Periodic Reporting for period 4 - CRISPR2.0 (Microbial genome defence pathways: from molecular mechanisms to next-generation molecular tools)
Período documentado: 2023-11-01 hasta 2025-04-30
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 report covers the first 2.5 years of the project from 01. May 2019 until 31. October 2021.
Within Aim 1, research has focused on structural and biochemical characterisation of type III CRISPR-Cas systems, specifically their RNA-guided targeting complexes and cyclic oligoadenylate-dependent effector nucleases. Initial studies determined the structural basis of cyclic-hexa-AMP (cA6) recognition by the CARF-HENP nuclease Csm6, revealing specific determinants of the Csm6-cA6 interaction (Garcia-Doval et al., Nature Communications, 2020). Subsequent studies have focused on other cOA-dependent effectors, including CARF-PD(D/E)xK and CARF-PIN nucleases, determining crystal structures of the respective proteins to shed light on their mechanisms of cOA activation and substrate nucleic acid binding. This work is still in progress and is complemented with biochemical and biophysical studies to validate structural observations. In a parallel effort, cryo-EM was used to determine multiple structures of the Cas10-Csm RNA-guided effector complex from E. italicus in the process of cA6 synthesis. These structure are currently being analyzed to generate an overview of the multi-step catalytic mechanism, particularly the molecular determinants responsible for product length specificity and cyclisation. This work is currently ongoing. Finally, initial steps were taken to translate the E. italicus Cas10-Csm6 system into a molecular RNA detection technology for viral diagnostics, exploiting the system for the detection of the SARS-CoV-2 virus to enable rapid detection without the need for enzymatic amplification. Despite initially promising results, the sensitivity could not be improved to reach femtomolar detection necessary for practical implementation. As other research groups published reports of successful development of CRISPR-based RNA detection technologies, this aspect of the project was discontinued.
Within Aim 2, research work focused on the structural and biochemical studies of CRISPR-associated transposons. Initial studies tackled type I CRISPR-transposon systems. However, the discovery of type V CRISPR-transposon systems in early 2019 broadened the scope of this subproject to include these systems as well. While research on type I systems has not yielded publishable results yet, a parallel effort focusing on type V systems led to the structural determination of the RNA-guided DNA targeting component Cas12k, revealing its mechanism of guide RNA- and PAM-dependent DNA targeting. This work was complemented by structural and biochemical studies of the transposon-associated AAA+ ATPase TnsC, which revealed that TnsC forms helical filaments in the presence of DNA and ATP. Follow-up studies confirmed the importance of filament formation for the RNA-guided DNA transposition activity of these systems in vivo. Overall, this work (published as Querques, Schmitz et al., Nature, 2021) provided important mechanistic insights into the molecular function of CRISPR-associated transposons and provided a framework for their engineering to develop RNA-guided DNA insertion technologies.
Within Aim 3, research was conducted on two initially selected novel host defence systems, namely the Shedu and Druantia systems. For both systems, the protein components (SduA for Shedu and DruE/DruH for Druantia) could be expressed and purified, and their structures determined using X-ray crystallography and cryo-EM. This structural work is in progress and is accompanied by biochemical studies to characterize the molecular activities of these proteins, and by functional studies to shed light on the interference mechanism of the defence systems and their modes of self vs. non-self discrimination.
Within Aim 1, research has focused on structural and biochemical characterisation of type III CRISPR-Cas systems, specifically their RNA-guided targeting complexes and cyclic oligoadenylate-dependent effector nucleases. Initial studies determined the structural basis of cyclic-hexa-AMP (cA6) recognition by the CARF-HENP nuclease Csm6, revealing specific determinants of the Csm6-cA6 interaction (Garcia-Doval et al., Nature Communications, 2020). Subsequent studies have focused on other cOA-dependent effectors, including CARF-PD(D/E)xK and CARF-PIN nucleases, determining crystal structures of the respective proteins to shed light on their mechanisms of cOA activation and substrate nucleic acid binding. This work is still in progress and is complemented with biochemical and biophysical studies to validate structural observations. In a parallel effort, cryo-EM was used to determine multiple structures of the Cas10-Csm RNA-guided effector complex from E. italicus in the process of cA6 synthesis. These structure are currently being analyzed to generate an overview of the multi-step catalytic mechanism, particularly the molecular determinants responsible for product length specificity and cyclisation. This work is currently ongoing. Finally, initial steps were taken to translate the E. italicus Cas10-Csm6 system into a molecular RNA detection technology for viral diagnostics, exploiting the system for the detection of the SARS-CoV-2 virus to enable rapid detection without the need for enzymatic amplification. Despite initially promising results, the sensitivity could not be improved to reach femtomolar detection necessary for practical implementation. As other research groups published reports of successful development of CRISPR-based RNA detection technologies, this aspect of the project was discontinued.
Within Aim 2, research work focused on the structural and biochemical studies of CRISPR-associated transposons. Initial studies tackled type I CRISPR-transposon systems. However, the discovery of type V CRISPR-transposon systems in early 2019 broadened the scope of this subproject to include these systems as well. While research on type I systems has not yielded publishable results yet, a parallel effort focusing on type V systems led to the structural determination of the RNA-guided DNA targeting component Cas12k, revealing its mechanism of guide RNA- and PAM-dependent DNA targeting. This work was complemented by structural and biochemical studies of the transposon-associated AAA+ ATPase TnsC, which revealed that TnsC forms helical filaments in the presence of DNA and ATP. Follow-up studies confirmed the importance of filament formation for the RNA-guided DNA transposition activity of these systems in vivo. Overall, this work (published as Querques, Schmitz et al., Nature, 2021) provided important mechanistic insights into the molecular function of CRISPR-associated transposons and provided a framework for their engineering to develop RNA-guided DNA insertion technologies.
Within Aim 3, research was conducted on two initially selected novel host defence systems, namely the Shedu and Druantia systems. For both systems, the protein components (SduA for Shedu and DruE/DruH for Druantia) could be expressed and purified, and their structures determined using X-ray crystallography and cryo-EM. This structural work is in progress and is accompanied by biochemical studies to characterize the molecular activities of these proteins, and by functional studies to shed light on the interference mechanism of the defence systems and their modes of self vs. non-self discrimination.
For Aim 1, the research has generated new insights into the cOA-dependent signalling in CRISPR-Cas systems, substantially advancing our knowledge of the mechanisms of cOA sensing. Ongoing work will focus on completing the structural and mechanistic studies of other CARF-nuclease effectors to shed light on their aOA sensing mechanisms and substrate nucleic acid specificity. These studies will on one hand shed light on the molecular mechanisms of these enzymes in prokaryotic genome defence pathways. On the other hand, it is envisioned that this work will widen the spectrum of enzymes that could be used to couple cOA sensing to a detectable signal, thereby building the foundation for other CRISPR-based nucleic acid detection technologies.
Within the scope of Aim 2, the research has yielded revolutionary insights into the molecular architecture and mechanism of CRISPR-transposon systems, setting the stage for their exploitation as gene insertion tools. Ongoing structure work (by cro-EM) on type V systems will examine how the RNA-guided effector Cas12k is coupled to the transposase components TnsC, TniQ and TnsB to bring about RNA-guided, site specific DNA integration (Schmitz, Querques et al., bioRxiv, 2022). This will be complemented by efforts to adapt these systems for RNA-guided DNA insertion in eukaryotic (esp. animal) cells. Finally, future work within this subproject will continue to investigate type I CRISPR-transposons to identify mechanistic parallels and differences between them and the type V systems. These insights are expected to guide further technology development to finally generate a functional system for eyukaryotic cells.
Ongoing research work within Aim 3 will follow up on the initial structural analysis of Druantia and Shedu systems to characterise their molecular mechanisms. This work is expected to provide new insights into the biological functions of these systems and identify potential activities/factors that could be repurposed as novel tools for genetic engineering or nucleic acid detection.
Within the scope of Aim 2, the research has yielded revolutionary insights into the molecular architecture and mechanism of CRISPR-transposon systems, setting the stage for their exploitation as gene insertion tools. Ongoing structure work (by cro-EM) on type V systems will examine how the RNA-guided effector Cas12k is coupled to the transposase components TnsC, TniQ and TnsB to bring about RNA-guided, site specific DNA integration (Schmitz, Querques et al., bioRxiv, 2022). This will be complemented by efforts to adapt these systems for RNA-guided DNA insertion in eukaryotic (esp. animal) cells. Finally, future work within this subproject will continue to investigate type I CRISPR-transposons to identify mechanistic parallels and differences between them and the type V systems. These insights are expected to guide further technology development to finally generate a functional system for eyukaryotic cells.
Ongoing research work within Aim 3 will follow up on the initial structural analysis of Druantia and Shedu systems to characterise their molecular mechanisms. This work is expected to provide new insights into the biological functions of these systems and identify potential activities/factors that could be repurposed as novel tools for genetic engineering or nucleic acid detection.