Extracellular Vesicle-Liposome Hybrid Nanoparticles Delivery of CRISPR/Cas9 Induces a Unique DNA Repair Pattern in the HGF Gene of Stem Cells from Apical Papilla.

Abstract

Extracellular vesicles (EVs) have been investigated due to their natural biocompatibility and targeting capabilities. The specific approach of combining EVs with liposomes to create hybrid nanoparticles (ELNPs) for the delivery of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas9) system for deletion of the HGF gene in stem cells, but their effectiveness in encapsulating large nucleic acids is limited due to their small size. This study aimed to knock out the HGF gene by the CRISPR/Cas9 system by ELNPs, and it was expected that the efficiency of the CRISPR/Cas9 system transfer would increase compared to the usual methods of using lipofectamine in stem cells from apical papilla (SCAPs). In this study, gRNA suitable for the HGF gene is designed first, and after insertion into the CRISPR/Cas9 vector, it enters Lipofectamine 2000. In the next step, ELNPs are prepared after collecting EVs and hybridizing them with liposomes containing CRISPR/Cas9 vector. Then, these integrated nanoparticles were presented to SCAPs, and the removal of HGF gene expression was evaluated at the level of RNA and protein. This study showed that the CRISPR/Cas9 system can be efficiently transferred to SCAP cells using ELNPs. Genomic DNA sequencing analyses of SCAP cells showed a unique pattern of mutation, highly likely mediated through EVs. Quantitative PCR and protein staining further showed a decrease in HGF gene expression in the knockout cells. Moreover, cell proliferation analysis showed a decrease in cell proliferation in KO-HGF adipose cells compared to the nonedited counterpart. In summary, this study highlights the supportive role of EVs in facilitating cell transfection and promoting a dominant DNA repair pattern, likely through an RNA-mediated mechanism, rather than the random insertions and deletions typically induced during CRISPR editing of the HGF gene in SCAPs.