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Rationally engineered Cas9 nucleases with improved specificity

Published on Jan 1, 2016in Science41.063
· DOI :10.1126/science.aad5227
Ian Slaymaker11
Estimated H-index: 11
,
Linyi Gao8
Estimated H-index: 8
(MIT: Massachusetts Institute of Technology)
+ 3 AuthorsFeng Zhang118
Estimated H-index: 118
Sources
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.
  • References (21)
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The physical size of the commonly used Cas9 from Streptococcus pyogenes poses challenges for CRISPR-Cas genome editing systems that use the adeno-associated virus as a delivery vehicle; here, smaller Cas9 orthologues are characterized, and Cas9 from Staphylococcus aureus allowed targeting of the cholesterol regulatory gene Pcsk9 in the mouse liver.
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