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


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.

Data availability

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


  1. 1.

    Ran, F. A. et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).

  2. 2.

    Nihongaki, Y., Otabe, T. & Sato, M. Emerging approaches for spatiotemporal control of targeted genome with inducible CRISPR-Cas9. Anal. Chem. 90, 429–439 (2018).

  3. 3.

    Brocken, D. J. W., Tark-Dame, M. & Dame, R. T. dCas9: a versatile tool for epigenome editing. Curr. Issues Mol. Biol. 26, 15–32 (2018).

  4. 4.

    Zetsche, B., Volz, S. E. & Zhang, F. A split-Cas9 architecture for inducible genome editing and transcription modulation. Nat. Biotechnol. 33, 139–142 (2015).

  5. 5.

    Davis, K. M., Pattanayak, V., Thompson, D. B., Zuris, J. A. & Liu, D. R. Small molecule-triggered Cas9 protein with improved genome-editing specificity. Nat. Chem. Biol. 11, 316–318 (2015).

  6. 6.

    Nihongaki, Y., Kawano, F., Nakajima, T. & Sato, M. Photoactivatable CRISPR-Cas9 for optogenetic genome editing. Nat. Biotechnol. 33, 755–760 (2015).

  7. 7.

    Dominguez, A. A., Lim, W. A. & Qi, L. S. Beyond editing: repurposing CRISPR-Cas9 for precision genome regulation and interrogation. Nat. Rev. Mol. Cell Biol. 17, 5–15 (2016).

  8. 8.

    Tanenbaum, M. E., Gilbert, L. A., Qi, L. S., Weissman, J. S. & Vale, R. D. A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell 159, 635–646 (2014).

  9. 9.

    Chavez, A. et al. Highly efficient Cas9-mediated transcriptional programming. Nat. Methods 12, 326–328 (2015).

  10. 10.

    Konermann, S. et al. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 517, 583–588 (2015).

  11. 11.

    Zalatan, J. G. et al. Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds. Cell 160, 339–350 (2015).

  12. 12.

    Zetsche, B. et al. Cpf1 is a single RNA-Guided endonuclease of a class 2 CRISPR-Cas system. Cell 163, 759–771 (2015).

  13. 13.

    Kim, H. K. et al. In vivo high-throughput profiling of CRISPR-Cpf1 activity. Nat. Methods 14, 153–159 (2017).

  14. 14.

    Zetsche, B. et al. Multiplex gene editing by CRISPR-Cpf1 using a single crRNA array. Nat. Biotechnol. 35, 31–34 (2017).

  15. 15.

    Kleinstiver, B. P. et al. Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells. Nat. Biotechnol. 34, 869–874 (2016).

  16. 16.

    Kim, D. et al. Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells. Nat. Biotechnol. 34, 863–868 (2016).

  17. 17.

    Singh, D. et al. Real-time observation of DNA target interrogation and product release by the RNA-guided endonuclease CRISPR Cpf1 (Cas12a). Proc. Natl Acad. Sci. USA 115, 5444–5449 (2018).

  18. 18.

    Nishimasu, H. et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156, 935–949 (2014).

  19. 19.

    Yamano, T. et al. Crystal structure of Cpf1 in complex with guide RNA and target DNA. Cell 165, 949–962 (2016).

  20. 20.

    Yamano, T. et al. Structural basis for the canonical and non-canonical PAM recognition by CRISPR-Cpf1. Mol. Cell 67, 633–645.e3 (2017).

  21. 21.

    DeRose, R., Miyamoto, T. & Inoue, T. Manipulating signaling at will: chemically-inducible dimerization (CID) techniques resolve problems in cell biology. Pflugers Arch. 465, 409–417 (2013).

  22. 22.

    Kawano, F., Suzuki, H., Furuya, A. & Sato, M. Engineered pairs of distinct photoswitches for optogenetic control of cellular proteins. Nat. Commun. 6, 6256 (2015).

  23. 23.

    Chavez, A. et al. Comparison of Cas9 activators in multiple species. Nat. Methods 13, 563–567 (2016).

  24. 24.

    Nihongaki, Y. et al. CRISPR–Cas9-based photoactivatable transcription systems to induce neuronal differentiation. Nat. Methods 14, 963–966 (2017).

  25. 25.

    Suzuki, K. et al. In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Nature 540, 144–149 (2016).

  26. 26.

    Wu, J., Corbett, A. H. & Berland, K. M. The intracellular mobility of nuclear import receptors and NLS cargoes. Biophys. J. 96, 3840–3849 (2009).

  27. 27.

    Tak, Y. E. et al. Inducible and multiplex gene regulation using CRISPR-Cpf1-based transcription factors. Nat. Methods 14, 1163–1166 (2017).

  28. 28.

    Liu, Y. et al. Engineering cell signaling using tunable CRISPR–Cpf1-based transcription factors. Nat. Commun. 8, 2095 (2017).

  29. 29.

    Zhang, X. et al. Gene activation in human cells using CRISPR/Cpf1-p300 and CRISPR/Cpf1-SunTag systems. Protein Cell 9, 380–383 (2018).

  30. 30.

    Wright, A. V. et al. Rational design of a split-Cas9 enzyme complex. Proc. Natl Acad. Sci. USA 112, 2984–2989 (2015).

  31. 31.

    Morita, S. et al. Targeted DNA demethylation in vivo using dCas9-peptide repeat and scFv-TET1 catalytic domain fusions. Nat. Biotechnol. 34, 1060–1065 (2016).

  32. 32.

    Cheng, A. W. et al. Casilio: a versatile CRISPR-Cas9-Pumilio hybrid for gene regulation and genomic labeling. Cell Res. 26, 254–257 (2016).

  33. 33.

    Truong, D.-J. J. et al. Development of an intein-mediated split-Cas9 system for gene therapy. Nucleic Acids Res. 43, 6450–6458 (2015).

  34. 34.

    Villiger, L. et al. Treatment of a metabolic liver disease by in vivo genome base editing in adult mice. Nat. Med. 24, 1519–1525 (2018).

  35. 35.

    Li, X. et al. Base editing with a Cpf1-cytidine deaminase fusion. Nat. Biotechnol. 36, 324–327 (2018).

  36. 36.

    Brinkman, E. K., Chen, T., Amendola, M. & Steensel, B. Van Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res. 42, e168 (2014).

  37. 37.

    Schmittgen, T. D. & Livak, K. J. Analyzing real-time PCR data by the comparative C(T) method. Nat. Protoc. 3, 1101–1108 (2008).

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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).

Author information

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.

Correspondence to Moritoshi Sato.

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

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