Rapid Genotyping of <i>FecB</i> Mutation in Sheep Using CRISPR-Cas12a Integrated with DNA Nanotree Biosensing Platform
The A-to-G mutation (<i>FecB</i>) in the <i>BMPR1B</i> gene is strongly linked to fertility in sheep, significantly increasing ovulation rates and litter sizes compared to wild-type populations. The rapid and reliable screening of the <i>FecB</i> gene is therefore...
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MDPI AG
2025-02-01
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| Series: | Biomolecules |
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| Online Access: | https://www.mdpi.com/2218-273X/15/3/315 |
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| author | Dongxia Pan Mukaddas Mijit Hui Wang Chaoqun Sun Bantan Pingcuo Zhixue Yu Benhai Xiong Xiangfang Tang |
| author_facet | Dongxia Pan Mukaddas Mijit Hui Wang Chaoqun Sun Bantan Pingcuo Zhixue Yu Benhai Xiong Xiangfang Tang |
| author_sort | Dongxia Pan |
| collection | DOAJ |
| description | The A-to-G mutation (<i>FecB</i>) in the <i>BMPR1B</i> gene is strongly linked to fertility in sheep, significantly increasing ovulation rates and litter sizes compared to wild-type populations. The rapid and reliable screening of the <i>FecB</i> gene is therefore critical for advancing sheep breeding programs. This study aimed to develop a fast and accurate method for detecting the <i>FecB</i> mutation and genotyping the gene to enhance sheep reproduction and productivity. To achieve this, we integrated the CRISPR-Cas12a system with an optimized amplification refractory mutation system (ARMS). A similar DNA origami technique-based fluorescence reporter nanotree structure was synthesized using gold nanomagnetic beads as carriers to amplify the fluorescence signal further. The resulting biosensing platform, termed CRISPR-ARMS, demonstrated excellent sensitivity for detecting <i>FecB</i> mutations, with a detection limit as low as 0.02 pmol. Therefore, this innovative approach shows great promise for single-base mutation detection and represents a pioneering tool for high-yield genetic screening. |
| format | Article |
| id | doaj-art-cce7b1d9d57a4f5f96817dbf32fc8641 |
| institution | Kabale University |
| issn | 2218-273X |
| language | English |
| publishDate | 2025-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biomolecules |
| spelling | doaj-art-cce7b1d9d57a4f5f96817dbf32fc86412025-08-20T03:43:26ZengMDPI AGBiomolecules2218-273X2025-02-0115331510.3390/biom15030315Rapid Genotyping of <i>FecB</i> Mutation in Sheep Using CRISPR-Cas12a Integrated with DNA Nanotree Biosensing PlatformDongxia Pan0Mukaddas Mijit1Hui Wang2Chaoqun Sun3Bantan Pingcuo4Zhixue Yu5Benhai Xiong6Xiangfang Tang7State Key Laboratory of Animal Nutrition and Feeding, Institute of Animal Science of CAAS, Beijing 100193, ChinaState Key Laboratory of Animal Nutrition and Feeding, Institute of Animal Science of CAAS, Beijing 100193, ChinaState Key Laboratory of Animal Nutrition and Feeding, Institute of Animal Science of CAAS, Beijing 100193, ChinaState Key Laboratory of Animal Nutrition and Feeding, Institute of Animal Science of CAAS, Beijing 100193, ChinaInstitute of Animal Husbandry and Veterinary, Tibet Autonomous Regional Academy of Agricultural Sciences, Lhasa 850004, ChinaState Key Laboratory of Animal Nutrition and Feeding, Institute of Animal Science of CAAS, Beijing 100193, ChinaState Key Laboratory of Animal Nutrition and Feeding, Institute of Animal Science of CAAS, Beijing 100193, ChinaState Key Laboratory of Animal Nutrition and Feeding, Institute of Animal Science of CAAS, Beijing 100193, ChinaThe A-to-G mutation (<i>FecB</i>) in the <i>BMPR1B</i> gene is strongly linked to fertility in sheep, significantly increasing ovulation rates and litter sizes compared to wild-type populations. The rapid and reliable screening of the <i>FecB</i> gene is therefore critical for advancing sheep breeding programs. This study aimed to develop a fast and accurate method for detecting the <i>FecB</i> mutation and genotyping the gene to enhance sheep reproduction and productivity. To achieve this, we integrated the CRISPR-Cas12a system with an optimized amplification refractory mutation system (ARMS). A similar DNA origami technique-based fluorescence reporter nanotree structure was synthesized using gold nanomagnetic beads as carriers to amplify the fluorescence signal further. The resulting biosensing platform, termed CRISPR-ARMS, demonstrated excellent sensitivity for detecting <i>FecB</i> mutations, with a detection limit as low as 0.02 pmol. Therefore, this innovative approach shows great promise for single-base mutation detection and represents a pioneering tool for high-yield genetic screening.https://www.mdpi.com/2218-273X/15/3/315CRISPR-Cas12aamplification retarded mutation system<i>FecB</i>Fe<sub>3</sub>O<sub>4</sub>@Au-Linker-Cy5-Nanotreerapid detection |
| spellingShingle | Dongxia Pan Mukaddas Mijit Hui Wang Chaoqun Sun Bantan Pingcuo Zhixue Yu Benhai Xiong Xiangfang Tang Rapid Genotyping of <i>FecB</i> Mutation in Sheep Using CRISPR-Cas12a Integrated with DNA Nanotree Biosensing Platform Biomolecules CRISPR-Cas12a amplification retarded mutation system <i>FecB</i> Fe<sub>3</sub>O<sub>4</sub>@Au-Linker-Cy5-Nanotree rapid detection |
| title | Rapid Genotyping of <i>FecB</i> Mutation in Sheep Using CRISPR-Cas12a Integrated with DNA Nanotree Biosensing Platform |
| title_full | Rapid Genotyping of <i>FecB</i> Mutation in Sheep Using CRISPR-Cas12a Integrated with DNA Nanotree Biosensing Platform |
| title_fullStr | Rapid Genotyping of <i>FecB</i> Mutation in Sheep Using CRISPR-Cas12a Integrated with DNA Nanotree Biosensing Platform |
| title_full_unstemmed | Rapid Genotyping of <i>FecB</i> Mutation in Sheep Using CRISPR-Cas12a Integrated with DNA Nanotree Biosensing Platform |
| title_short | Rapid Genotyping of <i>FecB</i> Mutation in Sheep Using CRISPR-Cas12a Integrated with DNA Nanotree Biosensing Platform |
| title_sort | rapid genotyping of i fecb i mutation in sheep using crispr cas12a integrated with dna nanotree biosensing platform |
| topic | CRISPR-Cas12a amplification retarded mutation system <i>FecB</i> Fe<sub>3</sub>O<sub>4</sub>@Au-Linker-Cy5-Nanotree rapid detection |
| url | https://www.mdpi.com/2218-273X/15/3/315 |
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