2. Academic Validation
  3. Chemogenetic System Demonstrates That Cas9 Longevity Impacts Genome Editing Outcomes

Chemogenetic System Demonstrates That Cas9 Longevity Impacts Genome Editing Outcomes

  • ACS Cent Sci. 2020 Dec 23;6(12):2228-2237. doi: 10.1021/acscentsci.0c00129.
Vedagopuram Sreekanth 1 2 3,  Qingxuan Zhou 1 2 3,  Praveen Kokkonda 1 2 3,  Heysol C Bermudez-Cabrera 3 4,  Donghyun Lim 1 2 3,  Benjamin K Law 1 2 3,  Benjamin R Holmes 5 6,  Santosh K Chaudhary 1 2 3,  Rajaiah Pergu 1 2 3,  Brittany S Leger 7,  James A Walker 7 8,  David K Gifford 5 6 9 10,  Richard I Sherwood 3 4 11,  Amit Choudhary 1 2 3
Affiliations

Affiliations

  • 1 Chemical Biology and Therapeutics Science Program, Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, United States.
  • 2 Divisions of Renal Medicine and Engineering, Brigham and Women's Hospital, Boston, Massachusetts 02115, United States.
  • 3 Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, United States.
  • 4 Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115, United States.
  • 5 McGovern Institute for Brain Research at MIT, Cambridge, Massachusetts 02142, United States.
  • 6 Computer Science and Artificial Intelligence Laboratory, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States.
  • 7 Center for Genomic Medicine, Massachusetts General Hospital, Boston, Massachusetts 02114, United States.
  • 8 Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, United States.
  • 9 Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States.
  • 10 Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States.
  • 11 Hubrecht Institute for Developmental Biology and Stem Cell Research, Royal Netherlands Academy of Arts and Sciences (KNAW), Utrecht 3584 CT, The Netherlands.
PMID: 33376784 DOI: 10.1021/acscentsci.0c00129
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.

Figures
Products
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  • HY-147098
    99.65%, FKBP12F36V PROTAC降解剂
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