Regulating cleavage activity and enabling microRNA detection with split sgRNA in Cas12b
Abstract The CRISPR-Cas12b system has revolutionized molecular diagnostics, yet its reliance on single guide RNAs (sgRNAs) exceeding 100 nt limits precise regulation and applications. We present a split sgRNA strategy for Cas12b, utilizing universal components with customizable Spacer to detect vari...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2025-07-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-025-61748-4 |
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| author | Jiaqi Wang Xiaofang Ye Yuanfang Liu Wentao Li Xue Zhang Wei Zhang Changqing Yi Chaoxing Liu |
| author_facet | Jiaqi Wang Xiaofang Ye Yuanfang Liu Wentao Li Xue Zhang Wei Zhang Changqing Yi Chaoxing Liu |
| author_sort | Jiaqi Wang |
| collection | DOAJ |
| description | Abstract The CRISPR-Cas12b system has revolutionized molecular diagnostics, yet its reliance on single guide RNAs (sgRNAs) exceeding 100 nt limits precise regulation and applications. We present a split sgRNA strategy for Cas12b, utilizing universal components with customizable Spacer to detect various nucleic acid targets by simply replacing Spacer. Glyoxal labeling of the universal split direct repeat (DR) region represses Cas12b activity, which is restored by elevated temperatures or prolonged incubation, enabling dynamic regulation. Incorporating a photo-cleavable linker into the DR allows UV-mediated modulation, ensuring compatibility with recombinase polymerase amplification. Successful detection of Epstein-Barr virus in clinical plasma samples matched the sensitivity of traditional qPCR. Importantly, microRNAs can replace the Spacer, enabling direct detection without reverse transcription or amplification. Supported by evidence from cultured cells and plasma from healthy individuals and colorectal cancer patients, this method yields consistent results with RT-qPCR while simplifying protocols. This split strategy enhances Cas12b systems, offering a promising approach for clinical nucleic acid analysis. |
| format | Article |
| id | doaj-art-b35756d8e46a41149d90a47f4e6195a5 |
| institution | DOAJ |
| issn | 2041-1723 |
| language | English |
| publishDate | 2025-07-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj-art-b35756d8e46a41149d90a47f4e6195a52025-08-20T03:05:10ZengNature PortfolioNature Communications2041-17232025-07-0116111410.1038/s41467-025-61748-4Regulating cleavage activity and enabling microRNA detection with split sgRNA in Cas12bJiaqi Wang0Xiaofang Ye1Yuanfang Liu2Wentao Li3Xue Zhang4Wei Zhang5Changqing Yi6Chaoxing Liu7Guangdong Provincial Key Laboratory of Digestive Cancer Research, Digestive Diseases Center, Scientific Research Center, The Seventh Affiliated Hospital of Sun Yat-sen University, ShenzhenGuangdong Provincial Key Laboratory of Digestive Cancer Research, Digestive Diseases Center, Scientific Research Center, The Seventh Affiliated Hospital of Sun Yat-sen University, ShenzhenGuangdong Provincial Key Laboratory of Digestive Cancer Research, Digestive Diseases Center, Scientific Research Center, The Seventh Affiliated Hospital of Sun Yat-sen University, ShenzhenDepartment of Clinical Laboratory, The Seventh Affiliated Hospital of Sun Yat-sen University, ShenzhenGuangdong Provincial Key Laboratory of Digestive Cancer Research, Digestive Diseases Center, Scientific Research Center, The Seventh Affiliated Hospital of Sun Yat-sen University, ShenzhenGuangdong Provincial Key Laboratory of Digestive Cancer Research, Digestive Diseases Center, Scientific Research Center, The Seventh Affiliated Hospital of Sun Yat-sen University, ShenzhenGuangdong Provincial Key Laboratory of Sensor Technology and Biomedical Instrument, School of Biomedical Engineering, Shenzhen Campus of Sun Yat-Sen University, ShenzhenGuangdong Provincial Key Laboratory of Digestive Cancer Research, Digestive Diseases Center, Scientific Research Center, The Seventh Affiliated Hospital of Sun Yat-sen University, ShenzhenAbstract The CRISPR-Cas12b system has revolutionized molecular diagnostics, yet its reliance on single guide RNAs (sgRNAs) exceeding 100 nt limits precise regulation and applications. We present a split sgRNA strategy for Cas12b, utilizing universal components with customizable Spacer to detect various nucleic acid targets by simply replacing Spacer. Glyoxal labeling of the universal split direct repeat (DR) region represses Cas12b activity, which is restored by elevated temperatures or prolonged incubation, enabling dynamic regulation. Incorporating a photo-cleavable linker into the DR allows UV-mediated modulation, ensuring compatibility with recombinase polymerase amplification. Successful detection of Epstein-Barr virus in clinical plasma samples matched the sensitivity of traditional qPCR. Importantly, microRNAs can replace the Spacer, enabling direct detection without reverse transcription or amplification. Supported by evidence from cultured cells and plasma from healthy individuals and colorectal cancer patients, this method yields consistent results with RT-qPCR while simplifying protocols. This split strategy enhances Cas12b systems, offering a promising approach for clinical nucleic acid analysis.https://doi.org/10.1038/s41467-025-61748-4 |
| spellingShingle | Jiaqi Wang Xiaofang Ye Yuanfang Liu Wentao Li Xue Zhang Wei Zhang Changqing Yi Chaoxing Liu Regulating cleavage activity and enabling microRNA detection with split sgRNA in Cas12b Nature Communications |
| title | Regulating cleavage activity and enabling microRNA detection with split sgRNA in Cas12b |
| title_full | Regulating cleavage activity and enabling microRNA detection with split sgRNA in Cas12b |
| title_fullStr | Regulating cleavage activity and enabling microRNA detection with split sgRNA in Cas12b |
| title_full_unstemmed | Regulating cleavage activity and enabling microRNA detection with split sgRNA in Cas12b |
| title_short | Regulating cleavage activity and enabling microRNA detection with split sgRNA in Cas12b |
| title_sort | regulating cleavage activity and enabling microrna detection with split sgrna in cas12b |
| url | https://doi.org/10.1038/s41467-025-61748-4 |
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