Establishment of Pancreatic β-Cell-Specific Gene Knockout System Based on CRISPR-Cas9 Technology With AAV8-Mediated gRNA Delivery.

The Cre-loxP system provides valuable resources to analyze the importance of tissue-specific gene knockout (KO), including pancreatic β-cells associated with the pathogenesis of diabetes. However, it is expensive and time consuming to generate transgenic mice harboring floxed genes of interest and cross them with cell-specific Cre expression mice. We establish a βCas9 system with mice expressing Cas9 in pancreatic β-cells and adeno-associated virus 8 (AAV8)-mediated guide RNA (gRNA) delivery based on CRISPR-Cas9 technology to overcome those shortcomings. Interbreeding CAG-loxP-STOP-loxP (LSL)-Cas9 with Ins1-Cre mice generates normal glucose-tolerant βCas9 mice expressing Cas9 with fluorescent reporter EGFP specifically in β-cells. We also show significant β-cell-specific gene KO efficiency with AAV8-mediated delivery of gRNA for EGFP reporter by intraperitoneal injection in the mice. As a proof of concept, we administered AAV8 to βCas9 mice for expressing gRNA for Pdx1, a culprit gene of maturity-onset diabetes of the young 4. As reported previously, we demonstrate that those mice show glucose intolerance with transdifferentiation of Pdx1 KO β-cells into glucagon-expressing cells. We successfully generated a convenient β-cell-specific gene KO system with βCas9 mice and AAV8-mediated gRNA delivery.