The ERC project CRISPR2.0 aimed to understand how microbes defend themselves against invading genetic material and how these natural systems can be turned into useful tools for biotechnology. The work focused on three main areas: type III CRISPR-Cas systems, CRISPR-associated transposons (CASTs), and newly discovered microbial defence systems. Across all three areas, the project combined structural biology, biochemistry, genetics, and microbiology to deliver major advances in both basic science and technology development.
In the first area, the project clarified how type III CRISPR-Cas systems detect foreign RNA and trigger an antiviral response. It showed how the enzyme Csm6 is activated by cyclic oligoadenylate signalling molecules and how this activity is later switched off to protect the host cell (Garcia-Doval et al., Nature Communications, 2020). Further work explained how other type III defence enzymes respond to the same signal (Jungfer et al., Nucleic Acids Research, 2023) and how the main type III CRISPR complex recognizes target RNA and activates immune signalling (Jungfer et al., Nucleic Acids Research, 2025). Together, these studies provided a complete molecular picture of how this branch of CRISPR immunity works.
A second major part of the project focused on CAST systems, which combine CRISPR-based targeting with the ability to insert DNA at chosen sites. CRISPR2.0 showed how type V CAST systems recognize target DNA and recruit the proteins needed for DNA insertion (Querques, Schmitz et al., Nature, 2021; Schmitz, Querques et al., Cell, 2022). It also revealed how type I CAST systems assemble their transposition machinery (Finocchio et al., Nucleic Acids Research, 2025). These findings provided the first detailed molecular explanation of RNA-guided DNA insertion and laid the groundwork for future genome engineering applications. During the final period, this knowledge was also used to support efforts to improve CAST performance in mammalian cells.
The third area of the project explored new microbial defence systems. The work revealed previously unknown ways in which microbes detect and eliminate foreign DNA. The Shedu system was shown to recognize free DNA ends as a sign of invasion (Loeff et al., Cell, 2025). The DdmDE system revealed how a guide-dependent Argonaute protein can direct a helicase-nuclease complex to destroy plasmids; the report highlights this as a major project output linked to a Science 2024 publication. The project also clarified the activation mechanism of the Argonaute-based system SPARTA (Finocchio et al., NAR, 2024). In addition, work on the Druantia system produced important mechanistic insights and is expected to lead to a future publication.
Beyond these discoveries, the project also delivered important methodological advances by combining cryo-electron microscopy, X-ray crystallography, biochemistry, and microbiology to study large and dynamic molecular machines. These approaches made it possible to analyse systems that had previously been difficult to access. The results also had clear technological relevance: CAST research created a basis for programmable DNA insertion tools, while type III signalling studies suggested new approaches for nucleic-acid detection.
Overall, the project’s main achievements were: a full mechanistic framework for type III CRISPR signalling; a molecular understanding of RNA-guided DNA insertion by CAST systems; and the discovery and explanation of new defence pathways including Shedu, DdmDE, and SPARTA. These results were widely disseminated through high-impact publications. In the final reporting period, the team completed key structural and biochemical studies, continued work on spacer acquisition and Druantia, and placed strong emphasis on exploitation and dissemination, especially by using CAST findings to guide the development of improved genome engineering tools.
