Rapid characterization of CRISPR-Cas9 protospacer adjacent motif sequence elements. Issue 1 (December 2015)

• Record Type: Journal Article
• Authors: Karvelis, Tautvydas; Gasiunas, Giedrius; Young, Joshua; Bigelyte, Greta; Silanskas, Arunas; Cigan, Mark; Siksnys, Virginijus
• Is Part Of: Genome biology. Volume 16:Issue 1(2015)
• Page Start: 1
• Page End: 13
• Publication Date: 2015-12
• Subjects: CRISPR -- Cas9 -- Nuclease -- PAM determination -- Guide RNA determination; Genomes -- Periodicals; Biology -- Periodicals; Molecular biology -- Periodicals; 572.8633
• DOI: 10.1186/s13059-015-0818-7
• Languages: English
• ISSNs: 1474-760X

Abstract

Abstract To expand the repertoire of Cas9s available for genome targeting, we present a newin vitro method for the simultaneous examination of guide RNA and protospacer adjacent motif (PAM) requirements. The method relies on thein 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 forStreptococcus pyogenes, Streptococcus thermophilus CRISPR3 (Sth3), and CRISPR1 (Sth1). Additionally, PAM and sgRNA solutions for a novel Cas9 protein fromBrevibacillus laterosporus are provided by the assay and are demonstrated to support functional activityin vitro and in plants.