Simplified pipelines for genetic engineering of mammalian embryos by CRISPR-Cas9 electroporation†
| العنوان: | Simplified pipelines for genetic engineering of mammalian embryos by CRISPR-Cas9 electroporation† |
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| المؤلفون: | De-Qiang Miao, Blanca Lopez-Biladeau, Mariana Ianello Giassetti, Michela Ciccarelli, Jon M. Oatley |
| المصدر: | Biol Reprod |
| بيانات النشر: | Oxford University Press (OUP), 2019. |
| سنة النشر: | 2019 |
| مصطلحات موضوعية: | Male, 0301 basic medicine, Mice, 129 Strain, Swine, Cloning, Organism, Mice, Transgenic, Computational biology, Biology, medicine.disease_cause, Embryo Culture Techniques, Mice, 03 medical and health sciences, Genome editing, medicine, Animals, CRISPR, Zona pellucida, Cells, Cultured, Gene Editing, Mutation, Zygote, Electroporation, Gene Transfer Techniques, 0402 animal and dairy science, RNA-Binding Proteins, Embryo, 04 agricultural and veterinary sciences, Cell Biology, General Medicine, Embryo Transfer, Embryo, Mammalian, 040201 dairy & animal science, Embryo transfer, Mice, Inbred C57BL, 030104 developmental biology, medicine.anatomical_structure, Reproductive Medicine, Cattle, Female, CRISPR-Cas Systems, Genetic Engineering, Research Article |
| الوصف: | Gene editing technologies, such as CRISPR-Cas9, have important applications in mammalian embryos for generating novel animal models in biomedical research and lines of livestock with enhanced production traits. However, the lack of methods for efficient introduction of gene editing reagents into zygotes of various species and the need for surgical embryo transfer in mice have been technical barriers of widespread use. Here, we described methodologies that overcome these limitations for embryos of mice, cattle, and pigs. Using mutation of the Nanos2 gene as a readout, we refined electroporation parameters with preassembled sgRNA-Cas9 RNPs for zygotes of all three species without the need for zona pellucida dissolution that led to high-efficiency INDEL edits. In addition, we optimized culture conditions to support maturation from zygote to the multicellular stage for all three species that generates embryos ready for transfer to produce gene-edited animals. Moreover, for mice, we devised a nonsurgical embryo transfer method that yields offspring at an efficiency comparable to conventional surgical approaches. Collectively, outcomes of these studies provide simplified pipelines for CRISPR-Cas9-based gene editing that are applicable in a variety of mammalian species. |
| تدمد: | 1529-7268 0006-3363 |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::3f27fb4b92c2025b1a2e6ac84392bf14 https://doi.org/10.1093/biolre/ioz075 |
| حقوق: | OPEN |
| رقم الأكسشن: | edsair.doi.dedup.....3f27fb4b92c2025b1a2e6ac84392bf14 |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 15297268 00063363 |
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