Increasing the Activity of the High-Fidelity SpyCas9 Form in Yeast by Directed Mutagenesis of the PAM-Interacting Domain

Abstract

CRISPR/Cas systems are used for genome editing, both in basic science and in biotechnology. However, CRISPR/Cas editors have several limitations, including insufficient specificity leading to “off-targets” and the dependence of activity on chromatin state. A number of highly specific Cas9 variants have now been obtained, but most of them are characterized by reduced activity on eukaryotic chromatin. We identified a spatial cluster of amino acid residues in the PAM-recognizing domain of Streptococcus pyogenes Cas9, whose mutations restore the activity of one of the highly specific forms of SpyCas9 without reducing its activity in Saccharomyces cerevisiae. In addition, one of these new mutations also increases the efficiency of SpyCas9-mediated editing of a site localized on the stable nucleosome. The improved Cas9 variants we obtained, which are capable of editing hard-to-reach regions of the yeast genome, may help in both basic research and yeast biotechnological applications.