Applying EVs-Encapsulated CRISPR Ribonucleoprotein Complex for Gene Editing in Vivo

The safe and efficient delivery of CRISPR/Cas9 remains a major challenge in advancing clinical gene-editing applications. This study investigates the use of extracellular vesicles (EVs) coated with VSV-G for CRISPR/Cas9 ribonucleoprotein complexes targeting the eGFP gene. HEK293T cells were engineered to produce EVs co-expressing mCas9/sgRNA and VSV-G. The isolated EVs achieved 14.67%±7.02 gene editing efficiency in HEK293T-eGFP cells. Notably, compared with VSV-G, SARS-CoV-2 spike(D614G-Δ21) protein inhibited the encapsulation of mCas9/sgRNA into EVs. SCID mice expressing EGFP or carrying EGFP xenograft tissues were administered EVs co-expressing VSV-G and mCas9/sgRNA targeting the eGFP gene by intravenous injection. No observable weight loss was detected, suggesting their safety and biocompatibility. Next-generation sequencing revealed 2% insertions and deletions in lung tissue and 1% in xenograft tissues. These results highlight EVs expressing VSV-G, but not spike protein, as an effective approach for CRISPR/Cas9 delivery; however, the delivery route needs further optimization of gene-editing efficiency in vivo.