CRISPR-Cas represent a groundbreaking tool in genetic engineering, and over the last decade, numerous Cas nucleases with novel properties have been identified for a variety of different applications.

Our metagenomics screen revealed a novel Cas12a2 nuclease variant, termed G-daseE, with an unexpected mode of action. In contrast to classical CRISPR systems, G-daseE induces collateral degradation of DNA and RNA upon gRNA-mediated recognition of a target RNA, which ultimately leads to cell death.

Here, we demonstrate the potential of G-daseE for therapeutic applications by selectively targeting and eliminating cancer cells. In cell culture studies with G-daseE ribonucleoprotein (RNP), we successfully achieved selective cell depletion by targeting the HPV oncogenes E6/E7 in cervical carcinoma cell lines, while sparing oncogene-negative cells.

Furthermore, we explored an additional, more therapeutically translational, delivery strategy using G-daseE-mRNA and synthetic gRNA. Recent advances in mRNA delivery technologies, such as lipid nanoparticle (LNPs), have shown great promise in cancer therapy.  We designed and tested different G-daseE mRNA constructs by varying stabilizing regulatory elements, codon optimizations, and nucleotide modifications to achieve optimal mRNA stability and translational efficiency. Using the optimal mRNA variant, we successfully induced programmed cell death in human cancer cells – an important step towards the development of LNP-based systems for in-vivo applications.

Overall, we have shown that G-daseE can be delivered as mRNA to induce selective cell elimination upon recognition of marker RNAs, providing an innovative strategy for targeted cancer therapies.