[Exosome-mediated CRISPR/Cas9 system targets to cut the intercellular transmission function of hepatitis B virus...

Objective: To determine whether single-stranded guided RNA (gRNA) and Cas9 protein exist in the exosome secreted by cells transfected with CRISPR/Cas9 expression plasmid. Furthermore, to explore whether CRISPR/Cas9 system can edit target genes of peripheral cells through the intercellular transmission of exosomes. Methods: (1) The CRISPR/Cas9 expression plasmid was transfected into HuH7 cells. The supernatant was collected and the exosomes were concentrated and purified by differential centrifugation. The morphology and particle size of exosomes were determined by electronic microscopy and Malvern laser scatter granulometry. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to detect the levels of gRNA and Cas9 protein. (2) HuH7 cells transfected with pBB4.5 1.2×HBV and HBV specific CRISPR/Cas9 expression plasmids were co-cultured. After 2 days, HBV DNA was extracted and sequenced by PCR. Results: There were not only full-length gRNA and Cas9 protein in the exosomes of Huh7 cells transfected with CRISPR / Cas9 expression plasmid. In addition, gene-editing functions were delivered between the cells to achieve the destruction of HBV genome of surrounding cells. Conclusion: The CRISPR-Cas9 gene-editing system has the potential to deliver exosomes between cells via carrying functional gRNA and Cas9 proteins. This phenomenon hints that we are in the process of using the CRISPR/Cas9 system for gene therapy. Therefore, it is necessary to consider the potential effects of exosomes-carried gRNA and Cas9 proteins on the surrounding cells of the distal tubules.