Rationally engineered Cas9 nucleases with improved specificity.

Science
Authors
Keywords
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.

Year of Publication
2016
Journal
Science
Volume
351
Issue
6268
Pages
84-8
Date Published
2016 Jan 01
ISSN
1095-9203
URL
DOI
10.1126/science.aad5227
PubMed ID
26628643
PubMed Central ID
PMC4714946
Links
Grant list
R01 MH110049 / MH / NIMH NIH HHS / United States
5DP1-MH100706 / DP / NCCDPHP CDC HHS / United States
DP1 MH100706 / MH / NIMH NIH HHS / United States
1R01MH110049 / MH / NIMH NIH HHS / United States
R01 DK097768 / DK / NIDDK NIH HHS / United States
T32 GM008313 / GM / NIGMS NIH HHS / United States
T32GM007753 / GM / NIGMS NIH HHS / United States
5R01DK097768-03 / DK / NIDDK NIH HHS / United States