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

doi:10.1038/nature16526

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

Jan 06 2016
By Kleinstiver, Benjamin P. ; Pattanayak, Vikram ; Prew, Michelle S. ; ...
http://dx.doi.org/10.1038/nature16526
Source: Nature. 529(7587):490-495.

[PDF-4.90 MB]

English

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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.
Subjects:

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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 GM107427/GM/NIGMS NIH HHS/United States

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 GM107427/GM/NIGMS NIH HHS/United States Less -
Volume:

529
Issue:

7587
Collection(s):

CDC Public Access
Main Document Checksum:

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urn:sha256:231615061d3384519def297f2d29efe34331dd93bf1cfff2f93d1e60e6d5e766
Download URL:

https://stacks.cdc.gov/view/cdc/39163/cdc_39163_DS1.pdf
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