Periodic Reporting for period 5 - CRISPR2.0 (Microbial genome defence pathways: from molecular mechanisms to next-generation molecular tools)
Periodo di rendicontazione: 2025-05-01 al 2025-10-31
The constant arms race between prokaryotic microbes and their molecular invaders has driven the evolution of complex genome defence mechanisms. The CRISPR-Cas defence systems provide adaptive RNA-guided immunity against invasive nucleic acid elements. CRISPR- associated proteins such as Cas9, Cas12a and Cas13, which function as RNA-guided enzymes that cleave target DNA or RNA molecules, have been engineered as powerful tools for precision genome editing, gene expression control and nucleic acid detection. These tools form the basis of revolutionary applications in molecular medicine, including gene therapy and viral diagnostics. However, these technologies suffer from drawbacks that limit their efficacy and versatility, necessitating the search for additional exploitable molecular activities. Building on our recent structural and biochemical studies, this project will investigate the molecular architectures and mechanisms of CRISPR-associated systems and other genome defence mechanisms, aiming to shed light on their biological roles and inform their technological development. Specifically, the proposed studies will examine (i) the molecular basis of cyclic oligoadenylate signalling in type III CRISPR-Cas systems (Aim 1), (ii) the mechanism of transposon-associated type I CRISPR-Cas systems and their putative function in RNA-guided DNA transposition (Aim 2) , and (iii) molecular activities associated with recently described non-CRISPR defence systems (Aim 3), aiming to translate discoveries in each of the areas into novel molecular technologies.
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.
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.
CRISPR2.0 advanced the field well beyond the state of the art. Before the project, the molecular basis of type III CRISPR immunity was only partly understood. By explaining how immune signals are produced, how they activate defence enzymes, and how these events are linked to target recognition, the project delivered a much more complete understanding of this system (Garcia-Doval et al., 2020; Jungfer et al., 2023; Jungfer et al., 2025).
The project also moved genome engineering forward by providing the first detailed framework for RNA-guided DNA insertion by CAST systems (Querques, Schmitz et al., 2021; Schmitz, Querques et al., 2022; Finocchio et al., 2025). This was a major advance because it opened the way to a new class of programmable DNA insertion tools that may eventually complement or improve on current genome editing approaches.
A further important advance was the discovery of new defence mechanisms. Shedu, DdmDE, and SPARTA showed that microbial immunity is broader and more inventive than previously known (Loeff et al., Cell, 2025; Science, 2024; Finocchio et al., NAR, 2024). These findings not only expanded basic knowledge, but also created new starting points for future biotechnology.
The final phase of the project aimed to deepen these advances by obtaining a more complete view of RNA-guided transposition, improving CAST systems for implementation in mammalian cells, and continuing work on spacer acquisition and the Druantia defence system. More broadly, the project established a strong foundation for future tools in programmable DNA insertion, molecular detection, and the wider use of microbial defence systems in biotechnology and, in the longer term, therapeutic genome engineering.
The project also moved genome engineering forward by providing the first detailed framework for RNA-guided DNA insertion by CAST systems (Querques, Schmitz et al., 2021; Schmitz, Querques et al., 2022; Finocchio et al., 2025). This was a major advance because it opened the way to a new class of programmable DNA insertion tools that may eventually complement or improve on current genome editing approaches.
A further important advance was the discovery of new defence mechanisms. Shedu, DdmDE, and SPARTA showed that microbial immunity is broader and more inventive than previously known (Loeff et al., Cell, 2025; Science, 2024; Finocchio et al., NAR, 2024). These findings not only expanded basic knowledge, but also created new starting points for future biotechnology.
The final phase of the project aimed to deepen these advances by obtaining a more complete view of RNA-guided transposition, improving CAST systems for implementation in mammalian cells, and continuing work on spacer acquisition and the Druantia defence system. More broadly, the project established a strong foundation for future tools in programmable DNA insertion, molecular detection, and the wider use of microbial defence systems in biotechnology and, in the longer term, therapeutic genome engineering.