Rationally engineered Cas9 nucleases with improved specificity
Top Cited Papers
Open Access
- 1 January 2016
- journal article
- other
- Published by American Association for the Advancement of Science (AAAS) in Science
- Vol. 351 (6268), 84-88
- https://doi.org/10.1126/science.aad5227
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.Keywords
Funding Information
- National Institute of General Medical Sciences (T32GM007753)
- Daisy Soros Fellowship
- National Institutes of Health
- NIMH (5DP1-MH100706, 1R01MH110049)
- NIDDK (5R01DK097768-03)
- National Science Foundation
- Keck
- New York Stem Cell
- Damon Runyon
- Searle Scholars
- Merkin
- Vallee Foundations
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