# US 12,006,520 B2 · Evaluation and improvement of nuclease cleavage specificity

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- Number: US 12,006,520 B2
- Document ID: US12006520B2
- Status: Granted
- Kind: B2
- Application number: 16/441,751
- Filed: 2019-06-14
- Published: 2020-01-09
- Granted: 2024-06-11
- Earliest claimed: 2011-07-22
- Assignees: President and Fellows of Harvard College
- Applicants: President and Fellows of Harvard College
- Inventors: David R. Liu; John Paul Guilinger; Vikram Pattanayak
- Web page: https://patentgrep.com/patent/US12006520B2

## CPC

- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- A61P 31/18: for HIV
- A61P 35/00: Antineoplastic agents
- C12Q 1/44: involving esterase
- C12Q 1/68: involving nucleic acids
- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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

## Abstract

Engineered nucleases are promising tools for genome manipulation and determining off-target cleavage sites of these enzymes is of great interest. This disclosure provides in vitro selection methods that interrogate 1011 DNA sequences for their ability to be cleaved by active nucleases, e.g., ZFNs and TALENs. The method revealed hundreds of thousands of DNA sequences that can be cleaved in vitro by two ZFNs, CCR5-224 and VF2468, which target the endogenous human CCR5 and VEGF-A genes, respectively. Analysis of the identified sites in cultured human cells revealed CCR5-224-induced mutagenesis at nine off-target loci. This disclosure provides an energy compensation model of ZFN specificity in which excess binding energy contributes to off-target ZFN cleavage. It was also observed that TALENs can achieve cleavage specificity similar to or higher than that observed in ZFNs.

## Claims

### Claim 1

A library of synthetic nucleic acid molecules comprising a plurality of nucleic acid molecules,
wherein each of the synthetic nucleic acid molecules comprises multiple copies of a DNA sequence linked in series, wherein each of the multiple copies of the DNA sequence linked in series in each of the synthetic nucleic acid molecules is the same and has the following structure:
5′-[(candidate nuclease target site)-(constant insert sequence)]x-3′, wherein x is at least 5;
wherein the nucleotide sequence of the constant insert sequence in each of the synthetic nucleic acid molecules is the same; and
wherein the candidate nuclease target site of each of the synthetic nucleic acid molecules of the library comprises the following structure:
5′-[left-half site]-[spacer sequence]-[right-half site]-3′ (LSR) structure, and wherein the nucleotide sequence of the LSR structure of each of the synthetic nucleic acid molecules is the same.

### Claim 2 (depends on 1)

The library of claim 1, wherein the left-half site and the right-half site comprise different nucleic acid sequences.

### Claim 3 (depends on 1)

The library of claim 1, wherein the left-half site and/or the right-half site are/is 10-18 nucleotides long.

### Claim 4 (depends on 1)

The library of claim 1, wherein some of the synthetic nucleic acid molecules of the library comprise the candidate nuclease target site that can be cleaved by a nuclease comprising a FokI cleavage domain.

### Claim 5 (depends on 1)

The library of claim 1, wherein some of the synthetic nucleic acid molecules of the library comprise the candidate nuclease target site that can be cleaved by a Zinc Finger Nuclease (ZFN) or a Transcription Activator-Like Effector Nuclease (TALEN).

### Claim 6 (depends on 1)

The library of claim 1, wherein the library comprises at least 105 different synthetic nucleic acid molecules.

### Claim 7 (depends on 1)

The library of claim 1, wherein the library of synthetic nucleic acid molecules comprises nucleic acid molecules having a molecular weight of at least 5 kDa.

### Claim 8 (depends on 1)

The library of claim 1, wherein the left-half site is a partially randomized left-half site, the right-half site is a partially randomized right-half site, and/or the spacer sequence is a partially randomized spacer sequence.

### Claim 9 (depends on 8)

The library of claim 8, wherein the partially randomized left-half site and/or the partially randomized right-half site are/is based on a known target site of a nuclease of interest.

### Claim 10 (depends on 9)

The library of claim 9, wherein the nuclease of interest is a ZEN, a TALEN, or a derivative thereof.

### Claim 11 (depends on 8)

The library of claim 8, wherein the partially randomized left-half site and/or the partially randomized right-half site differ/differs from a consensus site by more than 5% on average.

### Claim 12 (depends on 8)

The library of claim 8, wherein the partially randomized left-half site and/or the partially randomized right-half site differ/differs from a consensus site by no more than 10% on average.

### Claim 13 (depends on 8)

The library of claim 8, wherein the partially randomized left-half site and/or the partially randomized right-half site differ/differs from a consensus site by no more than 15% on average.

### Claim 14 (depends on 1)

The library of claim 1, wherein the spacer sequence is a randomized spacer sequence.

### Claim 15 (depends on 1)

The library of claim 1, wherein the candidate nuclease target site is located within a genomic sequence.

### Claim 16 (depends on 15)

The library of claim 15, wherein the genomic sequence is known to be associated with a disease or disorder.

### Claim 17 (depends on 16)

The library of claim 16, wherein the disease or disorder is human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS), or a proliferative disease.

### Claim 18 (depends on 15)

The library of claim 15, wherein the genomic sequence is a C-C chemokine receptor type 5 (CCRS5) sequence or vascular endothelial growth factor type A (VEGFA) sequence.

### Claim 19 (depends on 1)

The library of claim 1, wherein each of the synthetic nucleic acid molecules of the library comprises at least ten copies of the DNA sequence linked in series.

### Claim 20 (depends on 1)

The library of claim 1, wherein each of the synthetic nucleic acid molecules of the library comprises at least 100 copies of the DNA sequence linked in series.

### Claim 21 (depends on 8)

The library of claim 8, wherein the partially randomized left-half site and/or the partially randomized right-half site differ/differs from a consensus site by one, two, three, four, or five nucleotide residue/residues.

### Claim 22 (depends on 8)

The library of claim 8, wherein the partially randomized left-half site and/or the partially randomized right-half site differ/differs from a consensus site by three nucleotide residues.

### Claim 23 (depends on 1)

The library of claim 1, wherein the synthetic nucleic acid molecules of the library comprise other candidate nuclease target sites that can be cleaved by an enediyne, a dynemicin, a neocarzinostatin, a calicheamicin, an esperamicin, or a bleomycin.