Exosome-mediated intercellular delivery of CRISPR/Cas9 targeting the hepatitis B virus genome

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) protein 9 (CRISPR/Cas9) technology was adapted from the adaptive immunity mechanism of bacteria. It recognizes specific DNA sequences through a single guide RNA (gRNA) and guides the Cas9 protein, which has endonuclease activity, to cut the DNA double strands. Compared with gene editing technologies such as zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN), the improved CRISPR/Cas9 system consists of only two parts, the gRNA and the Cas9 protein, and has advantages such as simple operation, short experimental cycle and low cost.

To determine whether single guide RNA (gRNA) and Cas9 protein exist in exosomes secreted by cells transfected with CRISPR/Cas9 expression plasmids, and to explore whether the CRISPR/Cas9 system can achieve editing of target genes in surrounding cells through intercellular transfer of exosomes, Wang Jie, Chen Ran and Lu Fengmin of Peking University transfected CRISPR/Cas9 expression plasmids into HuH7 cells, collected the cell culture supernatant, concentrated and purified the exosomes by differential centrifugation, measured the morphology and particle size of the exosomes by electron microscopy and a Malvern laser scattering particle size analyzer, and detected gRNA and Cas9 protein levels by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, respectively. (2) HuH7 cells respectively transfected with 1.2x hepatitis B virus (HBV) expression plasmids and HBV-specific CRISPR/Cas9 expression plasmids were co-cultured. After 2 days, intracellular HBV DNA was extracted, PCR-amplified and sequenced.

The results showed that the exosomes produced by HuH7 cells transfected with CRISPR/Cas9 expression plasmids not only contained full-length gRNA and Cas9 protein, but could also transfer their gene editing function between cells, achieving destruction of the HBV genome in surrounding cells.

As a potential delivery system, exosomes can transfer the gene editing function of the CRISPR/Cas9 system between cells by carrying functional gRNA and Cas9 protein. This phenomenon also suggests that when using the CRISPR/Cas9 system for gene therapy, it is necessary to consider the potential impact of exosomes carrying gRNA and Cas9 protein on surrounding and distant tissue cells. Source: Chinese Journal of Hepatology.