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Engineered circular guide RNAs enhance miniature CRISPR/Cas12f-based gene activation and adenine base editing

Author

Listed:
  • Xin Zhang

    (Southern Medical University
    the First Affiliated Hospital of Guangzhou Medical University)

  • Mengrao Li

    (Southern Medical University)

  • Kechen Chen

    (Southern Medical University)

  • Yuchen Liu

    (Southern Medical University)

  • Jiawei Liu

    (Southern Medical University)

  • Jiahong Wang

    (Southern Medical University)

  • Hongxin Huang

    (Southern Medical University)

  • Yanqun Zhang

    (Southern Medical University)

  • Tao Huang

    (Southern Medical University)

  • Shufeng Ma

    (Southern Medical University
    the First Affiliated Hospital of Guangzhou Medical University)

  • Kaitong Liao

    (Southern Medical University)

  • Jiayi Zhou

    (Southern Medical University)

  • Mei Wang

    (Southern Medical University)

  • Ying Lin

    (Southern Medical University)

  • Zhili Rong

    (Southern Medical University)

Abstract

CRISPR system has been widely used due to its precision and versatility in gene editing. Un1Cas12f1 from uncultured archaeon (hereafter referred to as Cas12f), known for its compact size (529 aa), exhibits obvious delivery advantage for gene editing in vitro and in vivo. However, its activity remains suboptimal. In this study, we engineer circular guide RNA (cgRNA) for Cas12f and significantly improve the efficiency of gene activation about 1.9–19.2-fold. When combined with a phase separation system, the activation efficiency is further increased about 2.3–3.9-fold. In addition, cgRNA enhances the editing efficiency and narrows the editing window of adenine base editing about 1.2–2.5-fold. Importantly, this optimization strategy also boosts the Cas12f-induced gene activation efficiency in mouse liver. Therefore, we demonstrate that cgRNA is able to enhance Cas12f-based gene activation and adenine base editing, which holds great potential for gene therapy.

Suggested Citation

  • Xin Zhang & Mengrao Li & Kechen Chen & Yuchen Liu & Jiawei Liu & Jiahong Wang & Hongxin Huang & Yanqun Zhang & Tao Huang & Shufeng Ma & Kaitong Liao & Jiayi Zhou & Mei Wang & Ying Lin & Zhili Rong, 2025. "Engineered circular guide RNAs enhance miniature CRISPR/Cas12f-based gene activation and adenine base editing," Nature Communications, Nature, vol. 16(1), pages 1-9, December.
    • Handle: RePEc:nat:natcom:v:16:y:2025:i:1:d:10.1038_s41467-025-58367-4
    DOI: 10.1038/s41467-025-58367-4
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    References listed on IDEAS

    as
    1. R. Alexander Wesselhoeft & Piotr S. Kowalski & Daniel G. Anderson, 2018. "Engineering circular RNA for potent and stable translation in eukaryotic cells," Nature Communications, Nature, vol. 9(1), pages 1-10, December.
    2. Amy J. Heidersbach & Kristel M. Dorighi & Javier A. Gomez & Ashley M. Jacobi & Benjamin Haley, 2023. "A versatile, high-efficiency platform for CRISPR-based gene activation," Nature Communications, Nature, vol. 14(1), pages 1-10, December.
    3. F. Ann Ran & Le Cong & Winston X. Yan & David A. Scott & Jonathan S. Gootenberg & Andrea J. Kriz & Bernd Zetsche & Ophir Shalem & Xuebing Wu & Kira S. Makarova & Eugene V. Koonin & Phillip A. Sharp & , 2015. "In vivo genome editing using Staphylococcus aureus Cas9," Nature, Nature, vol. 520(7546), pages 186-191, April.
    4. Patricia A. Clow & Menghan Du & Nathaniel Jillette & Aziz Taghbalout & Jacqueline J. Zhu & Albert W. Cheng, 2022. "CRISPR-mediated multiplexed live cell imaging of nonrepetitive genomic loci with one guide RNA per locus," Nature Communications, Nature, vol. 13(1), pages 1-10, December.
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