Trabajo presentado en Current Trends in Biomedicine Workshop, celebrado en Baeza (España) del 02 al 04 de noviembre de 2021.
CRISPR-Cas13 is a family of class II type VI CRISPR systems which have been successfully used to induce RNA degradation in mammalian cells, plants and the fission yeast. We have recently optimized the use of CRISPR-RfxCas13d to efficiently induce mRNA knockdown in animal embryos, including zebrafish, medaka, killifish and mouse embryos. Since Cas13d is an efficient and specific RNA nuclease, it has been proposed the use of this system to target RNA viruses such as Influenza virus and the causative agent of Covid-19 pandemic, the novel coronavirus SARS-CoV-2. As a proof of principle, it has been demonstrated by other groups that the transgenic and constant expression of Cas13d with a combination of several gRNAs targeting nucleocapsid or RNA-dependent RNA polymerase genes from SARS-CoV-2 is able to reduce viral RNA in cells transfected with these gRNAs and challenged with SARS-CoV-2 virus. However, to use CRISPR-Cas13d as a potential antiviral therapy, a formulation able to be delivered in vivo and induce transient targeting is needed. Here, we show Cas13d-gRNA ribonucleoprotein (RNP) complex is able to efficiently target different RNAs from viruses belonging to the Nidovirales order including BEV torovirus, 229E coronavirus and SARS-CoV-2 when injected in zebrafish embryos as an in vivo model system. Finally, we have also achieved an efficient delivery approach based on fusogenic cellsomes which allows the entry of Cas13dgRNA RNPs in equine cell cultures infected with BEV torovirus leading to viral to a drastic reduction in viral protein expression and progeny virus production. We believe these preliminary but promising results support the future use of CRISPR-Cas13d RNPs as a potential antiviral therapy. Further analysis will show whether our optimized encapsulated RNP is able to degrade viral RNA in animal models infected with SARS-CoV-2 and other RNA viruses.