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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.

SUBMITTER: Karvelis T 

PROVIDER: S-EPMC4653880 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Rapid characterization of CRISPR-Cas9 protospacer adjacent motif sequence elements.

Karvelis Tautvydas T   Gasiunas Giedrius G   Young Joshua J   Bigelyte Greta G   Silanskas Arunas A   Cigan Mark M   Siksnys Virginijus V  

Genome biology 20151119


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 (  ...[more]

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