title: High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells.
abstract: Clustered, regularly interspaced, short palindromic repeat (CRISPR) RNA-guided nucleases (RGNs) have rapidly emerged as a facile and efficient platform for genome editing. Here, we use a human cell-based reporter assay to characterize off-target cleavage of CRISPR-associated (Cas)9-based RGNs. We find that single and double mismatches are tolerated to varying degrees depending on their position along the guide RNA (gRNA)-DNA interface. We also readily detected off-target alterations induced by four out of six RGNs targeted to endogenous loci in human cells by examination of partially mismatched sites. The off-target sites we identified harbored up to five mismatches and many were mutagenized with frequencies comparable to (or higher than) those observed at the intended on-target site. Our work demonstrates that RGNs can be highly active even with imperfectly matched RNA-DNA interfaces in human cells, a finding that might confound their use in research and therapeutic applications.
authors.affiliation: 1] Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, Massachusetts, USA. [2] Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts, USA. [3] Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA. [4] Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Yanfang
authors.initials: Y
authors.lastname: Fu
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Foden
authors.forename: Cyd
authors.initials: C
authors.lastname: Khayter
authors.forename: Morgan L
authors.initials: ML
authors.lastname: Maeder
authors.forename: Deepak
authors.initials: D
authors.lastname: Reyon
authors.forename: J Keith
authors.initials: JK
authors.lastname: Joung
authors.forename: Jeffry D
authors.initials: JD
authors.lastname: Sander
mesh_terms: D001483:Base Sequence; D064130:CRISPR-Associated Proteins / Q000235:genetics*; D004720:Endonucleases / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D020014:K562 Cells; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics*
chemical_list: D064130:CRISPR-Associated Proteins; D004720:Endonucleases