Trung Thanh Thach, Nam Hyeong Kim, Junho Hur and Yong Ho Kim
SAINT - Sungkyunkwan University, South Korea
Kyung Hee University, South Korea
Posters & Accepted Abstracts: J Proteomics Bioinform
A major limitation to expand RNA-guided CRISPR/Cas9 toolkit in genome editing is a sequence-specific recognition of the protospacer adjacent motif (PAM) at the target DNA site by the Cas9 protein. Exploration and characterization of new Cas9 ortholog binding distinct PAM sequence would beneficially expand the biological applications of the toolkit. Here, we identified new CRISPR/Cas9 system from uncharacterized bacterial strain. Analyses of the cleaved double-strand DNA (dsDNA) using deep sequencing showed that our system recognizes and cleaves target dsDNA with the distinct PAM sequence, yet previously described. Furthermore, structural analyses using small-angle x-ray scattering combined with binding affinity assays using bio-layer interferometry and PAM-based target DNA cleavage demonstrated that Arg-1083 and Arg-1116 residues in the Cas9-PAM interacting domain are key determinants of the PAM recognition. Collectively, our finding provides a novel CRIPRS/Cas9 system together with molecular basic understanding into a RNA-guided the distinct PAM-based target DNA cleavage, thus expand the target space for CRISPR/Cas9 toolkit applications.
