{"title":"影响通用光控CRISPR诊断中Cas12a crRNA活性的关键核苷酸的鉴定","authors":"Tian Tian, Hongrui Xiao, Xinyi Guo, Yuxin Chen, Zhiqiang Qiu, Ting Zhang, Meiyu Chen, Weiwei Qi, Peige Cai, Meng Cheng, Xiaoming Zhou","doi":"10.1038/s41467-025-62082-5","DOIUrl":null,"url":null,"abstract":"<p>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.</p>","PeriodicalId":19066,"journal":{"name":"Nature Communications","volume":"4 1","pages":""},"PeriodicalIF":15.7000,"publicationDate":"2025-07-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Identification of a key nucleotide influencing Cas12a crRNA activity for universal photo-controlled CRISPR diagnostics\",\"authors\":\"Tian Tian, Hongrui Xiao, Xinyi Guo, Yuxin Chen, Zhiqiang Qiu, Ting Zhang, Meiyu Chen, Weiwei Qi, Peige Cai, Meng Cheng, Xiaoming Zhou\",\"doi\":\"10.1038/s41467-025-62082-5\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p>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.</p>\",\"PeriodicalId\":19066,\"journal\":{\"name\":\"Nature Communications\",\"volume\":\"4 1\",\"pages\":\"\"},\"PeriodicalIF\":15.7000,\"publicationDate\":\"2025-07-21\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Nature Communications\",\"FirstCategoryId\":\"103\",\"ListUrlMain\":\"https://doi.org/10.1038/s41467-025-62082-5\",\"RegionNum\":1,\"RegionCategory\":\"综合性期刊\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"MULTIDISCIPLINARY SCIENCES\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Nature Communications","FirstCategoryId":"103","ListUrlMain":"https://doi.org/10.1038/s41467-025-62082-5","RegionNum":1,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"MULTIDISCIPLINARY SCIENCES","Score":null,"Total":0}
Identification of a key nucleotide influencing Cas12a crRNA activity for universal photo-controlled CRISPR diagnostics
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.
期刊介绍:
Nature Communications, an open-access journal, publishes high-quality research spanning all areas of the natural sciences. Papers featured in the journal showcase significant advances relevant to specialists in each respective field. With a 2-year impact factor of 16.6 (2022) and a median time of 8 days from submission to the first editorial decision, Nature Communications is committed to rapid dissemination of research findings. As a multidisciplinary journal, it welcomes contributions from biological, health, physical, chemical, Earth, social, mathematical, applied, and engineering sciences, aiming to highlight important breakthroughs within each domain.