Catalytically Enhanced Cas9 through Directed Protein Evolution

Abstract

<jats:title>Abstract</jats:title><jats:p>The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas9 system has found widespread applications in genome manipulations due to its simplicity and effectiveness. Significant efforts in enzyme engineering have been made to improve the CRISPR-Cas9 systems beyond their natural power with additional functionalities such as DNA modification, transcriptional regulation, and high target selectivity<jats:sup>1–10</jats:sup>. Relatively less attention, however, has been paid to improving the catalytic efficiency of CRISPR-Cas9. Increased catalytic efficiency may be desired in applications where the currently available CRISPR-Cas9 tools are either ineffective<jats:sup>4, 11–14</jats:sup> or of low efficiency such as with type II-C Cas9<jats:sup>15–18</jats:sup> or in non-mammals<jats:sup>19, 20</jats:sup>. We describe a directed protein evolution method that enables selection of catalytically enhanced CRISPR-Cas9 variants (CECas9). We demonstrate the effectiveness of this method with a previously characterized Type IIC Cas9 from <jats:italic>Acidothermus cellulolyticus</jats:italic> (AceCas9) with up to 4-fold improvement of in vitro catalytic efficiency, as well as the widely used <jats:italic>Streptococcus pyogenes</jats:italic> Cas9 (SpyCas9), which showed a 2-fold increase in homology directed repair (HDR)-based gene insertion in human colon cancer cells.</jats:p>