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High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects.

作者信息

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

机构信息

Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA.

Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA.

出版信息

Nature. 2016 Jan 28;529(7587):490-5. doi: 10.1038/nature16526. Epub 2016 Jan 6.


DOI:10.1038/nature16526
PMID:26735016
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC4851738/
Abstract

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.

摘要
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/01c1/4851738/649b077bf1f0/nihms744047f5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/01c1/4851738/b05ae093fd2a/nihms744047f6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/01c1/4851738/c17b53e806ac/nihms744047f7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/01c1/4851738/21bc78dc4070/nihms744047f8.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/01c1/4851738/6efe9713633c/nihms744047f9.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/01c1/4851738/0edc2744fc52/nihms744047f10.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/01c1/4851738/e1062fd4c514/nihms744047f11.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/01c1/4851738/53273d0e6c72/nihms744047f12.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/01c1/4851738/3de789ad984f/nihms744047f13.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/01c1/4851738/58dfab082145/nihms744047f14.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/01c1/4851738/558a058736f2/nihms744047f1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/01c1/4851738/f1a11e01b599/nihms744047f2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/01c1/4851738/6f592cae72bc/nihms744047f3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/01c1/4851738/73547aa6409f/nihms744047f4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/01c1/4851738/649b077bf1f0/nihms744047f5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/01c1/4851738/b05ae093fd2a/nihms744047f6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/01c1/4851738/c17b53e806ac/nihms744047f7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/01c1/4851738/21bc78dc4070/nihms744047f8.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/01c1/4851738/6efe9713633c/nihms744047f9.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/01c1/4851738/0edc2744fc52/nihms744047f10.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/01c1/4851738/e1062fd4c514/nihms744047f11.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/01c1/4851738/53273d0e6c72/nihms744047f12.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/01c1/4851738/3de789ad984f/nihms744047f13.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/01c1/4851738/58dfab082145/nihms744047f14.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/01c1/4851738/558a058736f2/nihms744047f1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/01c1/4851738/f1a11e01b599/nihms744047f2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/01c1/4851738/6f592cae72bc/nihms744047f3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/01c1/4851738/73547aa6409f/nihms744047f4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/01c1/4851738/649b077bf1f0/nihms744047f5.jpg

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[7]
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本文引用的文献

[1]
Rationally engineered Cas9 nucleases with improved specificity.

Science. 2016-1-1

[2]
Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems.

Mol Cell. 2015-11-5

[3]
Dynamics of CRISPR-Cas9 genome interrogation in living cells.

Science. 2015-11-13

[4]
Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition.

Nat Biotechnol. 2015-12

[5]
Conformational control of DNA target cleavage by CRISPR-Cas9.

Nature. 2015-11-5

[6]
Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system.

Cell. 2015-10-22

[7]
STRUCTURAL BIOLOGY. A Cas9-guide RNA complex preorganized for target DNA recognition.

Science. 2015-6-26

[8]
Engineered CRISPR-Cas9 nucleases with altered PAM specificities.

Nature. 2015-7-23

[9]
Dimeric CRISPR RNA-Guided FokI-dCas9 Nucleases Directed by Truncated gRNAs for Highly Specific Genome Editing.

Hum Gene Ther. 2015-7

[10]
In vivo genome editing using Staphylococcus aureus Cas9.

Nature. 2015-4-9

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