Summary/Abstract SARS-CoV-2 has caused the deaths of millions of people globally. Effective antiviral therapeutic treatment options are urgently needed. CRISPR-mediated genome editing has provided a very promising avenue for treatment of a variety of genetic diseases. Particularly, the CRISPR-Cas13 system has been demonstrated to possess the potential of inhibiting SARS-CoV-2 and influenza infections by degradation of viral genomic RNA and viral mRNA. However, it is still very challenging to deliver the CRISPR machinery to initiate genome editing efficiently in vivo. Extracellular vesicles (EVs) contain a variety of molecular components including lipids, mRNA, microRNAs, and proteins. A large body of studies has shown that EVs mediate cell-to-cell communication by transmitting their encapsulated contents. This proposal intends to construct EVs encapsulating the CRISPR machinery and deliver the EVs to respiratory epithelial cells to inhibit SARS-CoV-2 proliferation in vivo. We will therefore genetically engineer the Cas13d protein so that Cas13d/CRISPR-RNA (crRNA) ribonucleoprotein complex can be encapsulated into EVs. We will also engineer the membrane of EVs, such that EVs target respiratory epithelial cells and deliver Cas13d/crRNA for inhibiting SARS-CoV-2 viral assembly and proliferation, thereby inhibiting COVID-19. This study will allow us to understand the feasibility of an EVs-based vehicle to deliver genome editing machinery to inhibit SARS-CoV-2 proliferation in lung epithelial cells. This study will provide a treatment option for COVID-19 patients to reduce disease severity and mortality.