IDEAS home Printed from https://ideas.repec.org/a/nat/natcom/v16y2025i1d10.1038_s41467-025-62082-5.html
   My bibliography  Save this article

Identification of a key nucleotide influencing Cas12a crRNA activity for universal photo-controlled CRISPR diagnostics

Author

Listed:
  • Tian Tian

    (South China Normal University)

  • Hongrui Xiao

    (South China Normal University)

  • Xinyi Guo

    (South China Normal University)

  • Yuxin Chen

    (South China Normal University)

  • Zhiqiang Qiu

    (South China Normal University)

  • Ting Zhang

    (South China Normal University)

  • Meiyu Chen

    (South China Normal University)

  • Weiwei Qi

    (South China Normal University)

  • Peige Cai

    (South China Normal University)

  • Meng Cheng

    (The First Affiliated Hospital of Guangzhou Medical University)

  • Xiaoming Zhou

    (South China Normal University
    South China Normal University)

Abstract

Developing a one-pot assay is a critical strategy for enhancing the applicability of CRISPR-based molecular diagnostics; however, it is hindered by CRISPR cleavage interfering with nucleic acid amplification templates. Photo-regulation strategies provide an ideal solution to suppress undesired CRISPR cleavage while maintaining detection efficiency. However, existing photo-controlled CRISPR diagnostic methods face limitations in universality, cost, and detection efficiency. In this study, we systematically examine the impact of mutations in the repeat recognition sequence (RRS), a four-nucleotide segment within the Cas12a crRNA direct repeat (DR) region, on cleavage activity. We observe that mutations at positions 3 or 4 nearly abolished crRNA activity. Based on this discovery, we introduce 6-nitropiperonyloxymethyl (NPOM) photo-caging modifications at positions 3 and 4. Photo-caging at position 4 demonstrates the most effective suppression of enzymatic activity and optimal light-mediated activation. We leverage this finding to develop a photo-controlled CRISPR diagnostic method, enabling a universally adaptable one-pot detection strategy. Furthermore, by incorporating a crRNA splinting strategy, this pre-preparable reagent can be adapted for the detection of virtually any target gene.

Suggested Citation

  • Tian Tian & Hongrui Xiao & Xinyi Guo & Yuxin Chen & Zhiqiang Qiu & Ting Zhang & Meiyu Chen & Weiwei Qi & Peige Cai & Meng Cheng & Xiaoming Zhou, 2025. "Identification of a key nucleotide influencing Cas12a crRNA activity for universal photo-controlled CRISPR diagnostics," Nature Communications, Nature, vol. 16(1), pages 1-12, December.
    • Handle: RePEc:nat:natcom:v:16:y:2025:i:1:d:10.1038_s41467-025-62082-5
    DOI: 10.1038/s41467-025-62082-5
    as

    Download full text from publisher

    File URL: https://www.nature.com/articles/s41467-025-62082-5
    File Function: Abstract
    Download Restriction: no
    File URL: https://libkey.io/10.1038/s41467-025-62082-5?utm_source=ideas
    LibKey link: if access is restricted and if your library uses this service, LibKey will redirect you to where you can use your library subscription to access this item
    ---><---

    References listed on IDEAS

    as
    1. Santosh R. Rananaware & Emma K. Vesco & Grace M. Shoemaker & Swapnil S. Anekar & Luke Samuel W. Sandoval & Katelyn S. Meister & Nicolas C. Macaluso & Long T. Nguyen & Piyush K. Jain, 2023. "Programmable RNA detection with CRISPR-Cas12a," Nature Communications, Nature, vol. 14(1), pages 1-14, December.
    2. Yichuan Chen & Xinping Wang & Junqi Zhang & Qingyuan Jiang & Bin Qiao & Baoxia He & Wenhao Yin & Jie Qiao & Yi Liu, 2024. "Split crRNA with CRISPR-Cas12a enabling highly sensitive and multiplexed detection of RNA and DNA," Nature Communications, Nature, vol. 15(1), pages 1-12, December.
    3. Ines Fonfara & Hagen Richter & Majda Bratovič & Anaïs Le Rhun & Emmanuelle Charpentier, 2016. "The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA," Nature, Nature, vol. 532(7600), pages 517-521, April.
    4. Jared Carlson-Stevermer & Reed Kelso & Anastasia Kadina & Sahil Joshi & Nicholas Rossi & John Walker & Rich Stoner & Travis Maures, 2020. "CRISPRoff enables spatio-temporal control of CRISPR editing," Nature Communications, Nature, vol. 11(1), pages 1-7, December.
    5. Zhou-Hua Cheng & Xi-Yan Luo & Sheng-Song Yu & Di Min & Shu-Xia Zhang & Xiao-Fan Li & Jie-Jie Chen & Dong-Feng Liu & Han-Qing Yu, 2025. "Tunable control of Cas12 activity promotes universal and fast one-pot nucleic acid detection," Nature Communications, Nature, vol. 16(1), pages 1-16, December.
    Full references (including those not matched with items on IDEAS)

    Most related items

    These are the items that most often cite the same works as this one and are cited by the same works as this one.
    1. Jing Han & Yuan Min & Lan Hu & Jie-jie Chen & Shu-Xia Zhang & Xiao-Fan Li & Zhou-Hua Cheng & Dong-Feng Liu & Han-Qing Yu, 2025. "Tailoring Cas12a functionality with a user-friendly and versatile crRNA variant toolbox," Nature Communications, Nature, vol. 16(1), pages 1-15, December.
    2. Jiaqi Wang & Xiaofang Ye & Yuanfang Liu & Wentao Li & Xue Zhang & Wei Zhang & Changqing Yi & Chaoxing Liu, 2025. "Regulating cleavage activity and enabling microRNA detection with split sgRNA in Cas12b," Nature Communications, Nature, vol. 16(1), pages 1-14, December.
    3. Margot Karlikow & Evan Amalfitano & Xiaolong Yang & Jennifer Doucet & Abigail Chapman & Peivand Sadat Mousavi & Paige Homme & Polina Sutyrina & Winston Chan & Sofia Lemak & Alexander F. Yakunin & Adam, 2023. "CRISPR-induced DNA reorganization for multiplexed nucleic acid detection," Nature Communications, Nature, vol. 14(1), pages 1-11, December.
    4. Huiyou Chen & Fengge Song & Buhua Wang & Hui Huang & Yanchi Luo & Xiaosheng Han & Hewen He & Shaolu Lin & Liudang Wan & Zhengliang Huang & Zhaoyong Fu & Rodrigo Ledesma-Amaro & Dapeng Yin & Haimei Mao, 2025. "Ultrasensitive detection of clinical pathogens through a target-amplification-free collateral-cleavage-enhancing CRISPR-CasΦ tool," Nature Communications, Nature, vol. 16(1), pages 1-14, December.
    5. Jianli Tao & Daniel E. Bauer & Roberto Chiarle, 2023. "Assessing and advancing the safety of CRISPR-Cas tools: from DNA to RNA editing," Nature Communications, Nature, vol. 14(1), pages 1-16, December.
    6. Yichuan Chen & Xinping Wang & Junqi Zhang & Qingyuan Jiang & Bin Qiao & Baoxia He & Wenhao Yin & Jie Qiao & Yi Liu, 2024. "Split crRNA with CRISPR-Cas12a enabling highly sensitive and multiplexed detection of RNA and DNA," Nature Communications, Nature, vol. 15(1), pages 1-12, December.
    7. Peng Chen & Yankang Wu & Hongjian Wang & Huan Liu & Jin Zhou & Jingli Chen & Jun Lei & Zaiqiao Sun & Chonil Paek & Lei Yin, 2025. "Highly parallel profiling of the activities and specificities of Cas12a variants in human cells," Nature Communications, Nature, vol. 16(1), pages 1-13, December.
    8. Mei Lin & Zhiqiang Qiu & Mengen Hao & Weiwei Qi & Ting Zhang & Yuting Shen & Hongrui Xiao & Chaoyue Liang & Longxu Xie & Yongzhong Jiang & Meng Cheng & Tian Tian & Xiaoming Zhou, 2025. "Cas12a Cis-cleavage mediated lateral flow assay enables multiplex and ultra-specific nucleic acid detection," Nature Communications, Nature, vol. 16(1), pages 1-14, December.
    9. Ghanem El Kassem & Jasmine Hillmer & Michael Boettcher, 2025. "Evaluation of Cas13d as a tool for genetic interaction mapping," Nature Communications, Nature, vol. 16(1), pages 1-14, December.
    10. Shunsuke Kawasaki & Hiroki Ono & Moe Hirosawa & Takeru Kuwabara & Shunsuke Sumi & Suji Lee & Knut Woltjen & Hirohide Saito, 2023. "Programmable mammalian translational modulators by CRISPR-associated proteins," Nature Communications, Nature, vol. 14(1), pages 1-17, December.
    11. Pei Liu & Josquin Foiret & Yinglin Situ & Nisi Zhang & Aris J. Kare & Bo Wu & Marina N. Raie & Katherine W. Ferrara & Lei S. Qi, 2023. "Sonogenetic control of multiplexed genome regulation and base editing," Nature Communications, Nature, vol. 14(1), pages 1-11, December.
    12. Qichen Yuan & Xue Gao, 2022. "Multiplex base- and prime-editing with drive-and-process CRISPR arrays," Nature Communications, Nature, vol. 13(1), pages 1-13, December.
    13. Menglu Hu & Bingni Zhang & Yuanyue Shan & Feng Cao & Yihui Wang & Weiwei Qi & Xue Wang & Yuting Shen & Xinyi Guo & Mengmeng Zhang & Tian Tian & Wei Xie & Mingfeng Zhang & Fang Liang & Duanqing Pei & X, 2025. "Scalable modulation of CRISPR‒Cas enzyme activity using photocleavable phosphorothioate DNA," Nature Communications, Nature, vol. 16(1), pages 1-17, December.
    14. Santosh R. Rananaware & Emma K. Vesco & Grace M. Shoemaker & Swapnil S. Anekar & Luke Samuel W. Sandoval & Katelyn S. Meister & Nicolas C. Macaluso & Long T. Nguyen & Piyush K. Jain, 2023. "Programmable RNA detection with CRISPR-Cas12a," Nature Communications, Nature, vol. 14(1), pages 1-14, December.
    15. Wenhui Li & Xianyue Jiang & Wuke Wang & Liya Hou & Runze Cai & Yongqian Li & Qiuxi Gu & Qinchang Chen & Peixiang Ma & Jin Tang & Menghao Guo & Guohui Chuai & Xingxu Huang & Jun Zhang & Qi Liu, 2024. "Discovering CRISPR-Cas system with self-processing pre-crRNA capability by foundation models," Nature Communications, Nature, vol. 15(1), pages 1-14, December.
    16. Kevin D. P. Aris & Joshua C. Cofsky & Honglue Shi & Noor Al-Sayyad & Ivan E. Ivanov & Ashwin Balaji & Jennifer A. Doudna & Zev Bryant, 2025. "Dynamic basis of supercoiling-dependent DNA interrogation by Cas12a via R-loop intermediates," Nature Communications, Nature, vol. 16(1), pages 1-13, December.
    17. Wei Jin & Yexuan Deng & John E. La Marca & Emily J. Lelliott & Sarah T. Diepstraten & Christina König & Lin Tai & Valentina Snetkova & Kristel M. Dorighi & Luke Hoberecht & Millicent G. Hedditch & Lau, 2025. "Advancing the genetic engineering toolbox by combining AsCas12a knock-in mice with ultra-compact screening," Nature Communications, Nature, vol. 16(1), pages 1-15, December.
    18. Chunlei Jiao & Natalia L. Peeck & Jiaqi Yu & Mohammad Ghaem Maghami & Sarah Kono & Daphne Collias & Sandra L. Martinez Diaz & Rachael Larose & Chase L. Beisel, 2024. "TracrRNA reprogramming enables direct PAM-independent detection of RNA with diverse DNA-targeting Cas12 nucleases," Nature Communications, Nature, vol. 15(1), pages 1-15, December.

    More about this item

    Statistics

    Access and download statistics

    Corrections

    All material on this site has been provided by the respective publishers and authors. You can help correct errors and omissions. When requesting a correction, please mention this item's handle: RePEc:nat:natcom:v:16:y:2025:i:1:d:10.1038_s41467-025-62082-5. See general information about how to correct material in RePEc.

    If you have authored this item and are not yet registered with RePEc, we encourage you to do it here. This allows to link your profile to this item. It also allows you to accept potential citations to this item that we are uncertain about.

    If CitEc recognized a bibliographic reference but did not link an item in RePEc to it, you can help with this form .

    If you know of missing items citing this one, you can help us creating those links by adding the relevant references in the same way as above, for each refering item. If you are a registered author of this item, you may also want to check the "citations" tab in your RePEc Author Service profile, as there may be some citations waiting for confirmation.

    For technical questions regarding this item, or to correct its authors, title, abstract, bibliographic or download information, contact: Sonal Shukla or Springer Nature Abstracting and Indexing (email available below). General contact details of provider: http://www.nature.com .

    Please note that corrections may take a couple of weeks to filter through the various RePEc services.

    IDEAS is a RePEc service. RePEc uses bibliographic data supplied by the respective publishers.