CRSPR-Cas9-mediated alpha-Gal knockout in porcine fetal fibroblast cells

The CRISPR-Cas9 system has transformed genetic engineering by providing a precise and efficient tool for targeted genome editing. Pigs have emerged as valuable models in both biomedical and agricultural research due to their anatomical, physiological, and metabolic similarities to humans. In the context of xenotransplantation, however, hyperacute rejection (HAR) caused by the α-Gal epitope, which is synthesized by the α1,3 galactosyltransferase (α1,3GT) enzyme and encoded by the GGTA1 gene, remains a major immunological barrier. This study aimed to establish and optimize a CRISPR-Cas-9-mediated knockout protocol for the GGTA1 gene in porcine fetal fibroblast cells, a critical step toward generating engineered pigs with reduced risk of immune rejection. Fibroblast cells were isolated from porcine fetuses at approximately 36 days of gestation and cultured to develop stable cell lines. Additionally, PCR-based sex determination of fetal tail samples was performed. The first step of the project was to determine the optimal electroporation parameters for the obtained cell lines using a NEPA21 electroporator, testing multiple pulse conditions to maximize cell viability and transfection efficiency. Post-electroporation cell viability was assessed using Trypan blue exclusion, while Coomassie blue staining at 48 hours provided estimates of cell density and growth dynamics. Transfection efficiency was quantified by eGFP plasmid and Hoechst nuclear staining, followed by fluorescence microscopy. Lectin staining of wild-type fibroblasts was performed to detect the presence of α-Gal epitopes, enabling the future identification of cells lacking these epitopes because of successful CRISPR-Cas9-mediated edits. Optimization experiments identified electroporation conditions that enhanced cell survival and transfection efficiency, establishing a workflow for downstream GGTA1 knockout experiments. Following optimization, the CRISPR gRNA and Cas9 were transfected into the primary porcine fetal cell lines we generated using the best parameter identified. Electroporated cells were subjected to limiting dilution to promote colony formation, and individual colonies were isolated and expanded. Genomic DNA from wild-type cells was amplified by PCR and submitted for Sanger sequencing to confirm CRISPR-Cas9-mediated editing. Although the current experiment is not fully complete, the work described to date provides a reproducible framework for generating GGTA1-deficient porcine fibroblasts, which can serve as nuclear donors for somatic cell nuclear transfers (SCNT) to produce genetically engineered pigs. By addressing one of the primary barriers in pig-to-human organ transplantation, this research supports the development of xenotransplantation strategies aimed at alleviating the critical global shortage of transplantable organs, with the potential to benefit thousands of patients worldwide.