Abstract
Genetic engineering in the rat has been revolutionized by the development of CRISPR-based genome editing tools. Conventional methods for inserting genome editing elements such as CRISPR/Cas9 reagents into rat zygotes include cytoplasmic or pronuclear microinjections. These techniques are labor-intensive, require specialized micromanipulator equipment, and are technically challenging. Here, we describe a simple and effective method for zygote electroporation in which CRISPR/Cas9 reagents are introduced into rat zygotes via pores produced by precise electrical pulses applied to the cells. Zygote electroporation allows for high-throughput efficient genome editing in rat embryos.
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References
Li D et al (2013) Heritable gene targeting in the mouse and rat using a CRISPR-Cas system. Nat Biotechnol 31(8):681–683
Kaneko T et al (2014) Simple knockout by electroporation of engineered endonucleases into intact rat embryos. Sci Rep 4:6382
Chen S et al (2016) Highly efficient mouse genome editing by CRISPR ribonucleoprotein electroporation of zygotes. J Biol Chem 291(28):14457–14467
Hashimoto M, Yamashita Y, Takemoto T (2016) Electroporation of Cas9 protein/sgRNA into early pronuclear zygotes generates non-mosaic mutants in the mouse. Dev Biol 418(1):1–9
Wang W et al (2016) Delivery of Cas9 protein into mouse zygotes through a series of electroporation dramatically increases the efficiency of model creation. J Genet Genomics 43(5):319–327
Tröder SE et al (2018) An optimized electroporation approach for efficient CRISPR/Cas9 genome editing in murine zygotes. PLoS One 13(5):e0196891
Teixeira M et al (2018) Electroporation of mice zygotes with dual guide RNA/Cas9 complexes for simple and efficient cloning-free genome editing. Sci Rep 8(1):474
Remy S et al (2017) Generation of gene-edited rats by delivery of CRISPR/Cas9 protein and donor DNA into intact zygotes using electroporation. Sci Rep 7(1):16554
Tanihara F et al (2020) Efficient generation of GGTA1-deficient pigs by electroporation of the CRISPR/Cas9 system into in vitro-fertilized zygotes. BMC Biotechnol 20(1):40
Tanihara F et al (2021) Generation of CD163-edited pig via electroporation of the CRISPR/Cas9 system into porcine in vitro-fertilized zygotes. Anim Biotechnol 32(2):147–154
Camargo LSA et al (2020) Efficient one-step knockout by electroporation of ribonucleoproteins into zona-intact bovine embryos. Front Genet 11:570069
Lin JC, Van Eenennaam AL (2021) Electroporation-mediated genome editing of livestock zygotes. Front Genet 12:648482
Betters E, Charney RM, GarcÃa-Castro MI (2018) Electroporation and in vitro culture of early rabbit embryos. Data Brief 21:316–320
Acknowledgments
This work was done in collaboration with the Rat Resource and Research Center (RRRC) funded by NIH grant OD011062 and University of Missouri Animal Modeling Core (AMC).
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Davis, D.J., Men, H., Bryda, E.C. (2023). Electroporation-Mediated CRISPR/Cas9 Genome Editing in Rat Zygotes. 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_11
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DOI: https://doi.org/10.1007/978-1-0716-2990-1_11
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Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2989-5
Online ISBN: 978-1-0716-2990-1
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