing enzyme under conditions sufficient for conversion of deoxycytidine to deoxyuridine to occur on one strand and nick generation to occur on the opposite strand of one or more of the linear dsDNA and then incubating the modified linear dsDNA oligonucleotide(s) in the presence of a uracil-DNA glycosylase and Endonuclease VIII to generate a single-strand break at sites with uridine nucleotides, creating a staggered double-strand break in the modified linear dsDNA oligonucleotide(s), and thereby creating first and second dsDNA oligonucleotide fragments with 5′ phosphorylated overhangs;
(iii) incubating the dsDNA oligonucleotide fragments with a DNA polymerase that creates 5′ phosphorylated blunt ends from the 5′ phosphorylated overhangs;
(iv) ligating the 5′ phosphorylated blunt ends with double stranded DNA adapters comprising primer sequences;
(v) amplifying the first dsDNA oligonucleotide fragments using one primer specific to the primer sequence of the DNA adapter and one primer specific to the first common sequence and amplifying the second dsDNA oligonucleotide fragments using one primer specific to the primer sequence of the DNA adapter and one primer specific to the second common sequence;
and
(vi) determining the sequence of a barcode sequence of one or more of the amplified dsDNA oligonucleotide fragments thereby identifying double stranded DNA sequences that are modified by the base editing enzyme.

### Claim 2 (depends on 1)

The method of claim 1, wherein the linear dsDNA oligonucleotides comprise potential DNA substrate sequences comprising:
(i) a set of all potential off-target sequences for the cytidine deaminase base editing enzyme in a reference genome bearing up to a certain number of substitutions, single base pair deletions, and/or single base pair insertions relative to an identified on-target site for the cytidine deaminase base editing enzyme;
(ii) a comprehensive set of all potential off-target sequences for the cytidine deaminase base editing enzyme bearing up to a certain number of substitutions, single base pair deletions, and/or single base pair insertions relative to an identified on-target site for the cytidine deaminase base editing enzyme;
(iii) a set of potential off-target sequences bearing up to a certain number of substitutions, single base pair deletions, and/or single base pair insertions relative to an identified on-target site for the cytidine deaminase base editing enzyme;
(iv) a set of all potential off-target sequences for the cytidine deaminase base editing enzyme in the coding sequence of a human reference genome bearing up to a certain number of substitutions, single base pair deletions, and/or single base pair insertions relative to an identified on-target site for the cytidine deaminase base editing enzyme; or
(v) a set of all potential off-target sequences for the cytidine deaminase base editing enzyme in the sequence of an oncogene hotspot and/or tumor suppressor gene of a reference genome bearing up to a certain number of substitutions, single base pair deletions, and/or single base pair insertions relative to an identified on-target site for the cytidine deaminase base editing enzyme.

### Claim 3 (depends on 1)

The method of claim 1, wherein the linear dsDNA oligonucleotides are first synthesized as individual single-stranded DNA sequences and wherein the single-stranded DNA sequences are converted into double-stranded DNA molecules.

### Claim 4 (depends on 1)

The method of claim 1, wherein the linear dsDNA oligonucleotides comprise 1,000 to 1011 different sequences.

### Claim 5 (depends on 1)

The method of claim 1, wherein the linear dsDNA oligonucleotides comprise sequences that are 50 to 500 bp long.

### Claim 6 (depends on 1)

The method of claim 1, wherein the DNA polymerase is T4 DNA polymerase.

### Claim 7 (depends on 1)

The method of claim 1, wherein the cytidine deaminase base editing enzyme that converts deoxycytidine to deoxyuridine and generates a nick on the opposite strand is BE3.

### Claim 8

An in vitro method of identifying double stranded DNA sequences that are modified by an adenine base editing enzyme that converts deoxyadenine to deoxyinosine and generates a nick on the opposite strand, the method comprising:
(i) providing a library of defined linear dsDNA oligonucleotides of known sequences, each library member having a first strand comprising, from 5′ to 3′:
a first known common sequence common to each of the oligonucleotides in the library;
a first known barcode sequence unique to the library member;
one and only one copy of a known potential DNA substrate sequence for the adenine base editing enzyme comprising a cognate protospacer adjacent motif (PAM);
a second known barcode sequence identical to the first barcode sequence; and
a second known common sequence common to each of the oligonucleotides in the library; and
a second strand complementary to the known sequences of the first strand;
(ii) incubating the library of linear dsDNA oligonucleotides in the presence of the adenine base editing enzyme under conditions sufficient for conversion of deoxyadenine to deoxyinosine to occur on one strand and nick generation to occur on the opposite strand of one or more of the linear dsDNA oligonucleotides and then incubating the modified linear dsDNA oligonucleotide(s) in the presence of endonuclease V to generate a single-strand break at sites with inosine nucleotides, creating a staggered double-strand break in the modified linear dsDNA oligonucleotide(s), and thereby creating first and second dsDNA oligonucleotide fragments with 5′ phosphorylated overhangs;
(iii) incubating the dsDNA oligonucleotide fragments with a DNA polymerase that creates 5′ phosphorylated blunt ends from the 5′ phosphorylated overhangs;
(iv) ligating the 5′ phosphorylated blunt ends with double stranded DNA adapters comprising primer sequences;
(v) amplifying the first dsDNA oligonucleotide fragments using one primer specific to the primer sequence of the DNA adapter and one primer specific to the first common sequence and amplifying the second dsDNA oligonucleotide fragments using one primer specific to the primer sequence of the DNA adapter and one primer specific to the second common sequence;
and
(vi) determining the sequence of a barcode sequence of one or more of the amplified dsDNA oligonucleotide fragments thereby identifying double stranded DNA sequences that are modified by the base editing enzyme.

### Claim 9 (depends on 8)

The method of claim 8, wherein the linear dsDNA oligonucleotides comprise potential DNA substrate sequences comprising:
(i) a set of all potential off-target sequences for the adenine base editing enzyme in a reference genome bearing up to a certain number of substitutions, single base pair deletions, and/or single base pair insertions relative to an identified on-target site for the adenine base editing enzyme;
(ii) a comprehensive set of all potential off-target sequences for the adenine base editing enzyme bearing up to a certain number of substitutions, single base pair deletions, and/or single base pair insertions relative to an identified on-target site for the adenine base editing enzyme;
(iii) a set of potential off-target sequences bearing up to a certain number of substitutions, single base pair deletions, and/or single base pair insertions relative to an identified on-target site for the adenine base editing enzyme;
(iv) a set of all potential off-target sequences for the adenine base editing enzyme in the coding sequence of a human reference genome bearing up to a certain number of substitutions, single base pair deletions, and/or single base pair insertions relative to an identified on-target site for the adenine base editing enzyme; or
(v) a set of all potential off-target sequences for the adenine base editing enzyme in the sequence of an oncogene hotspot and/or tumor suppressor gene of a reference genome bearing up to a certain number of substitutions, single base pair deletions, and/or single base pair insertions relative to an identified on-target site for the adenine base editing enzyme.

### Claim 10 (depends on 8)

The method of claim 8, wherein the linear dsDNA oligonucleotides are first synthesized as individual single-stranded DNA sequences and wherein the single-stranded DNA sequences are converted into double-stranded DNA molecules.

### Claim 11 (depends on 8)

The method of claim 8, wherein the library members comprise 1,000 to 1011 different sequences.

### Claim 12 (depends on 8)

The method of claim 8, wherein the library members comprise sequences that are 50 to 500 bp long.

### Claim 13 (depends on 8)

The method of claim 8, wherein the DNA polymerase is T4 DNA polymerase.

### Claim 14 (depends on 8)

The method of claim 8, wherein the adenine base editing enzyme that converts deoxyadenine to deoxyinosine and generates a nick on the opposite strand is ABE.