Figure 2.

Overview and validation of the strategy to knockout genes in the immortalized EndoC-βH1 pancreatic β-cell line. (A) Schematic representation of the V2 lentiviral construct containing a RIP promoter driving Cas9 expression, a HpaI restriction site allowing for cloning of a desired gRNA and puromycin drug selection to enrich for infected cells. (B) Sequence of gRNAs used to target the INS, PDX1, and HNF1A genes. Red or green bars show the relative position of the gRNAs per gene. (C) Flow cytometry analysis of cells following CRISPR–CAS9 targeting of the INS gene to determine the efficiency of knockout with 2 different gRNAs (n = 3, representative FACS plot from a single experiment is shown). (D) Knock-out efficiency per gRNA in each targeted cell line using flow cytometry (n = 3).