A chemical-inducible CRISPR–Cas9 system for rapid control of genome editing

Abstract

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.

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Figure 1: Building and testing a 4-HT-inducible Cas9.
Figure 2: Optimization of the ERT2–Cas9–ERT2 architecture.
Figure 3: Optimization of 4-HT treatment conditions.
Figure 4: Comparison of iCas with an alternative inducible-promoter-based system.
Figure 5: Comparison of iCas with intein–Cas9 and split-Cas9.
Figure 6: Toggling the activity of iCas on and off.

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Acknowledgements

We thank R. Dasgupta for helpful discussions, Y. Wan for critical reading of the manuscript, and H.H. Ng (Genome Institute of Singapore), R. Dasgupta (Genome Institute of Singapore), and B. Lim (Genome Institute of Singapore) for reagents. STF3A cells were a gift from D. Virshup (Duke-National University of Singapore Graduate Medical School). This work was supported by core funding from the Genome Institute of Singapore (M.H.T.), an Agency for Science Technology and Research Joint Council Office grant 1431AFG103 (M.H.T.), and a startup grant from Nanyang Technological University (M.H.T.).

Author information

M.H.T. conceived the project, designed the experiments, performed the experiments, analyzed the data, supervised the research, and wrote the manuscript. K.I.L., M.N.B.R., C.W.A.W., Y.W., T.Z., B.Y.C., A.G., M.Z.H.C., J.J., and J.H.J.L. performed experiments and analyzed data. X.Z. analyzed deep-sequencing data. G.R.D.Y. provided input about the experimental design, performed the experiments, and analyzed the data.

Correspondence to Meng How Tan.

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Competing interests

The authors declare that a patent has been filed for iCas (Singapore Patent Application No. 10201509153Y). M.H.T. is an inventor on the patent.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–26 and Supplementary Tables 1–4. (PDF 2930 kb)

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Liu, K., Ramli, M., Woo, C. et al. A chemical-inducible CRISPR–Cas9 system for rapid control of genome editing. Nat Chem Biol 12, 980–987 (2016) doi:10.1038/nchembio.2179

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