Chemogenetic System Demonstrates That Cas9 Longevity Impacts Genome Editing Outcomes.
Chemogenetic System Demonstrates That Cas9 Longevity Impacts Genome Editing Outcomes.
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DOI：
10.1021/acscentsci.0c00129
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发表时间：
2020-12-23
影响因子：
18.2
通讯作者：
Choudhary A
中科院分区：
文献类型：
--
作者：
Sreekanth V;Zhou Q;Kokkonda P;Bermudez-Cabrera HC;Lim D;Law BK;Holmes BR;Chaudhary SK;Pergu R;Leger BS;Walker JA;Gifford DK;Sherwood RI;Choudhary A
Cas9活性延长可能会阻碍基因组工程，因为它会导致脱靶效应、遗传毒性、异质基因组编辑结果、免疫原性和胚胎编辑中的嵌合性——这些问题可以通过控制Cas9的寿命来解决。虽然已经开发了一些Cas9活性的时间控制，但只有繁琐的系统可以修改寿命。在这里，我们开发了一种化学发生系统，使Cas9接近泛素连接酶，使蛋白酶体能够快速泛素化和降解Cas9。尽管Cas9的大小很大，但我们能够证明在来自多个物种的细胞中有效降解。此外，通过控制Cas9寿命，我们能够对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进行偏置。最后，我们能够有效地控制Cas9的活性和特异性，以改善脱靶效应。该系统改变Cas9生命周期的能力，因此，在所需方向上的偏倚修复途径和特异性允许精确控制基因组编辑结果。一种Cas9和双功能降解分子对的化学发生表盘已经被开发出来，以剂量和时间依赖的方式调节特异性和DNA修复结果。
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. A chemogenetic dial of Cas9 and bifunctional degrader molecule pair has been developed to tune the specificity and DNA repair outcome in a dose- and time-dependent manner.
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影响因子：
4
作者：
Gallagher DN;Haber JE
通讯作者：
Haber JE
DOI：
10.1002/anie.201806976
发表时间：
2018-09-10
期刊：
Angewandte Chemie (International ed. in English)
影响因子：
--
作者：
Chen X;Venkatachalapathy M;Dehmelt L;Wu YW
通讯作者：
Wu YW
影响因子：
82.9
作者：
Charlesworth, Carsten T.;Deshpande, Priyanka S.;Porteus, Matthew H.
通讯作者：
Porteus, Matthew H.
影响因子：
64.5
作者：
Maji, Basudeb;Gangopadhyay, Soumyashree A.;Choudhary, Amit
通讯作者：
Choudhary, Amit
影响因子：
4
作者：
Buckley DL;Raina K;Darricarrere N;Hines J;Gustafson JL;Smith IE;Miah AH;Harling JD;Crews CM
通讯作者：
Crews CM