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A split CRISPR–Cpf1 platform for inducible genome editing and gene activation

Nature Chemical Biology volume 15, pages 882–888 (2019)Cite this article

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Abstract

The CRISPR–Cpf1 endonuclease has recently been demonstrated as a powerful tool to manipulate targeted gene sequences. Here, we performed an extensive screening of split Cpf1 fragments and identified a pair that, combined with inducible dimerization domains, enables chemical- and light-inducible genome editing in human cells. We also identified another split Cpf1 pair that is spontaneously activated. The newly generated amino and carboxyl termini of the spontaneously activated split Cpf1 can be repurposed as de novo fusion sites of artificial effector domains. Based on this finding, we generated an improved split dCpf1 activator, which has the potential to activate endogenous genes more efficiently than a previously established dCas9 activator. Finally, we showed that the split dCpf1 activator can efficiently activate target genes in mice. These results demonstrate that the present split Cpf1 provides an efficient and sophisticated genome manipulation in the fields of basic research and biotechnological applications.

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Fig. 1: Screening of split Cpf1 and development of rapamycin-inducible Cpf1.
Fig. 2: Photoactivatable Cpf1.
Fig. 3: Split dCpf1 activator offers potent endogenous gene activation.
Fig. 4: Comparisons of activation efficiency between dCpf1-SA2.0 and dCas9-SAM targeting different sites in promoter regions.
Fig. 5: In vivo gene activation using split dCpf1 activator.

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Data availability

The datasets generated during this study are available from the corresponding author upon request.

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Acknowledgements

This work was supported by START and CREST grants (no. JPMJCR1653) from Japan Science and Technology Agency (JST) and grants for Project for Cancer Research and Therapeutic Evolution (no. 17cm0106415h0002) from Japan Agency for Medical Research and Development (AMED) to M.S. This work was also supported by grants from Japan Society for the Promotion of Science (JSPS) to M.S. and JSPS Research Fellowships for Young Scientists to Y.N. (no. 15J05897).

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Author notes

  1. Yuta Nihongaki

    Present address: Department of Cell Biology and Center for Cell Dynamics, Johns Hopkins University School of Medicine, Baltimore, MD, USA

  2. These authors contributed equally: Yuta Nihongaki, Takahiro Otabe.

Authors and Affiliations

  1. Graduate School of Arts and Sciences, The University of Tokyo, Tokyo, Japan

    Yuta Nihongaki, Takahiro Otabe, Yoshibumi Ueda & Moritoshi Sato

  2. Japan Agency for Medical Research and Development-PRIME, Tokyo, Japan

    Yoshibumi Ueda

  3. Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Saitama, Japan

    Moritoshi Sato

Authors
  1. Yuta Nihongaki
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  2. Takahiro Otabe
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  3. Yoshibumi Ueda
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  4. Moritoshi Sato
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Contributions

Y.N., T.O. and M.S. conceived the project. Y.N., T.O. and Y.U. designed experiments. Y.N. and T.O. performed experiments. Y.N. and T.O. analyzed data. Y.N., T.O. and M.S. wrote the manuscript.

Corresponding author

Correspondence to Moritoshi Sato.

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The authors declare no competing interests.

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Supplementary Tables 1–13, Supplementary Figures 1–18, Supplementary Note 1.

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Nihongaki, Y., Otabe, T., Ueda, Y. et al. A split CRISPR–Cpf1 platform for inducible genome editing and gene activation. Nat Chem Biol 15, 882–888 (2019). https://doi.org/10.1038/s41589-019-0338-y

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