title: Distinct patterns of Cas9 mismatch tolerance in vitro and in vivo.
abstract: Cas9, a CRISPR-associated RNA-guided nuclease, has been rapidly adopted as a tool for biochemical and genetic manipulation of DNA. Although complexes between Cas9 and guide RNAs (gRNAs) offer remarkable specificity and versatility for genome manipulation, mis-targeted events occur. To extend the understanding of gRNA::target homology requirements, we compared mutational tolerance for a set of Cas9::gRNA complexes in vitro and in vivo (in Saccharomyces cerevisiae). A variety of gRNAs were tested with variant libraries based on four different targets (with varying GC content and sequence features). In each case, we challenged a mixture of matched and mismatched targets, evaluating cleavage activity on a wide variety of potential target sequences in parallel through high-throughput sequencing of the products retained after cleavage. These experiments evidenced notable and consistent differences between in vitro and S. cerevisiae (in vivo) Cas9 cleavage specificity profiles including (i) a greater tolerance for mismatches in vitro and (ii) a greater specificity increase in vivo with truncation of the gRNA homology regions.
authors.affiliation: Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA xuhua@stanford.edu.
authors.forename: Becky X H
authors.initials: BX
authors.lastname: Fu
authors.affiliation: Stanford Genome Technology Center, Palo Alto, CA 94304, USA Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305, USA.
authors.forename: Robert P
authors.initials: RP
authors.lastname: St Onge
authors.affiliation: Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA xuhua@stanford.edu.
authors.forename: Andrew Z
authors.initials: AZ
authors.lastname: Fire
authors.affiliation: Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA Stanford Genome Technology Center, Palo Alto, CA 94304, USA jdsmith4@stanford.edu.
authors.forename: Justin D
authors.initials: JD
authors.lastname: Smith
mesh_terms: D020137:Base Pair Mismatch; D001483:Base Sequence; D004720:Endonucleases / Q000378:metabolism*; D014644:Genetic Variation; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics* / Q000378:metabolism*; D011995:Recombination, Genetic; D013379:Substrate Specificity
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems; D004720:Endonucleases