• Nanoparticles Gold CRISPR/Cas9 Delivery

My thesis is part of a work aiming to use nanotechnology to both exalt CRISPR/Cas9 qualities and overcame the aforementioned criticisms related to this system. The considered nanotechnological approach consists in plasmonic GNPs conjugated with Cas9 molecules, referred to as GNP-Cas9. Specifically, my work is centered on characterizing the chemical and physical properties of the gold nanoparticles conjugated to the ribonucleoprotein to further study their toxicity and cellular internalization dynamics. The first part of my work has focused on assessing the dimensional and physical characteristics of the gold nanoparticles taken into account, for subsequently functionalizing them with Cas9 protein, taking advantage of a directed linkage between the NTA group present on nanoparticles and the 6xhistidine-tag fused with the Cas9 protein. I have then tested the so obtained nanoformulation for its ability of cleaving a specific target DNA molecule after addition of the appropriate gRNA. In addition, I have performed toxicity studies of GNP functionalized with 6xhis-
tag-HSA proteins with the same link chemistry used for Cas9, testing the nanoformulation viability on both human A375 melanoma cells and Danio rerio (zebrafish) animal model. I have then investigated the internalization and localization of GNP conjugated with Cas9:gRNA in the same cellular model, under both active and disactivated cellular energy-dependent uptake conditions. Finally, I have collaborated to fine-tune a protocol for the production of homemade 6xhis-tag-Cas9 (hmCas9), which was subsequently tested for its stability and catalytic activity. I have tested the targeting and cleavage efficiency of hmCas9 both in vitro and in vivo, injecting the ribonucleoprotein complex targeting the tyrosinase gene inside zebrafish embryos. The gene editing efficiency has been evaluated phenotypically by the quantification of embryos depigmentation, and genotypically, by the means of high-resolution melting analyses.
I can conclude that the nanoformulation I have characterized maintains the catalytic activity of CRISPR/Cas9 system highlighted in in vitro experiments, without showing toxic effects neither in cells, nor in zebrafish. Furthermore, the GNPs demonstrate to be a feasible tool for delivering CRISPR/Cas9 technology inside cells by spontaneous internalization, allowing the localization of the system in the nuclear compartment.