Establishment of Pancreatic Beta 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, including pancreatic beta cells associated with the pathogenesis of diabetes mellitus. 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 beta cells and adeno-associated virus 8 (AAV8) -mediated 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 beta cells. We also show significant beta cell-specific gene knockout efficiency with AAV8-mediated delivery of gRNA for EGFP reporter by intraperitoneal injection in the mice. As a proof of concept, we administer AAV8 to βCas9 mice for expressing gRNA for Pdx1, a culprit gene of maturity-onset diabetes of the young 4 (MODY4). As reported previously, we demonstrate that those mice show glucose intolerance with trans-differentiation of Pdx1 knockout beta cells into glucagon-expressing cells. We successfully generate a convenient beta cell-specific gene knockout system with βCas9 mice and AAV8-mediated gRNA delivery.
・ Generating pancreatic beta cell-specific gene knockout mice without time- and money-consuming breeding could be beneficial.
・ We establish a convenient βCas9 system, which enables the beta cell-specific gene knockout with mice expressing Cas9 in pancreatic beta cells and adeno-associated virus 8-mediated gRNA delivery by simple intraperitoneal injection.
・ A fluorescent reporter-based estimation shows the high efficiency of gene knockout in the βCas9 system.
・ Beta cell-specific Pdx1 knockout in the βCas9 system validates the concept of our procedure by demonstrating glucose intolerance and trans-differentiation.