TY  - JOUR
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
Y1  - 2025/05/01 
JF  - Genome Research 
JO  - Genome Research 
SP  - 1167 
EP  - 1178 
DO  - 10.1101/gr.279709.124 
VL  - 35 
IS  - 5 
UR  - http://genome.cshlp.org/content/35/5/1167.abstract 
N2  - 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. 
ER  -