Structure-Guided design of Cas12a variants improves detection of nucleic acids
CRISPR-Cas12a holds promising potential for pathogen detection. However, its performance is not optimal when combined with isothermal amplification. Hence, we engineered a mutant of LbCas12a (K595A) with reduced cis-cleavage activity, to minimize interference with isothermal amplification. Compared...
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| Format: | Article |
| Language: | English |
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Elsevier
2025-04-01
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| Series: | Cell Insight |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2772892725000021 |
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| author | Xiaohan Tong Tianle Li Kun Zhang Dongming Zhao Ying Zhang Hao Yin |
| author_facet | Xiaohan Tong Tianle Li Kun Zhang Dongming Zhao Ying Zhang Hao Yin |
| author_sort | Xiaohan Tong |
| collection | DOAJ |
| description | CRISPR-Cas12a holds promising potential for pathogen detection. However, its performance is not optimal when combined with isothermal amplification. Hence, we engineered a mutant of LbCas12a (K595A) with reduced cis-cleavage activity, to minimize interference with isothermal amplification. Compared to wild-type Cas12a, the K595A mutant exhibited a 2–3 times faster reaction speed and a 1,000–10,000 times increase in sensitivity in a one-pot reaction. We applied this mutant for detection of African Swine Fever Virus (ASFV). This K595A mutant successfully detected all 30 ASFV samples within 20 minutes. Our study suggests a universal approach to improve the performance of Cas12a for pathogen detection. |
| format | Article |
| id | doaj-art-da4a11b93e7e46ccb78e3902f2d809bb |
| institution | DOAJ |
| issn | 2772-8927 |
| language | English |
| publishDate | 2025-04-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Cell Insight |
| spelling | doaj-art-da4a11b93e7e46ccb78e3902f2d809bb2025-08-20T03:03:38ZengElsevierCell Insight2772-89272025-04-014210022810.1016/j.cellin.2025.100228Structure-Guided design of Cas12a variants improves detection of nucleic acidsXiaohan Tong0Tianle Li1Kun Zhang2Dongming Zhao3Ying Zhang4Hao Yin5Departments of Urology and Laboratory Medicine, Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan 430071, Hubei, China; State Key Laboratory of Virology, TaiKang Center for Life and Medical Sciences, TaiKang Medical School, Wuhan University, Wuhan 430071, Hubei, China; Department of Rheumatology and Immunology, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan 430071, Hubei, ChinaDepartments of Urology and Laboratory Medicine, Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan 430071, Hubei, China; State Key Laboratory of Virology, TaiKang Center for Life and Medical Sciences, TaiKang Medical School, Wuhan University, Wuhan 430071, Hubei, ChinaDepartments of Urology and Laboratory Medicine, Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan 430071, Hubei, China; State Key Laboratory of Virology, TaiKang Center for Life and Medical Sciences, TaiKang Medical School, Wuhan University, Wuhan 430071, Hubei, ChinaState Key Laboratory for Animal Disease Control and Prevention, National African Swine Fever Para-reference Laboratory, National High Containment Facilities for Animal Diseases Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, Heilongjiang, ChinaDepartments of Urology and Laboratory Medicine, Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan 430071, Hubei, China; State Key Laboratory of Virology, TaiKang Center for Life and Medical Sciences, TaiKang Medical School, Wuhan University, Wuhan 430071, Hubei, China; Department of Rheumatology and Immunology, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan 430071, Hubei, ChinaDepartments of Urology and Laboratory Medicine, Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan 430071, Hubei, China; State Key Laboratory of Virology, TaiKang Center for Life and Medical Sciences, TaiKang Medical School, Wuhan University, Wuhan 430071, Hubei, China; RNA Institute, Wuhan University, Wuhan 430071, Hubei, China; Corresponding author. Departments of Urology and Laboratory Medicine, Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan 430071, China.CRISPR-Cas12a holds promising potential for pathogen detection. However, its performance is not optimal when combined with isothermal amplification. Hence, we engineered a mutant of LbCas12a (K595A) with reduced cis-cleavage activity, to minimize interference with isothermal amplification. Compared to wild-type Cas12a, the K595A mutant exhibited a 2–3 times faster reaction speed and a 1,000–10,000 times increase in sensitivity in a one-pot reaction. We applied this mutant for detection of African Swine Fever Virus (ASFV). This K595A mutant successfully detected all 30 ASFV samples within 20 minutes. Our study suggests a universal approach to improve the performance of Cas12a for pathogen detection.http://www.sciencedirect.com/science/article/pii/S2772892725000021 |
| spellingShingle | Xiaohan Tong Tianle Li Kun Zhang Dongming Zhao Ying Zhang Hao Yin Structure-Guided design of Cas12a variants improves detection of nucleic acids Cell Insight |
| title | Structure-Guided design of Cas12a variants improves detection of nucleic acids |
| title_full | Structure-Guided design of Cas12a variants improves detection of nucleic acids |
| title_fullStr | Structure-Guided design of Cas12a variants improves detection of nucleic acids |
| title_full_unstemmed | Structure-Guided design of Cas12a variants improves detection of nucleic acids |
| title_short | Structure-Guided design of Cas12a variants improves detection of nucleic acids |
| title_sort | structure guided design of cas12a variants improves detection of nucleic acids |
| url | http://www.sciencedirect.com/science/article/pii/S2772892725000021 |
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