Deep mutational scanning of S. pyogenes Cas9 reveals important functional domains.

ABSTRACT: RNA-guided endonucleases (RGENs) have invigorated the field of site-specific nucleases. The success of Streptococcus pyogenes Cas9 (SpCas9) has led to the discovery of several other CRISPR-associated RGENs. As more RGENs become available, it will be necessary to refine their activity before they can be translated into the clinic. With this in mind, we sought to demonstrate how deep mutational scanning (DMS) could provide details about important functional regions in SpCas9 and speed engineering efforts. Consequently, we developed a nuclease screening platform which could distinguish active Cas9 mutants. We screened a library of 1.9 × 10⁷ with over 8500 possible non-synonymous mutations and inferred the effects of each mutation using DMS. We demonstrate that the RuvC and HNH doma

SUBMITTER: Spencer JM 

PROVIDER: S-EPMC5715146 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature