High-fidelity CRISPR-Cas9 variants with undetectable genome-wide off-targets

Kleinstiver, Benjamin P.; Pattanayak, Vikram; Prew, Michelle S.; Tsai, Shengdar Q.; Nguyen, Nhu; Zheng, Zongli; Joung, J. Keith

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High-fidelity CRISPR-Cas9 variants with undetectable genome-wide off-targets

Published Date:

Jan 06 2016

Source:

Nature. 529(7587):490-495.

[PDF-4.90 MB]

Details:

Alternative Title:

Nature

Personal Author:

Kleinstiver, Benjamin P. ; Pattanayak, Vikram ; Prew, Michelle S. ; Tsai, Shengdar Q. ; Nguyen, Nhu ; ... More ▼

Description:

CRISPR-Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. Existing strategies for reducing genome-wide off-target effects of the widely used Streptococcus pyogenes Cas9 (SpCas9) are imperfect, possessing only partial or unproven efficacies and other limitations that constrain their use. Here we describe SpCas9-HF1, a high-fidelity variant harbouring alterations designed to reduce non-specific DNA contacts. SpCas9-HF1 retains on-target activities comparable to wild-type SpCas9 with >85% of single-guide RNAs (sgRNAs) tested in human cells. Notably, with sgRNAs targeted to standard non-repetitive sequences, SpCas9-HF1 rendered all or nearly all off-target events undetectable by genome-wide break capture and targeted sequencing methods. Even for atypical, repetitive target sites, the vast majority of off-target mutations induced by wild-type SpCas9 were not detected with SpCas9-HF1. With its exceptional precision, SpCas9-HF1 provides an alternative to wild-type SpCas9 for research and therapeutic applications. More broadly, our results suggest a general strategy for optimizing genome-wide specificities of other CRISPR-RNA-guided nucleases.

Subject:

[+]

Pubmed ID:

26735016

Pubmed Central ID:

PMC4851738

Document Type:

Journal Article

Funding:

DP1 GM105378/DP/NCCDPHP CDC HHS/United States ; DP1 GM105378/GM/NIGMS NIH HHS/United States ; R01 GM088040/GM/NIGMS NIH HHS/United States ; R01 GM088040/GM/NIGMS NIH HHS/United States ; R01 GM107427/GM/NIGMS NIH HHS/United States ; ... More ▼

Collection(s):

CDC Public Access

Main Document Checksum:

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