Catalytically Enhanced Cas9 Through Directed Protein Evolution

Abstract

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 <i>Acidothermus cellulolyticus</i> (AceCas9) and show by enzyme kinetics an up to fourfold improvement of the <i>in vitro</i> catalytic efficiency by AceCECas9. We further evolved the more widely used <i>Streptococcus pyogenes</i> Cas9 (SpyCas9) and demonstrated a noticeable improvement in the SpyCECas9-facilitated homology directed repair-based gene insertion in human colon cancer cells.