| Title |
Rapid characterization of CRISPR-Cas9 protospacer adjacent motif sequence elements / |
| Authors |
Karvelis, Tautvydas ; Gasiūnas, Giedrius ; Young, Joshua ; Bigelytė, Greta ; Šilanskas, Arūnas ; Cigan, Mark ; Šikšnys, Virginijus |
| DOI |
10.1186/s13059-015-0818-7 |
| Full Text |
|
| Is Part of |
Genome biology.. London : BioMed Central Ltd.. 2015, vol. 16, art. no. 253, p. [1-13].. ISSN 1465-6906. eISSN 1474-760X |
| Keywords [eng] |
CRISPR ; Cas9 ; Nuclease ; PAM determination ; Guide RNA determination |
| Abstract [eng] |
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. |
| Published |
London : BioMed Central Ltd |
| Type |
Journal article |
| Language |
English |
| Publication date |
2015 |
| CC license |
|
