Chemogenetic System Demonstrates That Cas9 Longevity Impacts Genome Editing Outcomes.
Vedagopuram Sreekanth, Qingxuan Zhou, Praveen Kokkonda, Heysol C Bermudez-Cabrera, Donghyun Lim, Benjamin K Law, Benjamin R Holmes, Santosh K Chaudhary, Rajaiah Pergu, Brittany S Leger, James A Walker, David K Gifford, Richard I Sherwood, Amit Choudhary
Author Information
Vedagopuram Sreekanth: Chemical Biology and Therapeutics Science Program, Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, United States.
Qingxuan Zhou: Chemical Biology and Therapeutics Science Program, Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, United States.
Praveen Kokkonda: Chemical Biology and Therapeutics Science Program, Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, United States.
Heysol C Bermudez-Cabrera: Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, United States.
Donghyun Lim: Chemical Biology and Therapeutics Science Program, Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, United States.
Benjamin K Law: Chemical Biology and Therapeutics Science Program, Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, United States.
Benjamin R Holmes: McGovern Institute for Brain Research at MIT, Cambridge, Massachusetts 02142, United States.
Santosh K Chaudhary: Chemical Biology and Therapeutics Science Program, Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, United States.
Rajaiah Pergu: Chemical Biology and Therapeutics Science Program, Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, United States.
Brittany S Leger: Center for Genomic Medicine, Massachusetts General Hospital, Boston, Massachusetts 02114, United States.
James A Walker: Center for Genomic Medicine, Massachusetts General Hospital, Boston, Massachusetts 02114, United States.
David K Gifford: McGovern Institute for Brain Research at MIT, Cambridge, Massachusetts 02142, United States.
Richard I Sherwood: Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, United States.
Amit Choudhary: Chemical Biology and Therapeutics Science Program, Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, United States.
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.
