Chemogenetic System Demonstrates That Cas9 Longevity Impacts Genome Editing Outcomes
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acscentsci.0c00129.pdf
Description
Published version
Size
1.2 MB
Format
Adobe PDF
Checksum (MD5)
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Author(s)
Sreekanth, Vedagopuram
Zhou, Qingxuan
Kokkonda, Praveen
Bermudez-Cabrera, Heysol C.
Lim, Donghyun
Law, Benjamin K.
Holmes, Benjamin Ray
Chaudhary, Santosh K.
Pergu, Rajaiah
Leger, Brittany S.
Date Issued
November 2020
Journal
ACS Central Science
Publisher
American Chemical Society (ACS)
Citation
Sreekanth, Vedagopuram et al. "Chemogenetic System Demonstrates That Cas9 Longevity Impacts Genome Editing Outcomes." ACS Central Science 6, 12 (November 2020): 2228–2237 © American Chemical Society
Version
Final published version
Abstract
Prolonged Cas9 activity can hinder genome engineering as it causes off-target effects, genotoxicity, heterogeneous genome-editing outcomes, immunogenicity, and mosaicism in embryonic editing - issues which could be addressed by controlling the longevity of Cas9. Though some temporal controls of Cas9 activity have been developed, only cumbersome systems exist for modifying the lifetime. Here, we have developed a chemogenetic system that brings Cas9 in proximity to a ubiquitin ligase, enabling rapid ubiquitination and degradation of Cas9 by the proteasome. Despite the large size of Cas9, we were able to demonstrate efficient degradation in cells from multiple species. Furthermore, by controlling the Cas9 lifetime, we were able to bias the DNA repair pathways and the genotypic outcome for both templated and nontemplated genome editing. Finally, we were able to dosably control the Cas9 activity and specificity to ameliorate the off-target effects. The ability of this system to change the Cas9 lifetime and, therefore, bias repair pathways and specificity in the desired direction allows precision control of the genome editing outcome.
MIT Department
McGovern Institute for Brain Research at MIT
Massachusetts Institute of Technology. Computer Science and Artificial Intelligence Laboratory
Massachusetts Institute of Technology. Department of Electrical Engineering and Computer Science
Massachusetts Institute of Technology. Department of Biological Engineering
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DOI of Published Version
https://doi.org/10.1021/acscentsci.0c00129
