Rationally engineered Cas9 nucleases with improved specificity.
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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
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Journal | Science
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Volume | 351
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Issue | 6268
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Pages | 84-8
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Date Published | 2016 Jan 01
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ISSN | 1095-9203
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URL | |
DOI | 10.1126/science.aad5227
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PubMed ID | 26628643
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PubMed Central ID | PMC4714946
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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
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