本课题组前期研究发现转染CRISPR/Cas9表达质粒的细胞所释放的外泌体中存在全长导向RNA（gRNA）和Cas9蛋白，提示内源外泌体可能参与CRISPR/Cas9系统及其基因编辑功能的传递作用。本项目拟探究gRNA和Cas9蛋白经外泌体释放至细胞外的途径和方式，及其在外泌体中的存在形式，进而揭示外泌体传递CRISPR/Cas9系统的机制。然后，深入探讨内源外泌体可否将靶向乙型肝炎病毒（HBV）的gRNA和Cas9蛋白传递至带有HBV基因组的靶细胞，并通过靶向破坏HBV基因组抑制HBV复制，进而揭示内源外泌体在CRISPR/Cas9抑制HBV复制中的作用。此外，通过尾静脉水压动力注射HBV复制小鼠模型进一步阐明内源外泌体在CRISPR/Cas9抑制HBV复制中的作用及机制。本研究有可能为CRISPR/Cas9系统靶向清除HBV提供一种潜在的递送系统，并推动其在慢乙肝治疗中的临床应用。

Our previous study demonstrated that the full-length guide RNA (gRNA) and Cas9 protein were present in the endogenous exosomes secreted from the cells transfected with CRISPR/Cas9 expression plasmids, indicating that the endogenous exosomes might be involved in the transmission of CRISPR/Cas9 system and its gene editing function. This project intends to further explore the releasing pathway of gRNA and Cas9 protein via exosomes, the nature of gRNA and Cas9 protein in exosomes, and the way they enter the exosomes, so as to interpretate the mechanisms on the endogenous exosomes-mediated delivery of CRISPR/Cas9 system. Further, we will explore whether the endogenous exosomes can deliver gRNA targeting the hepatitis B virus (HBV) genome and Cas9 protein to the target cells containing HBV genome, and inhibit HBV replication by destroying HBV genome, which eradicates the role of endogenous exosomes in the CRISPR/Cas9-mediated inhibition of HBV replication. Additionally, the mechanism and role of endogenous exosomes in the CRISPR/Cas9-mediated inhibition of HBV replication will be further explored in the HBV replicative mouse model using vein hydrodynamic injection technology. This study may provide a potential delivery system for the clearance of HBV by CRISPR/Cas9 system, and promote the clinical application of CRISPR/Cas9 system in the treatment of chronic hepatitis B.