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

Abstract

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-specificCreex-pression mice. We establish a bCas9 system with mice expressing Cas9 in pancreatic β-Cells and adeno-associated virus8(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 bCas9 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.