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

Tian Tian (), Hongrui Xiao, Xinyi Guo, Yuxin Chen, Zhiqiang Qiu, Ting Zhang, Meiyu Chen, Weiwei Qi, Peige Cai, Meng Cheng and Xiaoming Zhou ()
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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

Nature Communications, 2025, vol. 16, issue 1, 1-12

Abstract: 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.

Date: 2025
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DOI: 10.1038/s41467-025-62082-5

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