title: DNA targeting specificity of RNA-guided Cas9 nucleases.
abstract: The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing. Here, we characterize SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. Our study evaluates >700 guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted genomic off-target loci in 293T and 293FT cells. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. To facilitate mammalian genome engineering applications, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses.
authors.affiliation: 1] Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. [2] McGovern Institute for Brain Research, Department of Brain and Cognitive Sciences, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. [3] Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA. [4].
authors.forename: Patrick D
authors.initials: PD
authors.lastname: Hsu
authors.forename: David A
authors.initials: DA
authors.lastname: Scott
authors.forename: Joshua A
authors.initials: JA
authors.lastname: Weinstein
authors.forename: F Ann
authors.initials: FA
authors.lastname: Ran
authors.forename: Silvana
authors.initials: S
authors.lastname: Konermann
authors.forename: Vineeta
authors.initials: V
authors.lastname: Agarwala
authors.forename: Yinqing
authors.initials: Y
authors.lastname: Li
authors.forename: Eli J
authors.initials: EJ
authors.lastname: Fine
authors.forename: Xuebing
authors.initials: X
authors.lastname: Wu
authors.forename: Ophir
authors.initials: O
authors.lastname: Shalem
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Cradick
authors.forename: Luciano A
authors.initials: LA
authors.lastname: Marraffini
authors.forename: Gang
authors.initials: G
authors.lastname: Bao
authors.forename: Feng
authors.initials: F
authors.lastname: Zhang
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D020137:Base Pair Mismatch; D001483:Base Sequence; D004247:DNA / Q000235:genetics*; D003851:Deoxyribonucleases / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D008969:Molecular Sequence Data; D013297:Streptococcus pyogenes / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics*; D058727:RNA, Small Untranslated
chemical_list: D001426:Bacterial Proteins; D014157:Transcription Factors; D004247:DNA; D003851:Deoxyribonucleases; D058727:RNA, Small Untranslated