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Spatially blocked split CRISPR-Cas12a system for ultra-sensitive and versatile small molecule activation and detection

Author

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  • Hao Hu

    (Huazhong University of Science and Technology
    Huazhong University of Science and Technology)

  • Songcheng Guo

    (Huazhong University of Science and Technology)

  • Yiyuan Li

    (Wuhan Polytechnic University)

  • Kejun Dong

    (Huazhong University of Science and Technology)

  • Yan Lu

    (Huazhong University of Science and Technology)

  • Keyi Ye

    (Huazhong University of Science and Technology)

  • Longjie Li

    (Wuhan Polytechnic University)

  • Xiaoyu Zhou

    (City University of Hong Kong)

  • Liming Cheng

    (Huazhong University of Science and Technology)

  • Xianjin Xiao

    (Huazhong University of Science and Technology
    Huazhong University of Science and Technology)

Abstract

Detecting small molecules is pivotal across fields like clinical diagnostics, environmental monitoring, and food safety. The CRISPR-Cas12a system, known for its simplicity and sensitivity, offers a promising basis for small molecule detection. However, current CRISPR-based detection methods face challenges, including complex design requirements, high background noise, and limited adaptability to different targets. In our study, we introduce the SBS-Cas system, leveraging a split crRNA mode to induce spatial hindrance on the scaffold strand through molecular binding. This approach prevents the assembly with Cas12a, effectively masking its trans-cleavage activity. By introducing small molecules that competitively bind to the macromolecule, we eliminate this spatial hindrance, activating Cas12a. Our results demonstrate high sensitivity, versatility, and adaptability in small molecule detection across multiple reactions, with successful intracellular imaging and responsive fluctuations in complex environments underscoring the system’s robustness. This innovative CRISPR-Cas12a-based approach establishes a low-background, highly sensitive platform for small molecule detection. SBS-Cas promises not only to enhance tools for clinical, environmental, and food safety applications but also to advance CRISPR research, providing insights and expanding possibilities in molecular detection science.

Suggested Citation

  • Hao Hu & Songcheng Guo & Yiyuan Li & Kejun Dong & Yan Lu & Keyi Ye & Longjie Li & Xiaoyu Zhou & Liming Cheng & Xianjin Xiao, 2025. "Spatially blocked split CRISPR-Cas12a system for ultra-sensitive and versatile small molecule activation and detection," Nature Communications, Nature, vol. 16(1), pages 1-14, December.
    • Handle: RePEc:nat:natcom:v:16:y:2025:i:1:d:10.1038_s41467-025-60265-8
    DOI: 10.1038/s41467-025-60265-8
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    1. Mindong Liang & Zilong Li & Weishan Wang & Jiakun Liu & Leshi Liu & Guoliang Zhu & Loganathan Karthik & Man Wang & Ke-Feng Wang & Zhong Wang & Jing Yu & Yuting Shuai & Jiaming Yu & Lu Zhang & Zhiheng , 2019. "A CRISPR-Cas12a-derived biosensing platform for the highly sensitive detection of diverse small molecules," Nature Communications, Nature, vol. 10(1), pages 1-9, December.
    2. Xiao Wang & Zhike Lu & Adrian Gomez & Gary C. Hon & Yanan Yue & Dali Han & Ye Fu & Marc Parisien & Qing Dai & Guifang Jia & Bing Ren & Tao Pan & Chuan He, 2014. "N6-methyladenosine-dependent regulation of messenger RNA stability," Nature, Nature, vol. 505(7481), pages 117-120, January.
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