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De novo design of hypercompact transcript degraders by engineering substrate-specific toxins and Cas6-CBS system

Author

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  • Pin-Ru Chen

    (Zhejiang University of Technology)

  • Pei-Pei Qin

    (Zhejiang University of Technology)

  • Ya-Nan Wang

    (Zhejiang University of Technology)

  • Peng-Fu Liu

    (Zhejiang University of Technology)

  • Xin-Yue Zhang

    (East China University of Science and Technology)

  • Tao Qian

    (Zhejiang University of Technology)

  • Bang-Ce Ye

    (East China University of Science and Technology)

  • Bin-Cheng Yin

    (East China University of Science and Technology
    Shihezi University)

Abstract

Artificial assembly of small functional proteins provides effective strategies for development of compact RNA degradation systems, which overcome the challenges associated with delivery. Here, we excavate and evolve three small toxin endoribonucleases with simple RNA cleavage motifs (barnase, MqsR, and MaZF), and integrate catalytically dead Cas6 (dCas6) along with its cognate stem-loop RNA (Cas6 binding site, termed CBS) from Escherichia coli (E. coli) to create hypercompact transcript degraders (317 ~ 430 amino acids), named STAR (small toxin- and dEcCas6-CBS-based RNA degraders). We experimentally find that CBS can be fine-tuned for EcCas6 processing but exhibits high conservatism in EcCas6 and dEcCas6 binding, laying a foundation for the design of CBS guides to effectively recruit dEcCas6-toxins. STAR exhibits high-efficiency knockdown of both cytoplasmic and nuclear transcripts in the tested mammalian cells, with significantly reduced off-target activities compared to established CRISPR and RNA interference (RNAi) technologies. Moreover, the small size of STAR enables delivery via a single adeno-associated virus (AAV) for ease of multiplex RNA knockdown, including effective silencing of the oncogenic RNA MYC in human cancer cells. Together, STAR unlocks new territory for employing toxin to design miniature, efficacious and safer RNA degraders.

Suggested Citation

  • Pin-Ru Chen & Pei-Pei Qin & Ya-Nan Wang & Peng-Fu Liu & Xin-Yue Zhang & Tao Qian & Bang-Ce Ye & Bin-Cheng Yin, 2025. "De novo design of hypercompact transcript degraders by engineering substrate-specific toxins and Cas6-CBS system," Nature Communications, Nature, vol. 16(1), pages 1-14, December.
    • Handle: RePEc:nat:natcom:v:16:y:2025:i:1:d:10.1038_s41467-025-63166-y
    DOI: 10.1038/s41467-025-63166-y
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