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

Hao Hu; Songcheng Guo; Yiyuan Li; Kejun Dong; Yan Lu; Keyi Ye; Longjie Li; Xiaoyu Zhou; Liming Cheng; Xianjin Xiao

doi:10.1038/s41467-025-60265-8

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

Hao Hu (Co-first Author)
, Songcheng Guo (Co-first Author)
, Yiyuan Li (Co-first Author)
, Kejun Dong
, Yan Lu
, Keyi Ye
, Longjie Li^*
, Xiaoyu Zhou^*
, Liming Cheng^*
, Xianjin Xiao^*

^*Corresponding author for this work

Research output: Journal Publications and Reviews › RGC 21 - Publication in refereed journal › peer-review

43 Citations (Scopus)

31 Downloads (CityUHK Scholars)

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. © The Author(s) 2025

Original language	English
Article number	5035
Journal	Nature Communications
Volume	16
DOIs	https://doi.org/10.1038/s41467-025-60265-8
Publication status	Published - 30 May 2025

Bibliographical note

© 2025. The Author(s).
Information for this record is supplemented by the author(s) concerned.

Funding

X.X. was supported by the National Key Research and Development Program of China [2023YFE0210200] and interdisciplinary research program of HUST. L.L. was supported by the Natural Science Foundation of Wuhan City (Chenguang Project) [2024040801020331]. X.Z. was supported by the central government-guided special funds for local scientific and technological development (no. 226Z2603G), and the Hong Kong Innovation and Technology Fund, the Mainland Hong Kong Joint Funding Scheme (Platform) (No. MHP/240/23). We are grateful to Biorender (https://app.biorender.com/) for offering the online illustration platform.

Research Keywords

CRISPR-Cas Systems/genetics
Endodeoxyribonucleases/metabolism
Humans
CRISPR-Associated Proteins/metabolism
Bacterial Proteins/metabolism

Publisher's Copyright Statement

This full text is made available under CC-BY-NC-ND 4.0. https://creativecommons.org/licenses/by-nc-nd/4.0/

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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. {\textcopyright} The Author(s) 2025",

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Spatially blocked split CRISPR-Cas12a system for ultra-sensitive and versatile small molecule activation and detection. / Hu, Hao (Co-first Author); Guo, Songcheng (Co-first Author); Li, Yiyuan (Co-first Author) et al.
In: Nature Communications, Vol. 16, 5035, 30.05.2025.

Research output: Journal Publications and Reviews › RGC 21 - Publication in refereed journal › peer-review

TY - JOUR

T1 - Spatially blocked split CRISPR-Cas12a system for ultra-sensitive and versatile small molecule activation and detection

AU - Hu, Hao

AU - Guo, Songcheng

AU - Li, Yiyuan

AU - Dong, Kejun

AU - Lu, Yan

AU - Ye, Keyi

AU - Li, Longjie

AU - Zhou, Xiaoyu

AU - Cheng, Liming

AU - Xiao, Xianjin

PY - 2025/5/30

Y1 - 2025/5/30

N2 - 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. © The Author(s) 2025

AB - 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. © The Author(s) 2025

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KW - Endodeoxyribonucleases/metabolism

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

Abstract

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Funding

Research Keywords

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