Davletshin, A.I.; Matveeva, A.A.; Bachurin, S.S.; Karpov, D.S.; Garbuz, D.G. Increasing the Activity of the High-Fidelity SpyCas9 Form in Yeast by Directed Mutagenesis of the PAM-Interacting Domain. Int. J. Mol. Sci.2024, 25, 444.
Davletshin, A.I.; Matveeva, A.A.; Bachurin, S.S.; Karpov, D.S.; Garbuz, D.G. Increasing the Activity of the High-Fidelity SpyCas9 Form in Yeast by Directed Mutagenesis of the PAM-Interacting Domain. Int. J. Mol. Sci. 2024, 25, 444.
Davletshin, A.I.; Matveeva, A.A.; Bachurin, S.S.; Karpov, D.S.; Garbuz, D.G. Increasing the Activity of the High-Fidelity SpyCas9 Form in Yeast by Directed Mutagenesis of the PAM-Interacting Domain. Int. J. Mol. Sci.2024, 25, 444.
Davletshin, A.I.; Matveeva, A.A.; Bachurin, S.S.; Karpov, D.S.; Garbuz, D.G. Increasing the Activity of the High-Fidelity SpyCas9 Form in Yeast by Directed Mutagenesis of the PAM-Interacting Domain. Int. J. Mol. Sci. 2024, 25, 444.
Abstract
Abstract: CRISPR/Cas systems are used for genome editing, both in basic science and in bio-technology. At the same time, 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 been obtained now, 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 cere-visiae. 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.
Keywords
Saccharomyces cerevisiae; genome editing; site-directed mutagenesis; CRISPR/Cas9; high-fidelity Cas9 forms
Subject
Biology and Life Sciences, Biology and Biotechnology
Copyright:
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
