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bioRxiv · 10.1101/2020.07.01.183194

Catalytically Enhanced Cas9 through Directed Protein Evolution

Hand, T. H.·Roth, M. O.·Smith, C. L.·Shiel, E.·Klein, K.·Gilbert, D. M.·Li, H.
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Abstract

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 selectivity1-10. 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 ineffective4, 11-14 or of low efficiency such as with type II-C Cas915-18 or in non-mammals19, 20. 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 Acidothermus cellulolyticus (AceCas9) with up to 4-fold improvement of in vitro catalytic efficiency, as well as the widely used Streptococcus pyogenes Cas9 (SpyCas9), which showed a 2-fold increase in homology directed repair (HDR)-based gene insertion in human colon cancer cells.

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Hand, T. H., Roth, M. O., Smith, C. L., Shiel, E., Klein, K., Gilbert, D. M., Li, H.. 2020-07-01. Catalytically Enhanced Cas9 through Directed Protein Evolution. https://doi.org/10.1101/2020.07.01.183194

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