title: Rationally engineered Cas9 nucleases with improved specificity.
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.
authors.affiliation: 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.
authors.forename: Ian M
authors.initials: IM
authors.lastname: Slaymaker
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Linyi
authors.initials: L
authors.lastname: Gao
authors.affiliation: 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.
authors.forename: Bernd
authors.initials: B
authors.lastname: Zetsche
authors.affiliation: 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.
authors.forename: David A
authors.initials: DA
authors.lastname: Scott
authors.affiliation: 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.
authors.forename: Winston X
authors.initials: WX
authors.lastname: Yan
authors.affiliation: 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.
authors.forename: Feng
authors.initials: F
authors.lastname: Zhang
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D053837:DNA Cleavage; D004720:Endonucleases / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D016296:Mutagenesis; D017354:Point Mutation; D011487:Protein Conformation; D015202:Protein Engineering; D013297:Streptococcus pyogenes / Q000201:enzymology*; D000094704:RNA, Guide, CRISPR-Cas Systems
chemical_list: D001426:Bacterial Proteins; D004720:Endonucleases; D000094704:RNA, Guide, CRISPR-Cas Systems