Improving the Efficiency of Precise Genome Editing with CRISPR/Cas9 to Generate Goats Overexpressing Human Butyrylcholinesterase

The CRISPR/Cas9 system is widely used for genome editing in livestock production, although off-target effects can occur. It the main method to produce genome-edited goats by somatic cell nuclear transfer (SCNT) of CRISPR/Cas9-mediated primary goat fetal fibroblast cells (GFFs). Improving double-strand break (DSB) efficiency Cas9 would improve homologous repair (HR) efficiency. low HR remains a major hurdle precise editing, increasing work required screen clones. In this study, we modified several essential parameters that affect knock-in GFF cloning system, including establishing high-efficiency transfection via nucleofection and optimizing homology arm (HA) length during HR. Here, specifically inserted recombinant human butyrylcholinesterase gene (rhBChE) into growth factor (FGF)-5 locus through thereby achieving simultaneous rhBChE insertion FGF5 knock-out. First, study introduced Cas9, knock-out small guide RNA, donors GFFs electroporation obtained positive clones without effects. Then, demonstrated expression verified its function. Finally, rhBChE-overexpression goat.