RT Journal
A1 Duan, Xiaoyue
A1 Chen, Chaolei
A1 Du, Chang
A1 Guo, Liang
A1 Liu, Jun
A1 Hou, Naipeng
A1 Li, Pan
A1 Qi, Xiaolan
A1 Gao, Fei
A1 Du, Xuguang
A1 Song, Jiangping
A1 Wu, Sen
T1 Homozygous editing of multiple genes for accelerated generation of xenotransplantation pigs
JF Genome Research 
JO Genome Research 
YR 2025 
FD May 01 
VO 35 
IS 5 
SP 1167 
OP 1178 
DO 10.1101/gr.279709.124 
UL http://genome.cshlp.org/content/35/5/1167.abstract 
AB 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. For simultaneous multigene editing, we targeted the most challenging site for selection, whereas other target sites did not require selection. Using this approach, we successfully obtained single-cell PFF clones (three of 10) with seven or more homozygously edited genes, including GGTA1, CMAH, B4GALNT2, CD46, CD47, THBD, and GHR. Importantly, cells edited using this strategy can be efficiently used for somatic cell nuclear transfer (SCNT) to generate healthy xenotransplantation pigs in <5 months, a process that previously required years of breeding or multiple rounds of SCNT.