Abstract
Electroporation of mouse embryos with CRISPR/Cas9 endonuclease tool is a facile and efficient method to edit endogenous genome sequences for generating genetically engineered mouse models (GEMMs). Common genome engineering projects, such as knock-out (KO), conditional knock-out (cKO), point mutation, and small foreign DNA (<1 Kb) knock-in (KI) alleles, can be effectively accomplished with a simple electroporation procedure. The use of electroporation in sequential gene editing at the one-cell (0.7 days post-coitum (dpc)) and at two-cell (1.5 dpc) embryonic stages provides a fast and compelling protocol to safely introduce multiple gene modifications on the same chromosome by limiting chromosomal fractures. In addition, the co-electroporation of the ribonucleoprotein (RNP) complex and single-stranded oligodeoxynucleotide (ssODN) donor DNA with the strand exchange protein Rad51 can significantly increase the number of homozygous founders. Here we describe a comprehensive guideline for mouse embryo electroporation to generate GEMMs and the implementation of Rad51 in RNP/ssODN complex EP medium protocol.
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We thank Dr. Thomas Saunders for kindly providing his feedback on this manuscript
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Garza, S., Paik, R. (2023). CRISPR/Cas9 Endonuclease-Mediated Mouse Genome Editing of One-Cell and/or Two-Cell Embryos by Electroporation, and the Use of Rad51 to Enhance Knock-In Allele Homozygosity via Interhomolog Repair Mechanism. In: Saunders, T.L. (eds) Transgenesis. Methods in Molecular Biology, vol 2631. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2990-1_10
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DOI: https://doi.org/10.1007/978-1-0716-2990-1_10
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Publisher Name: Humana, New York, NY
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Online ISBN: 978-1-0716-2990-1
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