An Episomal Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 System for Transgene-Free Multiplex Gene Editing in Pig Cells

Despite significant advancements in CRISPR/Cas-based genome editing technology over the past decade, achieving simultaneous homozygous gene editing at multiple targets in primary cells remains a major challenge. In this study, we developed and constructed a CRISPR multi-gene targeting system that integrates episomal vectors with tRNA–sgRNA array technology. This approach leverages scaffold/matrix attachment region (S/MAR) sequences to enable sustained episomal expression of both Cas9 and single-guide RNAs (sgRNAs) without genomic integration, thereby enhancing gene editing efficiency. For simultaneous editing of multiple loci, we used the tRNA–sgRNA architecture to process multiple sgRNAs from a single vector. Using this system in porcine fetal fibroblasts, we achieved concurrent editing of six genes, namely ANXA7, GSK3A, ENTPD6, SIRT3, CYP20A1, and SOCS2, in individual cells. These edited cells supported normal development following somatic cell nuclear transfer, yielding blastocysts with unaltered developmental competence. Collectively, our findings establish a framework for the application of CRISPR/Cas9 in gene-edited pigs, facilitating the generation of multi-gene-edited animals for biomedical and agricultural applications.