Production of β-lactoglobulin (BLG) gene knock- out blastocyst stage embryos of Indian water buffalo using CRISPR and SCNT technology
In several tropical countries, buffalo milk has a higher-value demand than cow milk due to its nutritional and economic value. In India, the buffalo is the main dairy animal and contributes 45% of the total milk produced in the country. Besides the nutritional value of milk, several allergen protei...
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Universidad del Zulia
2023-11-01
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| Series: | Revista Científica |
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| Online Access: | https://mail.produccioncientificaluz.org/index.php/cientifica/article/view/43492 |
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| author | Aseem Tara Priyanka Singh Devika Gautam Gaurav Tripathi Shreya Malhotra Sacchinandan De Manoj K. Singh Naresh L. Selokar |
| author_facet | Aseem Tara Priyanka Singh Devika Gautam Gaurav Tripathi Shreya Malhotra Sacchinandan De Manoj K. Singh Naresh L. Selokar |
| author_sort | Aseem Tara |
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In several tropical countries, buffalo milk has a higher-value demand than cow milk due to its nutritional and economic value. In India, the buffalo is the main dairy animal and contributes 45% of the total milk produced in the country. Besides the nutritional value of milk, several allergen proteins such as casein, α-lactalbumin, β- lactoglobulin (BLG), and immunoglobulins have been reported. Breeding strategies, nutritional management, and quantitative genetics have improved milk yield, but these approaches could not lead to significant changes in milk composition. With the development of biotechnology, especially genome editing tools (CRISPRs), it is possible to generate new value-added products such as designer hypoallergenic milk for human health benefits. Keeping this in mind, we planned to utilize the CRISPR tools to disrupt the buffalo β-lactoglobulin (BLG) gene to produce hypoallergenic milk in the long run. In pursuit of our objectives, we designed three single guide RNAs (sgRNAs) targeting the BLG locus in buffalo. Subsequently, we assessed their editing efficiency through a combination of Sanger sequencing, followed by TIDE and ICE analysis. Among three sgRNAs, the most efficient sgRNA was used to generate the clonal population of edited cells. Several single-cell clones were established and screened using the TA cloning (also known as rapid cloning or T cloning) and Sanger sequencing methods. Of 14 single-cell clones screened, eight were found to have BLG gene disruption events (57% editing rates). Using SCNT, we successfully produced cloned blastocyst stage embryos from 4 BLG-gene disrupted clonal cells. The cloned blastocyst production rates (25 to 30%) were similar to non-edited control cells. Efforts are ongoing to establish pregnancies from BLG-KO cloned embryos. This work can lead to the generation of designer buffaloes to produce hypoallergenic milk for human benefit.
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| format | Article |
| id | doaj-art-55aa8de364c04449bb14177498cfe97a |
| institution | Kabale University |
| issn | 0798-2259 2521-9715 |
| language | English |
| publishDate | 2023-11-01 |
| publisher | Universidad del Zulia |
| record_format | Article |
| series | Revista Científica |
| spelling | doaj-art-55aa8de364c04449bb14177498cfe97a2025-02-06T15:37:07ZengUniversidad del ZuliaRevista Científica0798-22592521-97152023-11-0133Suplemento10.52973/rcfcv-wbc124Production of β-lactoglobulin (BLG) gene knock- out blastocyst stage embryos of Indian water buffalo using CRISPR and SCNT technologyAseem Tara0Priyanka Singh1Devika Gautam2Gaurav Tripathi3Shreya Malhotra4Sacchinandan De5Manoj K. Singh6Naresh L. Selokar7Animal Biotechnology Division (ABTD), ICAR-National Dairy Research Institute, Karnal, Haryana, 132001, IndiaAnimal Biotechnology Division (ABTD), ICAR-National Dairy Research Institute, Karnal, Haryana, 132001, IndiaAnimal Biotechnology Division (ABTD), ICAR-National Dairy Research Institute, Karnal, Haryana, 132001, IndiaAnimal Biotechnology Division (ABTD), ICAR-National Dairy Research Institute, Karnal, Haryana, 132001, IndiaAnimal Biotechnology Division (ABTD), ICAR-National Dairy Research Institute, Karnal, Haryana, 132001, IndiaAnimal Biotechnology Division (ABTD), ICAR-National Dairy Research Institute, Karnal, Haryana, 132001, IndiaAnimal Biotechnology Division (ABTD), ICAR-National Dairy Research Institute, Karnal, Haryana, 132001, IndiaAnimal Biotechnology Division (ABTD), ICAR-National Dairy Research Institute, Karnal, Haryana, 132001, India In several tropical countries, buffalo milk has a higher-value demand than cow milk due to its nutritional and economic value. In India, the buffalo is the main dairy animal and contributes 45% of the total milk produced in the country. Besides the nutritional value of milk, several allergen proteins such as casein, α-lactalbumin, β- lactoglobulin (BLG), and immunoglobulins have been reported. Breeding strategies, nutritional management, and quantitative genetics have improved milk yield, but these approaches could not lead to significant changes in milk composition. With the development of biotechnology, especially genome editing tools (CRISPRs), it is possible to generate new value-added products such as designer hypoallergenic milk for human health benefits. Keeping this in mind, we planned to utilize the CRISPR tools to disrupt the buffalo β-lactoglobulin (BLG) gene to produce hypoallergenic milk in the long run. In pursuit of our objectives, we designed three single guide RNAs (sgRNAs) targeting the BLG locus in buffalo. Subsequently, we assessed their editing efficiency through a combination of Sanger sequencing, followed by TIDE and ICE analysis. Among three sgRNAs, the most efficient sgRNA was used to generate the clonal population of edited cells. Several single-cell clones were established and screened using the TA cloning (also known as rapid cloning or T cloning) and Sanger sequencing methods. Of 14 single-cell clones screened, eight were found to have BLG gene disruption events (57% editing rates). Using SCNT, we successfully produced cloned blastocyst stage embryos from 4 BLG-gene disrupted clonal cells. The cloned blastocyst production rates (25 to 30%) were similar to non-edited control cells. Efforts are ongoing to establish pregnancies from BLG-KO cloned embryos. This work can lead to the generation of designer buffaloes to produce hypoallergenic milk for human benefit. https://mail.produccioncientificaluz.org/index.php/cientifica/article/view/43492buffalomilk allergyβ-lactoglobulinCRISPRSCNThypoallergenic milk |
| spellingShingle | Aseem Tara Priyanka Singh Devika Gautam Gaurav Tripathi Shreya Malhotra Sacchinandan De Manoj K. Singh Naresh L. Selokar Production of β-lactoglobulin (BLG) gene knock- out blastocyst stage embryos of Indian water buffalo using CRISPR and SCNT technology Revista Científica buffalo milk allergy β-lactoglobulin CRISPR SCNT hypoallergenic milk |
| title | Production of β-lactoglobulin (BLG) gene knock- out blastocyst stage embryos of Indian water buffalo using CRISPR and SCNT technology |
| title_full | Production of β-lactoglobulin (BLG) gene knock- out blastocyst stage embryos of Indian water buffalo using CRISPR and SCNT technology |
| title_fullStr | Production of β-lactoglobulin (BLG) gene knock- out blastocyst stage embryos of Indian water buffalo using CRISPR and SCNT technology |
| title_full_unstemmed | Production of β-lactoglobulin (BLG) gene knock- out blastocyst stage embryos of Indian water buffalo using CRISPR and SCNT technology |
| title_short | Production of β-lactoglobulin (BLG) gene knock- out blastocyst stage embryos of Indian water buffalo using CRISPR and SCNT technology |
| title_sort | production of β lactoglobulin blg gene knock out blastocyst stage embryos of indian water buffalo using crispr and scnt technology |
| topic | buffalo milk allergy β-lactoglobulin CRISPR SCNT hypoallergenic milk |
| url | https://mail.produccioncientificaluz.org/index.php/cientifica/article/view/43492 |
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