Rationally engineered Cas9 nucleases with improved specificity

Ian M Slaymaker; Linyi Gao; Bernd Zetsche; David A Scott; Winston X Yan; Feng Zhang

doi:10.1126/science.aad5227

Rationally engineered Cas9 nucleases with improved specificity

Science. 2016 Jan 1;351(6268):84-8. doi: 10.1126/science.aad5227. Epub 2015 Dec 1.

Authors

Ian M Slaymaker¹, Linyi Gao², Bernd Zetsche¹, David A Scott¹, Winston X Yan³, Feng Zhang⁴

Affiliations

¹ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
² Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
³ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. Graduate Program in Biophysics, Harvard Medical School, Boston, MA 02115, USA. Harvard-MIT Division of Health Sciences and Technology, Harvard Medical School, Boston, MA 02115, USA.
⁴ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. zhang@broadinstitute.org.

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.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Bacterial Proteins / chemistry*
Bacterial Proteins / genetics
DNA Cleavage*
Endonucleases / chemistry*
Endonucleases / genetics
Humans
Mutagenesis
Point Mutation
Protein Conformation
Protein Engineering*
RNA, Guide, CRISPR-Cas Systems
Streptococcus pyogenes / enzymology*

Substances

Bacterial Proteins
Endonucleases

Abstract

Publication types

MeSH terms

Substances

Grants and funding