RT Journal Article
SR Electronic
T1 Catalytically Enhanced Cas9 through Directed Protein Evolution
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2020.07.01.183194
DO 10.1101/2020.07.01.183194
A1 Travis H. Hand
A1 Mitchell O. Roth
A1 Chardasia L. Smith
A1 Emily Shiel
A1 Kyle N. Klein
A1 David M. Gilbert
A1 Hong Li
YR 2020
UL http://biorxiv.org/content/early/2020/07/01/2020.07.01.183194.abstract
AB 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 StatementThe authors have declared no competing interest.