CRISPR/Cas9-Mediated α-ENaC Knockout in a Murine Pancreatic β-Cell Line

Many ion channels participate in controlling insulin synthesis and secretion of pancreatic beta-cells. Epithelial sodium channel (ENaC) expressed in human pancreatic tissue, but the biological role of ENaC in pancreatic beta-cells is still unclear. Here, we applied the CRISPR/Cas9 gene editing technique to knockout alpha-ENaC gene in a murine pancreatic beta-cell line (MIN6 cell). Four single-guide RNA (sgRNA) sites were designed for the exons of alpha-ENaC. The sgRNA1 and sgRNA3 with the higher activity were constructed and co-transfected into MIN6 cells. Through processing a series of experiment flow included drug screening, cloning, and sequencing, the alpha-ENaC gene-knockout (alpha-ENaC-/-) in MIN6 cells were obtained. Compared with the wild-type MIN6 cells, the cell viability and insulin content were significantly increased in alpha-ENaC-/- MIN6 cells. Therefore, alpha-ENaC-/- MIN6 cells generated by CRISPR/Cas9 technology added an effective tool to study the biological function of alpha-ENaC in pancreatic beta-cells.