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

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.

Claims23

Dependent claims

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.

3 more elements

Dependencies are inferred from the claim text.

Evaluation and improvement of nuclease cleavage specificity

Life of this patent

Evaluation and improvement of nuclease cleavage specificity

Life of this patent

Abstract

Claims23