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Abstract: Objective This study aims to utilize the CRISPR / Cas9 system to achieve β-cell specific gene knockout in the pancreas through adeno-associated virus ( AAV8 ) carrying guide RNA ( gRNA ) targeting the Adra2a gene. Methods  RIP2 ( rat insulin Ⅱ promoter ) -Cre; Cas9KI / + mice were used to achieve β-cell-specific gene editing. In this model, the RIP2 promoter drives Cre recombinase expression selectively in pancreatic β cells, resulting in targeted activation of Cas9 in these cells. To enable efficient editing of the Adra2a gene, adeno-associated virus serotype 8, a low-immunogenic vector, was used to deliver gene-specific guide RNAs. AAV8 particles carrying gRNAs targeting Adra2a were injected via the pancreatic duct, enabling precise geneknockout in β cells. Results   Successful β-cell-specific gene knockout was achieved in the pancreas, confirmed by tissue analysis, PCR, and protein expression analysis. GFP expression was observed in the pancreas, and the targeted gene was successfully disrupted in β cells. Conclusion  The use of AAV carrying gRNA in the RIP2-Cre; Cas9KI / + mouse model provides an effective method for β-cell-specific gene knockout, offering a valuable tool for future studies of islet gene function.

Key words: gene editing, CRISPR-Cas9, adeno-associated virus, β-cells