Chemogenetic System Demonstrates That Cas9 Longevity Impacts Genome Editing Outcomes,ACS Central Science

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Chemogenetic System Demonstrates That Cas9 Longevity Impacts Genome Editing Outcomes
ACS Central Science ( IF 12.7 ) Pub Date : 2020-11-18 , 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}

Affiliation

Chemical Biology and Therapeutics Science Program, Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, United States
Divisions of Renal Medicine and Engineering, Brigham and Women’s Hospital, Boston, Massachusetts 02115, United States
Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, United States
Division of Genetics, Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts 02115, United States
McGovern Institute for Brain Research at MIT, Cambridge, Massachusetts 02142, United States
Computer Science and Artificial Intelligence Laboratory, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States
Center for Genomic Medicine, Massachusetts General Hospital, Boston, Massachusetts 02114, United States
Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, United States
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States
Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States
Hubrecht Institute for Developmental Biology and Stem Cell Research, Royal Netherlands Academy of Arts and Sciences (KNAW), Utrecht 3584 CT, The Netherlands

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.

中文翻译：

化学生成系统表明，Cas9寿命会影响基因组编辑结果。

Cas9的延长活性会阻碍基因组工程化，因为它会导致脱靶效应，遗传毒性，基因组编辑异质性，免疫原性和胚胎编辑中的镶嵌现象，这些问题可以通过控制Cas9的寿命来解决。尽管已经开发了一些Cas9活性的时间控制方法，但是只有繁琐的系统才能改变生命周期。在这里，我们开发了一种化学生成系统，可使Cas9接近泛素连接酶，从而使蛋白酶体快速泛素化和降解Cas9。尽管Cas9的大小很大，我们仍然能够证明多种物种细胞的有效降解。此外，通过控制Cas9的寿命，我们能够偏向模板化和非模板化基因组编辑的DNA修复途径和基因型结果。最后，我们能够切实控制Cas9的活性和特异性，以改善脱靶效应。该系统改变Cas9寿命的能力，以及因此在所需方向上偏向修复途径和特异性的能力，可以精确控制基因组编辑结果。

更新日期：2020-12-23

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