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The cell cycle stage of bovine zygotes electroporated with CRISPR/Cas9-RNP affects frequency of Loss-of-heterozygosity editing events
At the embryonic level, CRISPR technologies have been used to edit genomes reliably and efficiently in various mammalian models, with Ribonucleoprotein (RNP) electroporation potentially representing a superior delivery method into mammalian zygotes. However, detailed insights of the interactions bet...
Autores principales: | , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Nature Publishing Group UK
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9232522/ https://www.ncbi.nlm.nih.gov/pubmed/35750764 http://dx.doi.org/10.1038/s41598-022-14699-5 |
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author | Miskel, Dennis Poirier, Mikhael Beunink, Luisa Rings, Franca Held, Eva Tholen, Ernst Tesfaye, Dawit Schellander, Karl Salilew-Wondim, Dessie Blaschka, Carina Große-Brinkhaus, Christine Brenig, Bertram Hoelker, Michael |
author_facet | Miskel, Dennis Poirier, Mikhael Beunink, Luisa Rings, Franca Held, Eva Tholen, Ernst Tesfaye, Dawit Schellander, Karl Salilew-Wondim, Dessie Blaschka, Carina Große-Brinkhaus, Christine Brenig, Bertram Hoelker, Michael |
author_sort | Miskel, Dennis |
collection | PubMed |
description | At the embryonic level, CRISPR technologies have been used to edit genomes reliably and efficiently in various mammalian models, with Ribonucleoprotein (RNP) electroporation potentially representing a superior delivery method into mammalian zygotes. However, detailed insights of the interactions between varying technical settings as well as the time point of electroporation in a bovine zygote’s cell cycle on developmental metrics and the frequency and type of editing events are largely unknown. The present study uncovers that increasing pulse lengths result in higher Full Edit rates, with Mosaicism in Full-Edit embryos being significantly affected by adjusting RNP-electroporation relative to zygote cell cycle. A considerable proportion of Full Edit embryos demonstrated loss-of-heterozygosity after RNP-electroporation prior to S-phase. Some of these loss-of-heterozygosity events are a consequence of chromosomal disruptions along large sections of the target chromosomes making it necessary to check for their presence prior use of this technique in animal breeding. One out of 2 of these loss-of-heterozygosity events, however, was not associated with loss of an entire chromosome or chromosomal sections. Whether analysed loss-of-heterozygosity in these cases, however, was a false negative result due to loss of PCR primer sequences after INDEL formation at the target side or indeed due to interhomolog recombination needs to be clarified in follow up studies since the latter would for sure offer attractive options for future breeding schedules. |
format | Online Article Text |
id | pubmed-9232522 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-92325222022-06-26 The cell cycle stage of bovine zygotes electroporated with CRISPR/Cas9-RNP affects frequency of Loss-of-heterozygosity editing events Miskel, Dennis Poirier, Mikhael Beunink, Luisa Rings, Franca Held, Eva Tholen, Ernst Tesfaye, Dawit Schellander, Karl Salilew-Wondim, Dessie Blaschka, Carina Große-Brinkhaus, Christine Brenig, Bertram Hoelker, Michael Sci Rep Article At the embryonic level, CRISPR technologies have been used to edit genomes reliably and efficiently in various mammalian models, with Ribonucleoprotein (RNP) electroporation potentially representing a superior delivery method into mammalian zygotes. However, detailed insights of the interactions between varying technical settings as well as the time point of electroporation in a bovine zygote’s cell cycle on developmental metrics and the frequency and type of editing events are largely unknown. The present study uncovers that increasing pulse lengths result in higher Full Edit rates, with Mosaicism in Full-Edit embryos being significantly affected by adjusting RNP-electroporation relative to zygote cell cycle. A considerable proportion of Full Edit embryos demonstrated loss-of-heterozygosity after RNP-electroporation prior to S-phase. Some of these loss-of-heterozygosity events are a consequence of chromosomal disruptions along large sections of the target chromosomes making it necessary to check for their presence prior use of this technique in animal breeding. One out of 2 of these loss-of-heterozygosity events, however, was not associated with loss of an entire chromosome or chromosomal sections. Whether analysed loss-of-heterozygosity in these cases, however, was a false negative result due to loss of PCR primer sequences after INDEL formation at the target side or indeed due to interhomolog recombination needs to be clarified in follow up studies since the latter would for sure offer attractive options for future breeding schedules. Nature Publishing Group UK 2022-06-24 /pmc/articles/PMC9232522/ /pubmed/35750764 http://dx.doi.org/10.1038/s41598-022-14699-5 Text en © The Author(s) 2022, corrected publication 2022 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Article Miskel, Dennis Poirier, Mikhael Beunink, Luisa Rings, Franca Held, Eva Tholen, Ernst Tesfaye, Dawit Schellander, Karl Salilew-Wondim, Dessie Blaschka, Carina Große-Brinkhaus, Christine Brenig, Bertram Hoelker, Michael The cell cycle stage of bovine zygotes electroporated with CRISPR/Cas9-RNP affects frequency of Loss-of-heterozygosity editing events |
title | The cell cycle stage of bovine zygotes electroporated with CRISPR/Cas9-RNP affects frequency of Loss-of-heterozygosity editing events |
title_full | The cell cycle stage of bovine zygotes electroporated with CRISPR/Cas9-RNP affects frequency of Loss-of-heterozygosity editing events |
title_fullStr | The cell cycle stage of bovine zygotes electroporated with CRISPR/Cas9-RNP affects frequency of Loss-of-heterozygosity editing events |
title_full_unstemmed | The cell cycle stage of bovine zygotes electroporated with CRISPR/Cas9-RNP affects frequency of Loss-of-heterozygosity editing events |
title_short | The cell cycle stage of bovine zygotes electroporated with CRISPR/Cas9-RNP affects frequency of Loss-of-heterozygosity editing events |
title_sort | cell cycle stage of bovine zygotes electroporated with crispr/cas9-rnp affects frequency of loss-of-heterozygosity editing events |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9232522/ https://www.ncbi.nlm.nih.gov/pubmed/35750764 http://dx.doi.org/10.1038/s41598-022-14699-5 |
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