Nanoparticles for Enhanced Gene Editing: Improving Delivery, Activity, and Safety of CRISPR/Cas Proteins

CRISPR-Cas systems are used by researchers worldwide for gene editing and diagnostics. CRISPR-Cas9 is the most widely used and studied in several fields. However, researchers have discovered several CRISPR-Cas systems in bacteria over the years. Among these, a novel RNA-targeting CRISPR-Cas13 system has recently been used as an antiviral strategy against RNA viruses. Researchers observed that CRISPR reduced the replication of SARS-CoV-2 and ZIKV by up to 90% compared to controls. Despite the potential of CRISPR-Cas systems, the use of Cas9 and Cas13d faces several challenges that limit their effectiveness before reaching target cells. In this PhD project, we proposed and evaluated 12 nm gold nanoparticles (Au_NPs) for delivering SpCas9 and Cas13d in two contexts, melanoma cancer and viral infections, respectively. This approach aimed at addressing delivery challenges. Initially, we characterized the Au_NPs for cytotoxicity, confirming they were non-toxic and did not affect cell replication. We then assessed their ability to conjugate SpCas9 to their surface, demonstrating efficient protein binding via affinity binding between the NTA-Ni2+ groups on the nanoparticle and the 6x His tag of the protein (AuNP Cas9). We showed that the AuNP Cas9 could cleave DNA in vitro. In addition, we characterized the entry time, localization, and gene-editing capabilities of Au_NPs Cas9. We showed that Au_NPs Cas9 can spontaneously enter cells and perform gene editing in human A375 melanoma cell lines without the need for any transfection reagents. However, we observed that Au_NPs Cas9 mainly localized to the cytoplasm and cytoplasmic vesicles rather than nuclei, which explains its lower efficiency in performing gene editing in cells. In parallel, we tested the same Au_NPs to deliver CRISPR-Cas13d in Huh-7 cells infected with SARS-CoV-2 and ZIKV. The Au_NPs-Cas13d complexes were administered to Huh-7 cells infected with either virus in single or multiple doses. The study showed that Au_NPs-Cas13d cleaved target RNAs with comparable efficiency to lipofected ribonucleoprotein. In addition, we found that Au_NPs-Cas13d nanoparticles can spontaneously enter cells by endocytosis or diffusion before the first 4 hours of treatment. 7 Au_NPs-Cas13d colocalized with SARS-CoV-2 virions in early endosomes and reduced SARS-CoV-2 replication after a single administration, unlike RNPs, which exhibited no antiviral activity. However, Au_NPs-Cas13d was less effective at reducing ZIKV replication than lipofected Cas13d RNPs, likely due to a difference in intracellular localization. Therefore, our system demonstrated the ability to spontaneously deliver different CRISPR/Cas systems into cells while maintaining the cleavage capacity of Cas enzymes. In particular, the Cas13d approach reduces SARS-CoV-2 replication by 5-fold compared to lipofected Cas13 counterparts. Future experiments will involve humanized ACE-2 mice infected with SARS-CoV-2. infected with SARS-CoV-2 and treated with a breathable formulation of Au_NPs Cas13d. In addition, we are trying to evaluate whether our nanoformulation can also be delivered to the CRISPR-Cas12 system.