title: Rapid characterization of CRISPR-Cas9 protospacer adjacent motif sequence elements.
abstract: To expand the repertoire of Cas9s available for genome targeting, we present a new in vitro method for the simultaneous examination of guide RNA and protospacer adjacent motif (PAM) requirements. The method relies on the in vitro cleavage of plasmid libraries containing a randomized PAM as a function of Cas9-guide RNA complex concentration. Using this method, we accurately reproduce the canonical PAM preferences for Streptococcus pyogenes, Streptococcus thermophilus CRISPR3 (Sth3), and CRISPR1 (Sth1). Additionally, PAM and sgRNA solutions for a novel Cas9 protein from Brevibacillus laterosporus are provided by the assay and are demonstrated to support functional activity in vitro and in plants.
authors.affiliation: Institute of Biotechnology, Vilnius University, Graiciuno 8, LT-02241, Vilnius, Lithuania.
authors.forename: Tautvydas
authors.initials: T
authors.lastname: Karvelis
authors.affiliation: Institute of Biotechnology, Vilnius University, Graiciuno 8, LT-02241, Vilnius, Lithuania.
authors.forename: Giedrius
authors.initials: G
authors.lastname: Gasiunas
authors.affiliation: DuPont Pioneer, 7300 NW 62nd Avenue, Johnston, IA, 50131, USA.
authors.forename: Joshua
authors.initials: J
authors.lastname: Young
authors.affiliation: Institute of Biotechnology, Vilnius University, Graiciuno 8, LT-02241, Vilnius, Lithuania.
authors.forename: Greta
authors.initials: G
authors.lastname: Bigelyte
authors.affiliation: Institute of Biotechnology, Vilnius University, Graiciuno 8, LT-02241, Vilnius, Lithuania.
authors.forename: Arunas
authors.initials: A
authors.lastname: Silanskas
authors.affiliation: DuPont Pioneer, 7300 NW 62nd Avenue, Johnston, IA, 50131, USA. mark.cigan@pioneer.com.
authors.forename: Mark
authors.initials: M
authors.lastname: Cigan
authors.affiliation: Institute of Biotechnology, Vilnius University, Graiciuno 8, LT-02241, Vilnius, Lithuania. siksnys@ibt.lt.
authors.forename: Virginijus
authors.initials: V
authors.lastname: Siksnys
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D058675:Brevibacillus / Q000201:enzymology / Q000235:genetics; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D018390:Gene Targeting / Q000379:methods*; D008969:Molecular Sequence Data; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D013291:Streptococcus / Q000201:enzymology / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D004720:Endonucleases