CRISPR/Cas9 gene-editing tools construction

Abstract

Type 1-diabetes is a disease where the body destroys its own insulin-producing, pancreatic beta cells. The pancreatic alpha cells produce the blood sugar-elevating hormone glucagon from the precursor protein proglucagon. Marking the proglucagon gene with CRISPR/Cas9 mediated knock-in of green fluorescent protein (GFP) might allow for visualising the dynamics within the cell under differentiation from stem cell to somatic cell. To perform the preferred knock-in, the gene editing tools were constructed. A combined vector of single guide RNA, Cas9 nuclease and a reporter for the Cas9 nuclease was constructed. A donor DNA template vector consisting of the GFP between sequences homologous to each side of the Cas9 cut site was cloned with In-Fusion multi-fragment cloning. Sanger sequencing confirmed that both vectors were successfully cloned before the combined vector was successfully transfected into human embryonic kidney cells. T7 Endonuclease I assay estimated an indel occurrence of 14,4% for the combined vector. The combined vector showed that it did perform cutting, and future research will reveal if the constructed donor DNA template vector results in a knock-in of GFP in the proglucagon gene.