期刊
GENOME BIOLOGY
卷 16, 期 -, 页码 -出版社
BMC
DOI: 10.1186/s13059-015-0818-7
关键词
CRISPR; Cas9; Nuclease; PAM determination; Guide RNA determination
资金
- Agency for Science, Innovation and Technology (MITA) [31v-13, 31v-36]
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.
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