Rationally engineered Cas9 nucleases with improved specificity
Name
Zhang_Rationally engineered.pdf
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Author(s)
Slaymaker, Ian
Gao, Linyi
Zetsche, Bernd
Scott, David Arthur
Yan, Winston Xia
Zhang, Feng
Date Issued
December 2015
Journal
Science
Publisher
American Association for the Advancement of Science (AAAS)
Citation
Slaymaker, I. M., L. Gao, B. Zetsche, D. A. Scott, W. X. Yan, and F. Zhang. “Rationally Engineered Cas9 Nucleases with Improved Specificity.” Science 351, no. 6268 (January 1, 2016): 84–88.
Version
Author's final manuscript
Abstract
The RNA-guided endonuclease Cas9 is a versatile genome-editing tool with a broad range of applications from therapeutics to functional annotation of genes. Cas9 creates double-strand breaks (DSBs) at targeted genomic loci complementary to a short RNA guide. However, Cas9 can cleave off-target sites that are not fully complementary to the guide, which poses a major challenge for genome editing. Here, we use structure-guided protein engineering to improve the specificity of Streptococcus pyogenes Cas9 (SpCas9). Using targeted deep sequencing and unbiased whole-genome off-target analysis to assess Cas9-mediated DNA cleavage in human cells, we demonstrate that “enhanced specificity” SpCas9 (eSpCas9) variants reduce off-target effects and maintain robust on-target cleavage. Thus, eSpCas9 could be broadly useful for genome-editing applications requiring a high level of specificity.
MIT Department
Harvard University--MIT Division of Health Sciences and Technology
Massachusetts Institute of Technology. Department of Biological Engineering
Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences
McGovern Institute for Brain Research at MIT
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Creative Commons Attribution-Noncommercial-Share Alike
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DOI of Published Version
https://doi.org/10.1126/science.aad5227
