Novel antiviral strategies based on CRISPR-Cas13d

This thesis explores the expanding potential of the CRISPR-Cas13 system for RNA editing, focusing specifically on the Cas13d enzyme as a basis for developing antiviral strategies targeting RNA viruses. The thesis begins with a detailed overview of recent advancements in CRISPR-Cas technology, emphasizing therapeutic and diagnostic applications in virology. A central focus of this work is identifying conserved RNA sequences across human coronaviruses to ensure broad-spectrum applicability. Targeting these conserved regions, less susceptible to genetic variability, enables the selection of crRNAs that effectively inhibit SARS-CoV-2 and other human coronaviruses, highlighting their potential as a broad-spectrum antiviral tool. Experimental validation revealed significant reductions in viral replication, confirming Cas13d’s ability to target coronaviruses. Structural analyses of RNA targets showed that Cas13d effectively cleaves stably folded RNA structures, highlighting its utility against viruses with structured RNA genomes. The thesis also investigates Cas13d's ability to target HIV-1, further extending its therapeutic potential. Therapeutic use, however, still faces challenges, including optimizing in vivo delivery and minimizing off-target effects. Beyond therapeutic applications, the CRISPR-Cas13d system offers powerful diagnostic capabilities. Tools like SHERLOCK utilize Cas13’s collateral cleavage activity for rapid and specific virus detection, making this approach particularly suitable for low-resource and pandemic scenarios due to its simplicity, minimal equipment needs, and high specificity. Overall, this thesis positions CRISPR-Cas13d as a transformative tool for RNA-targeted antiviral and diagnostic applications, with substantial promise for addressing both existing and emerging viral threats.