Scalable Production of Genome-Edited Livestock Embryos
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Scalable Production of Genome-Edited Livestock Embryos

Abstract

Genetic improvement of livestock is critical for animal production and wellbeing. Traditional breeding approaches however are slow and often result in unwanted genetic linkage drag. Genome-editing technologies offers an alternative that can quickly and precisely introduce useful traits into animals without linkage drag. Editing of mammalian livestock species has been achieved by somatic cell nuclear transfer of an edited cell or direct microinjection of zygotes. However, the efficiency of cloning in large livestock species is low and perinatal abnormalities are common, and introducing editing reagents through microinjection is a time-consuming task with a high technical barrier. Electroporation is a widely used technique for delivering gene-editing reagents into cells and poses as a possible high throughput approach to generate genome-edited animals through the treatment of early-stage embryos. Electroporators work by directing pulses of electrical current to create transient pores in the lipid bilayer of the plasma membrane, allowing the passage of reagents into cells. Upwards of 100 zygotes can be processed with the push of a button making electroporation a scalable and simple approach to producing genome edited livestock. Here, various electroporation parameters for generating gene-edited bovine, ovine, and caprine embryos were tested for producing targeted mutations.Targeted genetic knock-ins of over 1kb however have not been produced with electroporation alone as nucleic acids larger than 1kb are unable to pass the zona pellucida. To develop a high throughput approach to producing large template targeted knock-in livestock, another delivery system for large DNA repair templates must be employed. Non-pathogenic viruses such as rAAV can transport nucleic acid fragments of up to 4.9kb into cells. Here, a scalable approach was developed to transduce large DNA repair templates into bovine zygotes prior to electroporation for the production of 2.7kb knock-in blastocysts.

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