A CRISPR/Cas9 genome editing pipeline in the EndoC-βH1 cell line to study genes implicated in beta cell function.

摘要: Type 2 diabetes (T2D) is a global pandemic with strong genetic component, but most causal genes influencing the disease risk remain unknown. It clear, however, that pancreatic beta cell central to T2D pathogenesis. In vitro gene-knockout (KO) models study have so far focused on rodent cells. However, there are important structural and functional differences between human lines. With in mind, we developed robust pipeline create stable CRISPR/Cas9 KO an authentic line (EndoC-βH1). The consists of dual lentiviral sgRNA strategy targeted three ( INS , IDE PAM ) as proof concept. We achieved significant reduction mRNA levels complete protein depletion all target genes. Using this strategy, up 94 kb DNA were cut out editing efficiency each exceeded >87.5%. Sequencing off-targets showed no unspecific editing. Most importantly, did not affect glucose-responsive insulin secretion Interestingly, comparison lines for NEUROD1 SLC30A8 siRNA-mediated knockdown (KD) approaches demonstrate phenotypic differences. NEUROD1- cells viable displayed elevated markers ER stress apoptosis. -KD, only had modest elevation, by 34%, pro-apoptotic transcription factor CHOP gene expression profile indicative chronic without evidence death. On other hand, -KO demonstrated K ATP channel contrast siRNA silencing. Overall, efficiently EndoC-βH1 will allow better understanding involved dysfunction, their underlying mechanisms