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Catalytically Enhanced Cas9 through Directed Protein Evolution

View ORCID ProfileTravis H. Hand, Mitchell O. Roth, Chardasia L. Smith, Emily Shiel, Kyle N. Klein, View ORCID ProfileDavid M. Gilbert, Hong Li
doi: https://doi.org/10.1101/2020.07.01.183194
Travis H. Hand
1Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306, USA
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Mitchell O. Roth
1Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306, USA
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Chardasia L. Smith
2Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL 32306, USA
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Emily Shiel
2Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL 32306, USA
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Kyle N. Klein
3Department of Biological Sciences, Florida State University, Tallahassee, FL 32306, USA
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David M. Gilbert
3Department of Biological Sciences, Florida State University, Tallahassee, FL 32306, USA
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Hong Li
1Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306, USA
2Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL 32306, USA
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  • For correspondence: hong.li@fsu.edu
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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.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted July 01, 2020.
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Catalytically Enhanced Cas9 through Directed Protein Evolution
Travis H. Hand, Mitchell O. Roth, Chardasia L. Smith, Emily Shiel, Kyle N. Klein, David M. Gilbert, Hong Li
bioRxiv 2020.07.01.183194; doi: https://doi.org/10.1101/2020.07.01.183194
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Catalytically Enhanced Cas9 through Directed Protein Evolution
Travis H. Hand, Mitchell O. Roth, Chardasia L. Smith, Emily Shiel, Kyle N. Klein, David M. Gilbert, Hong Li
bioRxiv 2020.07.01.183194; doi: https://doi.org/10.1101/2020.07.01.183194

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