title: PAM multiplicity marks genomic target sites as inhibitory to CRISPR-Cas9 editing.
abstract: In CRISPR-Cas9 genome editing, the underlying principles for selecting guide RNA (gRNA) sequences that would ensure for efficient target site modification remain poorly understood. Here we show that target sites harbouring multiple protospacer adjacent motifs (PAMs) are refractory to Cas9-mediated repair in situ. Thus we refine which substrates should be avoided in gRNA design, implicating PAM density as a novel sequence-specific feature that inhibits in vivo Cas9-driven DNA modification.
authors.affiliation: Department of Biochemistry, McGill University, Montreal, Quebec H3G 1Y6, Canada.
authors.forename: Abba
authors.initials: A
authors.lastname: Malina
authors.affiliation: McGill Centre for Bioinformatics and School of Computer Science, McGill University, Montreal, Quebec H3G 0B1, Canada.
authors.forename: Christopher J F
authors.initials: CJF
authors.lastname: Cameron
authors.affiliation: Department of Biochemistry, McGill University, Montreal, Quebec H3G 1Y6, Canada.
authors.forename: Francis
authors.initials: F
authors.lastname: Robert
authors.affiliation: McGill Centre for Bioinformatics and School of Computer Science, McGill University, Montreal, Quebec H3G 0B1, Canada.
authors.forename: Mathieu
authors.initials: M
authors.lastname: Blanchette
authors.affiliation: Department of Biochemistry, McGill University, Montreal, Quebec H3G 1Y6, Canada.|The Rosalind and Morris Goodman Cancer Research Centre, McGill University, Montreal, Quebec H3G 1Y6, Canada.
authors.forename: Josée
authors.initials: J
authors.lastname: Dostie
authors.affiliation: Department of Biochemistry, McGill University, Montreal, Quebec H3G 1Y6, Canada.|The Rosalind and Morris Goodman Cancer Research Centre, McGill University, Montreal, Quebec H3G 1Y6, Canada.|Department of Oncology, McGill University, Montreal, Quebec H3G 1Y6, Canada.
authors.forename: Jerry
authors.initials: J
authors.lastname: Pelletier
mesh_terms: D015152:Blotting, Northern; D015153:Blotting, Western; D064130:CRISPR-Associated Proteins; D064113:CRISPR-Cas Systems; D053837:DNA Cleavage; D024202:Electrophoretic Mobility Shift Assay; D016678:Genome; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D061986:MCF-7 Cells; D059372:Nucleotide Motifs; D012313:RNA / Q000378:metabolism; D017393:RNA Editing; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000737:chemistry*
chemical_list: D064130:CRISPR-Associated Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D012313:RNA