Optimization of Electroporation and Adeno-Associated Virus-Mediated Generation of 2.7 kb Knock-In Livestock Blastocysts

Abstract

The production of genome-edited livestock has traditionally relied on either somatic cell nuclear transfer cloning of an edited cell or microinjection of gene-editing reagents into developing embryos. Electroporation of gene-editing reagents into zygotes offers an alternative that is both high throughput and easy to use. Transfer of large (>1 kb) homology-directed repair (HDR) donor nucleic acid templates into mammalian zygotes is hindered by the thick extracellular zona pellucida (ZP) glycoprotein layer. A potentially scalable approach to deliver HDR templates of up to 4.9 kb into zygotes is to use recombinant adeno-associated virus (rAAV) vectors. In this study, electroporation conditions were optimized to efficiently deliver CRISPR-associated protein 9 (Cas9):single guide RNA (sgRNA) ribonucleoprotein (RNP) complexes to bovine and ovine zygotes, while retaining embryo viability. rAAV vectors were then used to transduce a 3.9-kb HDR template in combination with electroporation of Cas9:sgRNA RNP editing reagents 6 h postinsemination to generate bovine blastocysts with a targeted 2.7 kb knock-in (KI) at the H11 safe harbor locus. With this approach, there was no need to remove or weaken the ZP of the blastocysts that developed, and a KI rate of up to ∼38% was observed. However, to circumvent transduction of the cumulus cells, the oocytes were denuded before transduction that decreased the blastocyst development rate. In addition, genetic mosaicism was observed in the blastocysts. These results highlight that further optimization of editing approaches will be required to achieve nonmosaic embryos with a targeted KI at scale for livestock applications.