Catalytically Enhanced Cas9 through Directed Protein Evolution

Guided by the extensive knowledge of CRISPR-Cas9 molecular mechanisms, protein engineering can be an effective method in improving CRISPR-Cas9 toward desired traits different from those of their natural forms. Here, we describe a directed protein evolution method that enables selection of catalytically enhanced CRISPR-Cas9 variants (CECas9) by targeting a shortened protospacer within a toxic gene. We demonstrate the effectiveness of this method with a previously characterized Type II-C Cas9 from Acidothermus cellulolyticus (AceCas9) and show by enzyme kinetics an up to fourfold improvement of the in vitro catalytic efficiency by AceCECas9. We further evolved the more widely used Streptococcus pyogenes Cas9 (SpyCas9) and demonstrated a noticeable improvement in the SpyCECas9-facilitated homology directed repair-based gene insertion in human colon cancer cells.

Automated summary

This study introduces a protein engineering method for selecting catalytically enhanced CRISPR-Cas9 variants by targeting a shortened protospacer within a toxic gene. By improving AceCas9 and SpyCas9, significant enhancements in catalytic efficiency were achieved, with noticeable improvement in gene insertion in human colon cancer cells facilitated by SpyCECas9.