Details:

Alternative Title:
Nature
Personal Author:
Kleinstiver, Benjamin P. ; Pattanayak, Vikram ; Prew, Michelle S. ; Tsai, Shengdar Q. ; Nguyen, Nhu ; ... More +
Kleinstiver, Benjamin P. ; Pattanayak, Vikram ; Prew, Michelle S. ; Tsai, Shengdar Q. ; Nguyen, Nhu ; Zheng, Zongli ; Joung, J. Keith Less -
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:
[+]
Article
Base Sequence
Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR-Associated Proteins
CRISPR-Cas Systems
DNA
DNA-Binding Proteins
Endonucleases
Genetic Engineering
Genome, Human
Humans
Mutation
Protein Binding
Reproducibility Of Results
RNA
Sequence Analysis, DNA
Streptococcus Pyogenes
Substrate Specificity

Source:
Nature. 529(7587):490-495.
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 +
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 ; R01 GM107427/GM/NIGMS NIH HHS/United States Less -
Collection(s):
CDC Public Access
Main Document Checksum:
[+]
urn:sha256:231615061d3384519def297f2d29efe34331dd93bf1cfff2f93d1e60e6d5e766
File Type:

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