Exploring the functional role of POU5F1 using CRISPR RNP electroporation of zygote (CRISPR-EZ) in buffaloes
POU5F1, a key transcription factor, plays a pivotal role in maintaining pluripotency and cellular differentiation, making it of immense interest in developmental biology and animal husbandry. In the present study, we employ state-of-the-art CRIS-PR ribonucleoprotein (RNP) electroporation of zygotes...
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Universidad del Zulia
2023-11-01
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| Series: | Revista Científica |
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| Online Access: | https://www.produccioncientificaluz.org/index.php/cientifica/article/view/43526 |
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| author | Meeti Punetha Dharmendra Kumar Suman Choudhary Sheetal Saini Kamlesh Kumari Bajwa Surabhi Sharma Manu Mangal P. S. Yadav T. K. Datta |
| author_facet | Meeti Punetha Dharmendra Kumar Suman Choudhary Sheetal Saini Kamlesh Kumari Bajwa Surabhi Sharma Manu Mangal P. S. Yadav T. K. Datta |
| author_sort | Meeti Punetha |
| collection | DOAJ |
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POU5F1, a key transcription factor, plays a pivotal role in maintaining pluripotency and cellular differentiation, making it of immense interest in developmental biology and animal husbandry. In the present study, we employ state-of-the-art CRIS-PR ribonucleoprotein (RNP) electroporation of zygotes (CRIS-PR-EZ) methodology to precisely manipulate the POU5F1 gene in buffalo zygotes for exploring the phenotypic and functional outcomes of embryonic development. Through a combination of strategies, we found that electroporation of buffalo zygote at 20V/mm, five pulses, 3 msec at 10 h post-insemination (hpi) resulted in greater membrane permeability and higher editing efficiency (88.71%) without affecting embryonic developmental potential. We targeted the buffalo POU5F1 gene using the abovementioned parameters, which caused nonsense-mediated mRNA decay and led to complete knockout (KO) of the POU5F1 gene. We analyzed the mutation rates and mosaic mutations using tracking of Indels by decomposition (TIDE) software. We observed no difference in the embryonic developmental competence at cleavage or blastocyst rate between control, POU5F1-KO, and electroporated control embryos. We determined the expression of SOX2, Nanog, and GATA2 in POU5F1 intact (Control) and POU5F1-KO confirmed blastocyst to better understand the impact of POU5F1-KO on other pluripotent genes. POU5F1-KO significantly (p<0.05) altered SOX2, Nanog, and GATA2 expression in blastocyst- stage embryos. In conclusion, direct electroporation of the CRISPR RNP component to the earlier stage of the zygote efficiently created mutations in the POU5F1 gene. We developed an easy and straightforward protocol for gene editing, which could serve as a valuable method for studying the functional genomics of the buffalo embryos.
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| format | Article |
| id | doaj-art-2edb3baf872a40e881668ec90f4f3d17 |
| institution | Kabale University |
| issn | 0798-2259 2521-9715 |
| language | English |
| publishDate | 2023-11-01 |
| publisher | Universidad del Zulia |
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| series | Revista Científica |
| spelling | doaj-art-2edb3baf872a40e881668ec90f4f3d172025-02-07T15:37:10ZengUniversidad del ZuliaRevista Científica0798-22592521-97152023-11-0133Suplemento10.52973/rcfcv-wbc132Exploring the functional role of POU5F1 using CRISPR RNP electroporation of zygote (CRISPR-EZ) in buffaloesMeeti Punetha0Dharmendra Kumar1Suman Choudhary2Sheetal Saini3Kamlesh Kumari Bajwa4Surabhi Sharma5Manu Mangal6P. S. Yadav7T. K. Datta8Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffaloes, Hisar 125001, Haryana, IndiaAnimal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffaloes, Hisar 125001, Haryana, IndiaAnimal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffaloes, Hisar 125001, Haryana, IndiaAnimal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffaloes, Hisar 125001, Haryana, IndiaAnimal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffaloes, Hisar 125001, Haryana, IndiaAnimal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffaloes, Hisar 125001, Haryana, IndiaAnimal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffaloes, Hisar 125001, Haryana, IndiaAnimal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffaloes, Hisar 125001, Haryana, IndiaAnimal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffaloes, Hisar 125001, Haryana, India POU5F1, a key transcription factor, plays a pivotal role in maintaining pluripotency and cellular differentiation, making it of immense interest in developmental biology and animal husbandry. In the present study, we employ state-of-the-art CRIS-PR ribonucleoprotein (RNP) electroporation of zygotes (CRIS-PR-EZ) methodology to precisely manipulate the POU5F1 gene in buffalo zygotes for exploring the phenotypic and functional outcomes of embryonic development. Through a combination of strategies, we found that electroporation of buffalo zygote at 20V/mm, five pulses, 3 msec at 10 h post-insemination (hpi) resulted in greater membrane permeability and higher editing efficiency (88.71%) without affecting embryonic developmental potential. We targeted the buffalo POU5F1 gene using the abovementioned parameters, which caused nonsense-mediated mRNA decay and led to complete knockout (KO) of the POU5F1 gene. We analyzed the mutation rates and mosaic mutations using tracking of Indels by decomposition (TIDE) software. We observed no difference in the embryonic developmental competence at cleavage or blastocyst rate between control, POU5F1-KO, and electroporated control embryos. We determined the expression of SOX2, Nanog, and GATA2 in POU5F1 intact (Control) and POU5F1-KO confirmed blastocyst to better understand the impact of POU5F1-KO on other pluripotent genes. POU5F1-KO significantly (p<0.05) altered SOX2, Nanog, and GATA2 expression in blastocyst- stage embryos. In conclusion, direct electroporation of the CRISPR RNP component to the earlier stage of the zygote efficiently created mutations in the POU5F1 gene. We developed an easy and straightforward protocol for gene editing, which could serve as a valuable method for studying the functional genomics of the buffalo embryos. https://www.produccioncientificaluz.org/index.php/cientifica/article/view/43526RNPelectroporationzygotesbuffaloPOU5F1 |
| spellingShingle | Meeti Punetha Dharmendra Kumar Suman Choudhary Sheetal Saini Kamlesh Kumari Bajwa Surabhi Sharma Manu Mangal P. S. Yadav T. K. Datta Exploring the functional role of POU5F1 using CRISPR RNP electroporation of zygote (CRISPR-EZ) in buffaloes Revista Científica RNP electroporation zygotes buffalo POU5F1 |
| title | Exploring the functional role of POU5F1 using CRISPR RNP electroporation of zygote (CRISPR-EZ) in buffaloes |
| title_full | Exploring the functional role of POU5F1 using CRISPR RNP electroporation of zygote (CRISPR-EZ) in buffaloes |
| title_fullStr | Exploring the functional role of POU5F1 using CRISPR RNP electroporation of zygote (CRISPR-EZ) in buffaloes |
| title_full_unstemmed | Exploring the functional role of POU5F1 using CRISPR RNP electroporation of zygote (CRISPR-EZ) in buffaloes |
| title_short | Exploring the functional role of POU5F1 using CRISPR RNP electroporation of zygote (CRISPR-EZ) in buffaloes |
| title_sort | exploring the functional role of pou5f1 using crispr rnp electroporation of zygote crispr ez in buffaloes |
| topic | RNP electroporation zygotes buffalo POU5F1 |
| url | https://www.produccioncientificaluz.org/index.php/cientifica/article/view/43526 |
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