Rationally engineered Cas9 nucleases with improved specificity.
Science
; 351(6268): 84-8, 2016 Jan 01.
Article
in En
| MEDLINE
| ID: mdl-26628643
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.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Streptococcus pyogenes
/
Bacterial Proteins
/
Protein Engineering
/
Endonucleases
/
DNA Cleavage
Limits:
Humans
Language:
En
Journal:
Science
Year:
2016
Document type:
Article