Homozygous editing of multiple genes for accelerated generation of xenotransplantation pigs

Although CRISPR-Cas based genome editing has made significant strides over the past decade, achieving simultaneous homozygous gene editing of multiple targets in primary cells remains a significant challenge. In this study, we optimized a coselection strategy to enhance homozygous gene editing rates in the genomes of primary porcine fetal fibroblasts (PFFs). The strategy utilizes the expression of a surrogate reporter (eGFP) to select for cells with the highest reporter expression, thereby improving editing efficiency. When applied to simultaneous multigene editing, we targeted the most challenging site for selection, while other target sites did not require selection. Using this approach, we successfully obtained single-cell PFF clones (3/10) with seven or more homozygously edited genes, including GGTA1, CMAH, B4GALNT2, CD46, CD47, THBD, and GHR. Importantly, cells edited using this strategy were efficiently used for somatic cell nuclear transfer (SCNT) to generate healthy xenotransplantation pigs in less than five months, a process that previously required years of breeding or multiple rounds of SCNT.