Electroporation of CRISPR/Cas9 Targeting Neurogenin 3 (NGN3) in Porcine Embryos and Its Effects on Mosaicism and Off-Target Effects by Next Generation Sequencing (NGS)
Introduction: CRISPR/Cas9-mediated editing of embryos through microinjection is limited in efficiency because of high rates of mosaicism and off-target mutations. Electroporation has been proposed as an easier and faster procedure compared to single embryo injection. Methods: Using a synthetic guide...
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| Format: | Article |
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Mary Ann Liebert
2024-01-01
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| Series: | Re:GEN Open |
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| Online Access: | https://www.liebertpub.com/doi/full/10.1089/REGEN.2023.0021 |
| _version_ | 1797272646362071040 |
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| author | Insung Park Sergio Navarro-Serna Sergio Navarro-Serna Raquel M. Pinho Trish Berger Elizabeth A. Maga Joaqu?n Gadea Joaqu?n Gadea Seung K. Kim Seung K. Kim Seung K. Kim Pablo J. Ross |
| author_facet | Insung Park Sergio Navarro-Serna Sergio Navarro-Serna Raquel M. Pinho Trish Berger Elizabeth A. Maga Joaqu?n Gadea Joaqu?n Gadea Seung K. Kim Seung K. Kim Seung K. Kim Pablo J. Ross |
| author_sort | Insung Park |
| collection | DOAJ |
| description | Introduction: CRISPR/Cas9-mediated editing of embryos through microinjection is limited in efficiency because of high rates of mosaicism and off-target mutations. Electroporation has been proposed as an easier and faster procedure compared to single embryo injection.
Methods: Using a synthetic guide-RNA targeting porcine NGN3, we optimized the electroporation conditions to effectively introduce CRISPR/Cas9 in porcine zygotes and evaluate its effects on mosaicism and off-targets.
Results: A numerical increase in the mutation rates was observed as embryos were electroporated with increasing concentrations of gRNA (5 ng/?l, 10 ng/?l, and 25 ng/?l) and Cas9 protein (10 ng/?l, 20 ng/?l, and 50 ng/?l) (1:2ratio) without compromising embryo development. Mosaicism results assessed by long-read targeted sequencing of embryos revealed that electroporation with the highest concentration of CRISPR/Cas9 resulted in 57.14% mosaic embryos, accompanied by a significant reduction in the average allele variants (2.43 alleles per embryo) compared to the lowest concentration (4.56 alleles per embryo). Off-target results suggested that no unintended indels were observed when electroporation with the highest CRISPR/Cas9 concentration (25 ng/?l:50 ng/?l) was used.
Conclusion: Embryo electroporation with 25 ng/?l:50 ng/?l of CRISPR/Cas9 targeting porcine NGN3 resulted in a very high mutation efficiency, minimal mosaicism, and no off-target mutations. |
| first_indexed | 2024-03-07T14:32:21Z |
| format | Article |
| id | doaj.art-ca79aef459f941d493f6c8f1535e9b85 |
| institution | Directory Open Access Journal |
| issn | 2766-2705 |
| language | English |
| last_indexed | 2024-03-07T14:32:21Z |
| publishDate | 2024-01-01 |
| publisher | Mary Ann Liebert |
| record_format | Article |
| series | Re:GEN Open |
| spelling | doaj.art-ca79aef459f941d493f6c8f1535e9b852024-03-06T04:00:34ZengMary Ann LiebertRe:GEN Open2766-27052024-01-014192010.1089/REGEN.2023.0021Electroporation of CRISPR/Cas9 Targeting Neurogenin 3 (NGN3) in Porcine Embryos and Its Effects on Mosaicism and Off-Target Effects by Next Generation Sequencing (NGS)Insung Park0Sergio Navarro-Serna1Sergio Navarro-Serna2Raquel M. Pinho3Trish Berger4Elizabeth A. Maga5Joaqu?n Gadea6Joaqu?n Gadea7Seung K. Kim8Seung K. Kim9Seung K. Kim10Pablo J. Ross11Department of Animal Science, University of California DavisDepartment of Physiology, International Excellence Campus for Higher Education and Research (Campus Mare Nostrum), University of MurciaInstitute for Biomedical Research of Murcia IMIB-ArrixacaDepartment of Animal Science, University of California DavisDepartment of Animal Science, University of California DavisDepartment of Animal Science, University of California DavisDepartment of Physiology, International Excellence Campus for Higher Education and Research (Campus Mare Nostrum), University of MurciaInstitute for Biomedical Research of Murcia IMIB-ArrixacaDepartment of Developmental Biology, Stanford University School of MedicineDepartment of Medicine, Stanford University School of MedicineStanford Diabetes Research Center, Stanford University School of MedicineDepartment of Animal Science, University of California DavisIntroduction: CRISPR/Cas9-mediated editing of embryos through microinjection is limited in efficiency because of high rates of mosaicism and off-target mutations. Electroporation has been proposed as an easier and faster procedure compared to single embryo injection. Methods: Using a synthetic guide-RNA targeting porcine NGN3, we optimized the electroporation conditions to effectively introduce CRISPR/Cas9 in porcine zygotes and evaluate its effects on mosaicism and off-targets. Results: A numerical increase in the mutation rates was observed as embryos were electroporated with increasing concentrations of gRNA (5 ng/?l, 10 ng/?l, and 25 ng/?l) and Cas9 protein (10 ng/?l, 20 ng/?l, and 50 ng/?l) (1:2ratio) without compromising embryo development. Mosaicism results assessed by long-read targeted sequencing of embryos revealed that electroporation with the highest concentration of CRISPR/Cas9 resulted in 57.14% mosaic embryos, accompanied by a significant reduction in the average allele variants (2.43 alleles per embryo) compared to the lowest concentration (4.56 alleles per embryo). Off-target results suggested that no unintended indels were observed when electroporation with the highest CRISPR/Cas9 concentration (25 ng/?l:50 ng/?l) was used. Conclusion: Embryo electroporation with 25 ng/?l:50 ng/?l of CRISPR/Cas9 targeting porcine NGN3 resulted in a very high mutation efficiency, minimal mosaicism, and no off-target mutations.https://www.liebertpub.com/doi/full/10.1089/REGEN.2023.0021Pig embryosCRISPR/Cas9 electroporationmosaicismoff-target effects |
| spellingShingle | Insung Park Sergio Navarro-Serna Sergio Navarro-Serna Raquel M. Pinho Trish Berger Elizabeth A. Maga Joaqu?n Gadea Joaqu?n Gadea Seung K. Kim Seung K. Kim Seung K. Kim Pablo J. Ross Electroporation of CRISPR/Cas9 Targeting Neurogenin 3 (NGN3) in Porcine Embryos and Its Effects on Mosaicism and Off-Target Effects by Next Generation Sequencing (NGS) Re:GEN Open Pig embryos CRISPR/Cas9 electroporation mosaicism off-target effects |
| title | Electroporation of CRISPR/Cas9 Targeting Neurogenin 3 (NGN3) in Porcine Embryos and Its Effects on Mosaicism and Off-Target Effects by Next Generation Sequencing (NGS) |
| title_full | Electroporation of CRISPR/Cas9 Targeting Neurogenin 3 (NGN3) in Porcine Embryos and Its Effects on Mosaicism and Off-Target Effects by Next Generation Sequencing (NGS) |
| title_fullStr | Electroporation of CRISPR/Cas9 Targeting Neurogenin 3 (NGN3) in Porcine Embryos and Its Effects on Mosaicism and Off-Target Effects by Next Generation Sequencing (NGS) |
| title_full_unstemmed | Electroporation of CRISPR/Cas9 Targeting Neurogenin 3 (NGN3) in Porcine Embryos and Its Effects on Mosaicism and Off-Target Effects by Next Generation Sequencing (NGS) |
| title_short | Electroporation of CRISPR/Cas9 Targeting Neurogenin 3 (NGN3) in Porcine Embryos and Its Effects on Mosaicism and Off-Target Effects by Next Generation Sequencing (NGS) |
| title_sort | electroporation of crispr cas9 targeting neurogenin 3 ngn3 in porcine embryos and its effects on mosaicism and off target effects by next generation sequencing ngs |
| topic | Pig embryos CRISPR/Cas9 electroporation mosaicism off-target effects |
| url | https://www.liebertpub.com/doi/full/10.1089/REGEN.2023.0021 |
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