UC Davis

The CRISPR-Cas9 system has revolutionized genome editing due to its simplicity, efficiency, and specificity; making genome-modified large animals’ important components in sustainable food production and biomedical systems. Small ruminant species are essential elements of such systems due to their size, anatomy, productivity, and adaptability. However, many limitations should be overcome before adopting genome editing technologies at a large scale. For example, mosaicism, a common feature of genome editing, represents a real impediment in the production of stable genome-modified animals. Moreover, costs, technical difficulties, and outcomes inconsistency of commonly used microinjection of CRISPR-Cas9, represents another challenge. Therefore, replacing microinjection with an alternative that can reduce costs, technical difficulties, and mosaicism is one of the essential steps before successfully embedding genome editing into the production system. Electroporation was used effectively to deliver genome editors into in vitro and in vivo cells. In my research, I optimized the electroporation of CRISPR-Cas9 parameter and time to overcome several obstacles faced by one-step zygote gene editing. Using dual sgRNAs, I targeted two sheep genomic loci: SOCS2 and PDX1 and one goat genomic locus: OTX2. I compared the efficiency of microinjection with three different electroporation settings at four different times of development. I found that electroporation of sheep and goat zygotes 6 hours after fertilization in an electroporation parameter that involved short-high voltage (poring) and long-low voltage (transfer) pulses was efficient to produce non-mosaic knock-out blastocysts (chapter 2). I transferred the SOCS2 knock-out embryos (chapter 3) to estrus synchronized recipients to extend my investigation beyond the preimplantation stages. I obtained eight knock-out fetuses, and lambs, of which 3 grew to become three healthy adults. All fetuses and lambs were SOCS2 biallelic mutants. The knock-out phenotype was confirmed by the absence of SOCS2 mRNA and protein in the healthy lambs. In conclusion, embryo electroporation of CRISPR-Cas9 is an efficient method to produce knock-out sheep.