A chemical-inducible CRISPR-Cas9 system for rapid control of genome editing.
CRISPR-Cas9 has emerged as a powerful technology that enables ready modification of the mammalian genome. The ability to modulate Cas9 activity can reduce off-target cleavage and facilitate precise genome engineering. Here we report the development of a Cas9 variant whose activity can be switched on and off in human cells with 4-hydroxytamoxifen (4-HT) by fusing the Cas9 enzyme with the hormone-binding domain of the estrogen receptor (ERT2). The final optimized variant, termed iCas, showed low endonuclease activity without 4-HT but high editing efficiency at multiple loci with the chemical. We also tuned the duration and concentration of 4-HT treatment to reduce off-target genome modification. Additionally, we benchmarked iCas against other chemical-inducible methods and found that it had the fastest on rate and that its activity could be toggled on and off repeatedly. Collectively, these results highlight the utility of iCas for rapid and reversible control of genome-editing function.
Genome Institute of Singapore, Agency for Science Technology and Research, Singapore.
Kaiwen Ivy
KI
Liu
Genome Institute of Singapore, Agency for Science Technology and Research, Singapore.
Muhammad Nadzim Bin
MN
Ramli
Genome Institute of Singapore, Agency for Science Technology and Research, Singapore.
Cheok Wei Ariel
http://orcid.org/0000-0002-6765-6632
CW
Woo
Genome Institute of Singapore, Agency for Science Technology and Research, Singapore.|School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore.
Yuanming
Y
Wang
Genome Institute of Singapore, Agency for Science Technology and Research, Singapore.
Tianyun
T
Zhao
Genome Institute of Singapore, Agency for Science Technology and Research, Singapore.|School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore.
Xiujun
X
Zhang
Genome Institute of Singapore, Agency for Science Technology and Research, Singapore.
Guo Rong Daniel
http://orcid.org/0000-0001-6153-4725
GR
Yim
Genome Institute of Singapore, Agency for Science Technology and Research, Singapore.|School of Biological Sciences, Nanyang Technological University, Singapore.
Bao Yi
BY
Chong
Genome Institute of Singapore, Agency for Science Technology and Research, Singapore.
Ali
A
Gowher
Genome Institute of Singapore, Agency for Science Technology and Research, Singapore.|School of Life Sciences and Chemical Technology, Ngee Ann Polytechnic, Singapore.
Mervyn Zi Hao
MZ
Chua
Genome Institute of Singapore, Agency for Science Technology and Research, Singapore.
Jonathan
http://orcid.org/0000-0003-0674-9066
J
Jung
Genome Institute of Singapore, Agency for Science Technology and Research, Singapore.
Jia Hui Jane
JH
Lee
Genome Institute of Singapore, Agency for Science Technology and Research, Singapore.|School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore.
Meng How
MH
Tan
D064113:CRISPR-Cas Systems / Q000187:drug effects* / Q000235:genetics*; D000072669:Gene Editing / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D013629:Tamoxifen / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology
D013629:Tamoxifen; C016601:afimoxifene