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, Mei‐Yu Chen, Weiwei Qi, Peige Cai, Meng Cheng, Xiaoming Zhou
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. Existing photo-controlled CRISPR diagnostic methods suffer from limited universality, high cost, and low detection efficiency. Here, the authors develop a photo-caging strategy designed for the direct repeat (DR) region of Cas12a crRNA, enabling a broadly applicable one-pot detection platform