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Highly efficient and specific regulation of gene expression using enhanced CRISPR-Cas12f system

Gene Therapy (2024)Cite this article

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Abstract

The recently developed CRISPR activator (CRISPRa) system uses a CRISPR-Cas effector-based transcriptional activator to effectively control the expression of target genes without causing DNA damage. However, existing CRISPRa systems based on Cas9/Cas12a necessitate improvement in terms of efficacy and accuracy due to limitations associated with the CRISPR-Cas module itself. To overcome these limitations and effectively and accurately regulate gene expression, we developed an efficient CRISPRa system based on the small CRISPR-Cas effector Candidatus Woesearchaeota Cas12f (CWCas12f). By engineering the CRISPR-Cas module, linking activation domains, and using various combinations of linkers and nuclear localization signal sequences, the optimized eCWCas12f-VPR system enabled effective and target-specific regulation of gene expression compared with that using the existing CRISPRa system. The eCWCas12f-VPR system developed in this study has substantial potential for controlling the transcription of endogenous genes in living organisms and serves as a foundation for future gene therapy and biological research.

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Fig. 1: Optimization of the dCWCas12f-based CRISPR activator for targeted transcriptional activation.
Fig. 2: Targeted transcriptional activation with eCWCas12f-VPR across various endogenous target sites in human cell line.
Fig. 3: Comparative evaluation of off-target effects induced by eCWCas12f-VPR and dLbCas12a-VPR.
Fig. 4: Activation of endogenous gene expression using eCWCas12f-VPR-loaded rAAV delivery vehicles.

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Data availability

All relevant data supporting the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

This research was supported by grants from the Korean Fund for Regenerative Medicine (KFRM) grant funded by the Korea government (the Ministry of Science and ICT, the Ministry of Health & Welfare) (22A0203L1). This study was also supported by the Chung-Ang University Research Scholarship Grants in 2023.

Author information

Authors and Affiliations

  1. Department of Life Science, Chung-Ang University, Seoul, 06974, Republic of Korea

    Yeounsun Oh & Seung Hwan Lee

  2. National Primate Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju, 28116, Republic of Korea

    Lee Wha Gwon

  3. KRIBB School of Bioscience, Korea University of Science and Technology, Daejeon, 34113, Republic of Korea

    Lee Wha Gwon & Kwang-Hyun Park

  4. Major in Medical Genetics, Department of Medicine, Hanyang University, Seoul, 04763, Republic of Korea

    Hyomin K. Lee

  5. Department of Genetics, College of Medicine, Hanyang University, Seoul, 04763, Republic of Korea

    Junho K. Hur

  6. Department of Medicine, HY Institute of Bioscience and Biotechnology, Hanyang University, Seoul, 04763, Republic of Korea

    Junho K. Hur

  7. Center for Translational Research in Neuroimaging and Data Science (TReNDS), Georgia State University, Atlanta, GA, 30303, USA

    Junho K. Hur

  8. Critical Diseases Diagnostics Convergence Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 34141, Republic of Korea

    Kwang-Hyun Park

  9. Department of Medical Life Sciences, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea

    Kee-Pyo Kim

  10. Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Münster, Germany

    Kee-Pyo Kim

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  1. Yeounsun Oh
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Contributions

Conceptualization, YO, KHP, KPK, and SHL; Methodology, YO, LWG, HKL, JKH, KHP, KPK, and SHL; Software, YO, HKL, JKH, and SHL; Validation, YO, and SHL; Formal Analysis, YO and SHL; Investigation, YO and SHL; Resources, YO, KPK, and SHL; Data Curation, YO, KPK, and SHL; Writing-Original Draft, YO and SHL; Writing-Review & Editing, YO, KHP, KPK and SHL; Visualization, YO and SHL; Supervision, YO, KHP, KPK, and SHL; Project Administration, YO, KPK, and SHL; Funding Acquisition, KPK and SHL.

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Correspondence to Kwang-Hyun Park, Kee-Pyo Kim or Seung Hwan Lee.

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Oh, Y., Gwon, L.W., Lee, H.K. et al. Highly efficient and specific regulation of gene expression using enhanced CRISPR-Cas12f system. Gene Ther (2024). https://doi.org/10.1038/s41434-024-00458-w

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