Journal article
Electroporation and genetic supply of Cas9 increase the generation efficiency of CRISPR/Cas9 knock-in alleles in C57BL/6J mouse zygotes
- Abstract:
- CRISPR/Cas9 machinery delivered as ribonucleoprotein (RNP) to the zygote has become a standard tool for the development of genetically modified mouse models. In recent years, a number of reports have demonstrated the effective delivery of CRISPR/Cas9 machinery via zygote electroporation as an alternative to the conventional delivery method of microinjection. In this study, we have performed side-by-side comparisons of the two RNP delivery methods across multiple gene loci and conclude that electroporation compares very favourably with conventional pronuclear microinjection, and report an improvement in mutagenesis efficiency when delivering CRISPR via electroporation for the generation of simple knock-in alleles using single-stranded oligodeoxynucleotide (ssODN) repair templates. In addition, we show that the efficiency of knock-in mutagenesis can be further increased by electroporation of embryos derived from Cas9-expressing donor females. The maternal supply of Cas9 to the zygote avoids the necessity to deliver the relatively large Cas9 protein, and high efficiency generation of both indel and knock-in allele can be achieved by electroporation of small single-guide RNAs and ssODN repair templates alone. Furthermore, electroporation, compared to microinjection, results in a higher rate of embryo survival and development. The method thus has the potential to reduce the number of animals used in the production of genetically modified mouse models.
- Publication status:
- Published
- Peer review status:
- Peer reviewed
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(Preview, Version of record, 2.1MB, Terms of use)
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- Publisher copy:
- 10.1038/s41598-020-74960-7
Authors
- Publisher:
- Springer Nature
- Journal:
- Scientific Reports More from this journal
- Volume:
- 10
- Article number:
- 17912
- Publication date:
- 2020-10-21
- Acceptance date:
- 2020-10-08
- DOI:
- EISSN:
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2045-2322
- Language:
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English
- Keywords:
- Subjects:
- Pubs id:
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1136437
- Local pid:
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pubs:1136437
- Deposit date:
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2020-10-08
Terms of use
- Copyright holder:
- Alghadban, S et al.
- Copyright date:
- 2020
- Rights statement:
- © The Author(s) 2020. 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.
- Licence:
- CC Attribution (CC BY)
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