HIT-Cas9: A CRISPR/Cas9 Genome-Editing Device under Tight and Effective Drug Control

Chen Zhao; Yingze Zhao; Jingfang Zhang; Jia Lu; Li Chen; Yue Zhang; Yue Ying; Junjun Xu; Shixian Wei; Yu Wang

doi:10.1016/j.omtn.2018.08.022

HIT-Cas9: A CRISPR/Cas9 Genome-Editing Device under Tight and Effective Drug Control

Mol Ther Nucleic Acids. 2018 Dec 7:13:208-219. doi: 10.1016/j.omtn.2018.08.022. Epub 2018 Sep 1.

Authors

Chen Zhao¹, Yingze Zhao¹, Jingfang Zhang¹, Jia Lu², Li Chen², Yue Zhang¹, Yue Ying¹, Junjun Xu³, Shixian Wei², Yu Wang⁴

Affiliations

¹ State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.
² State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China.
³ College of Biological Sciences, China Agricultural University, Beijing 100193, China.
⁴ State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China; Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China. Electronic address: yuwang@post.harvard.edu.

Abstract

The CRISPR/Cas9 enabled efficient gene editing in an easy and programmable manner. Controlling its activity in greater precision is desired for biomedical research and potential therapeutic translation. Here, we engrafted the CRISPR/Cas9 system with a mutated human estrogen receptor (ER^T2), which renders it 4-hydroxytamoxifen (4-OHT) inducible for the access of genome, and a nuclear export signal (NES), which lowers the background activity. Tight and efficient drug-inducible genome editing was achieved across several human cell types, including embryonic stem cells (ESCs) and mesenchymal stem cells (MSCs), upon vigorous optimization. Optimized terminal device, which we named hybrid drug inducible CRISPR/Cas9 technology (HIT-Cas9), delivered advantageous performances over several existing designs. Such architecture was also successfully applied to an orthogonal Cas9. The HIT-Cas9 system developed in this study will find broad utility in controlled editing of potentially any genomic loci.

Keywords: CRISPR/Cas9; drug inducible; genome editing; stem cells.