Projects (in alphabetical order)
Prof. Dr. Gert Bange, Philipps-University Marburg
Structure, mechanism and function of class I CRISPR-Cas systems
Homepage Bange Laboratory
CRISPR-Cas systems are structurally and mechanistically highly diverse prokaryotic adaptive immune systems that protect against mobile genetic elements. Class I CRISPR-Cas systems feature multisubunit effector complexes. We could recently demonstrate that even closely related subtypes, i.e. the type I-F and I-F variant (I-Fv), differ substantially in their structure and DNA surveillance mechanism. Furthermore, CRISPR-Cas systems have been implied to function in non-canonical processes, such as e.g. transcriptional control, stress response and pathogenicity development. Others and we could show that the CRISPR adaptation protein Cas1 interacts with the redox-stress sensing and signaling two-component system YedVW. The aim of this proposal is to produce an en-detail structural, mechanistic and functional understanding of poorly understood class I CRISPR-Cas systems, such as e.g. the Shewanella putrefaciens type I-Fv and the plasmid borne Aromatoleum aromaticum type IV system. Moreover, we aim at a structural, mechanistic and functional understanding of the interaction of Cas1 and YedVW in the redox-stress induced stress response of Escherichia coli.
Dr. Franz Baumdicker, Albert-Ludwigs-Universität, Freiburg
Evolutionary dynamics and phylogenetic inference of CRISPR systems in prokaryotic populations
Homepage Baumdicker Laboratory
The revolutionary CRISPR/Cas technology to alter genomes has its origin in the natural CRISPR systems in bacteria and archaea. Many of these systems can act as an adaptive defense system targeting specific viral sequences. Beyond this defensive function, CRISPR systems can influence the evolution of bacterial populations without viruses being involved. The overarching goal of this project is to develop and apply new state of the art models for the evolution of CRISPR in prokaryotes. Many studies have focused on the coevolutionary aspects between CRISPR possessing bacteria and the corresponding viruses. In contrast, we will consider the intrinsic evolution of CRISPR Cas systems in bacteria and archaea. In particular, we aim to answer the following questions:
a) To identify specific targets the CRISPR system contains an array of spacers that align with the targets. This spacer array provides a unique insight into the evolutionary history since spacers in this array are arranged in chronological order. Can we explain the observed pattern in spacer arrays and identify spacers under selection? Can we reconstruct ancestral spacer insertion times and link them to their ancestral history?
b) Accidental self-targeting usually leads to death such that potential self-targets in the bacterial genome are presumably under strong selection. Can we model the selective effect of self-targeting CRISPR systems from the genome sequences of bacteria possessing CRISPR?
c) CRISPR systems that target closely related species are capable to block recombination between bacterial strains and can modulate the frequency of genes in the population. Moreover, bacteria can use such systems as an offensive weapon targeting closely related competitors. How does CRISPR influence the frequency of genes in bacteria? Can we understand within population dynamics of CRISPR arrays targeting related species? Are CRISPR systems targeting subpopulations a promising new anti-microbial treatment?
We will address these questions with a mixture of mathematical and computational approaches
Prof. Dr. Chase Beisel, HIRI, Würzburg
Functional characterization of extensively self-targeting CRISPR-Cas systems in the bacterial plant pathogen Xanthomonas albilineans
Homepage Beisel Laboratory
CRISPR-Cas systems are widely recognized as RNA-guided, adaptive immune systems in prokaryotes that detect and eradicate foreign genetic material. However, recent work highlighting alternative roles in gene regulation and immune avoidance are challenging the singular definition of CRISPR as an immune system. Still unclear are the inherent roles of CRISPR and how they impact the overall physiology and behavior of prokaryotic life. We previously showed that the most abundant Type I CRISPR-Cas systems can be readily converted into transcriptional repressors by disrupting the system’s endonuclease Cas3. We hypothesized that Type I systems may perform a similar function naturally. By searching prokaryotic genomes for genome-targeting CRISPR arrays, we identified the plant pathogen Xanthomonas albilineans as an extremely promising case. The genome of this bacterium contains two complete Type I CRISPR-Cas systems, a I-C system and a I-F system, encoding a total of 64 genome-targeting spacers. Further analysis showed that the target sites are flanked by known protospacer-adjacent motifs (PAMs), and the I-C system appears to have a defective Cas3. These preliminary results strongly suggest that the CRISPR-Cas systems in this agriculturally important bacterium could act as transcriptional repressors and regulate a collection of cellular processes. The goal of this proposal is to characterize the properties of the two CRISPR-Cas systems in X. albilineans. We hypothesize that both systems are functionally expressed and regulate transcription of target sites through lack of Cas3 activity. To test this hypothesis, we propose the following two objectives:
Objective 1: Determine the outcome of DNA targeting by each CRISPR-Cas system using cell-free transcription-translation systems.
Objective 2: Determine the expression pattern and function of each CRISPR-Cas system in X. albilineans.
If successful, the proposed work could reveal novel roles of CRISPR-Cas systems that extend beyond adaptive immunity and provide the basis to identify similar functions across the diversity of Type I CRISPR-Cas systems found in the prokaryotic world. The PI has extensive experience with CRISPR-Cas systems and bacterial genetics, and the project directly aligns with the goals of SPP 2141.
Dr. Ulrike Endesfelder, Max Planck Institute for Terrestrial Microbiology, Marburg
Studying protein organization and dynamics of the Type I-Fv CRISPR-Cas system of Shewanella putrefaciensCN-32 at a high spatiotemporal resolution in living cells
Homepage Endesfelder Laboratory
Studying protein organization and dynamics of the Type I-Fv CRISPR-Cas system of Shewanella putrefaciensCN-32 at a high spatiotemporal resolution in living cells. The various CRISPR-Cas (Clustered regularly interspaced short palindromic repeats and CRISPR associated proteins) systems share some conserved features but are in general highly variable. While the organization of the Type II system and its signature protein Cas9 have already been studied in living cells using single-molecule methods, information on single-molecule (inter)dynamics of proteins of the Type I system stem from in vitro assays. There is, however, accumulating evidence that the interactions of CRISPR-Cas systems with their intracellular environment are much more complex than previously thought, going far beyond those of a monofunctional anti-viral defense mechanism. It is therefore becoming increasingly clear, that to fully understand their functionality, CRISPR-Cas systems cannot be studied in isolation. In this project, we will study the minimal CRISPR-Cas Type I-Fv system from Shewanella putrefaciensCN-32 in vivo using single-molecule localization microscopy (SMLM) and single-particle tracking (SPT). SMLM allows us to localize individual Cas molecules at a high spatiotemporal resolution and to observe their diffusion dynamics under different conditions and target affinities by SPT. In particular we aim to• characterize the kinetics of the Cascade Typ I-Fv complex in a spatiotemporal manner in living cells,• determine the kinetics and molecule interactions in PAM recognition and R-loop stabilization,• and to explore the significance of interactions between Cascade complexes and other proteins and molecular machineries. Furthermore, we aim to establish robust SPT and SMLM imaging in various less accommodating microorganisms, to be able to study CRISPR-Cas systems in their native host.
Prof. Dr. Uri Gophna, Tel Aviv University, Israel
The effects of CRISPR-Cas systems on microbial genetic diversity
Homepage Gophna Laboratory
Since CRISPR-Cas systems can impede lateral gene transfer, it is often assumed that they reduce genetic diversity in prokaryotes. However, data from our lab suggest the exact opposite: namely that these systems generate a high level of genomic diversity within populations. We have recently combined genomics of environmental strains and experimental genetics of halophilic archaea to show that they frequently acquire CRISPR spacers from chromosomes of related species in the environment, and similar observations have been made for Neisseria strains.The presence of these spacers then reduces gene exchange between lineages, indicating that CRISPR-Cas contributes to diversification. We have also shown, jointly with the Marchfelder lab, that inter-species mating events induce the acquisition of spacers against a strain's own replicons, supporting a role for CRISPR-Cas systems in generating deletions in natural plasmids and non-essential genomic loci, again increasing genome diversity within populations.To test our hypothesis that CRISPR-Cas systems increase within-population diversity, we will analyze hundreds of genomes of halophilic archaea as well as human-associated Neisseria strains comparing CRISPR-positive and CRISPR-negative lineages. We will then infer in which cases did CRISPR-Cas systems cause the deletion of genomic islands or other integrated selfish elements. Additionally,we will perform laboratory experiments attempting to induce such deletions, and explore the role of some CRISPR-associated genes in recombination processes. Taken together these analyses will reveal new roles for CRISPR-Cas systems in shaping the mobile dispensable part of microbial pan-genomes.
Prof. Dr. Dina Grohmann, Institut für Biochemie, Genetik und Mikrobiologie, Regensburg
Single-molecule analysis of non-canonical Cas1 and Cas9 complexes involved DNA repair and posttranscriptional gene regulation
Homepage Grohmann Laboratory
There is growing evidence that CRISPR-Cas machineries fulfil functions beyond their canonical role as defence systems. Among others, components of CRISPR-Cas are thought to be implicated in endogenous gene regulation, regulation of bacterial virulence, DNA repair, cell dormancy and infection and genome evolution. We aim to contribute to the understanding of the molecular principals that govern non-canonical CRISPR-Cas functions exploiting the power of fluorescence-based single molecule methods that are established in my laboratory. In context of the CRISPR-Cas SPP2141, we envisage to carry out the following projects: (i) Cas9 variants are usually involved in crRNA-directed DNA cleavage. Cas9 from Franciscella novicida (Fn) can form an unusual complex composed of scaRNA, tracrRNA and the blp mRNA which eventually leads to blp mRNA degradation. Similarly, the Cas9 variant from Campylobacter jejuni – the smallest Cas9 variant characterised so far – was recently shown to be also associated with mRNAs that are posttrancriptionally downregulated. How do the canonical crRNA/dsDNA/Cas9 complexes differ from the caRNA/tracrRNA/mRNA/ FnCas9 and crRNA/tracrRNA/mRNA/CjCAs9 complexes, respectively, in terms of structure and dynamics? Single molecule FRET measurements will be employed to answer this burning question. (ii) Cas1 from E. coli and P. furiosus have been shown to interact with proteins involved in DNA repair (e.g. RecB, RecC, RvuB and UvrC). Using a combination of the single-molecule pulldown (SimPull) and smFRET, we can directly isolate these complexes from E.coli without the need for in vitro reconstitution, are able to determine the stoichiometry of these complexes and can determine the mechanism of action using smFRET. State-of-the-art super-resolution microscopy and particle tracking experiments we will determine the localisation and interactions of Cas1 in vivo.
Prof. Dr. Wolfgang Hess, Albert-Ludwigs-Universität, Freiburg
Cross-talk and extensive rewiring of CRISPR-Cas systems with the cellular regulatory machinery in Synechocystis sp. PCC 6803
Hompepage Hess Lab
With this proposal we aim at analyzing the CRISPR-Cas systems in cyanobacteria with a focus on their interaction with the regulatory machinery of the host. As model organism we have chosen the unicellular strain Synechocystis sp. PCC 6803. This strain carries three CRISPR-Cas systems. Two of them were classified as subtype I-D and subtype III-D. The third constitutes a III-B variant (III-Bv), characterized by a Cmr6_Cmr1 fusion, a replaced Cmr5 protein, the possible involvement of a peptidase and the lack of an associated Cas6 maturation endoribonuclease. Bioinformatic predictions suggest similar variant systems to exist also in other prokaryotes. Instead of Cas6 we found that the host-encoded endoribonuclease E was recruited as the major maturation enzyme, which raises questions about the involved molecular and evolutionary mechanisms. It directly suggests that there are additional host-and CRISPR-encoded factors that need to be identified, e.g., in loading mature crRNAs into the surveillance complexes, in performing the 3’ end maturation of crRNAs, in modulating RNase specificity as well as the type of nucleic acid targeted by the CRISPR-Cas complexes. We will address these questions using pull-down assays, the characterization of deletions for respectivecandidate genes in interference assays and by molecular analyses, the elucidation of an RNase E CRISPR-repeat-RNA cocrystal structure and determination of the entire CRISPR3 complex structure.However, the possible interplay between CRISPR-associated RNases and the processes governing the maturation and stability of transcripts appears more complex than previously anticipated also with regard to the Cas6-dependent other two CRISPR-Cas systems. Therefore, we will also address the possible impact on the host transcriptome as well as redox control through the host-encoded RpaBNblStwo-component system. In both cases, the involvement of additional enzymes and the possible consequences for the post-transcriptional regulation of gene expression will be followed. The project work will be complemented by the metagenomic analysis of cyanobacterial mass developments to understand the evolving architecture of the studied types of CRISPR-Cas systems and their integration into and interaction with the cellular regulatory apparatus.
Prof. Dr. Gabriele Klug, Justus-Liebig-Universität, Gießen
CRISPR-Cas functions in the stress response of Rhodobacter capsulatus
Homepage Klug Laboratory
The genome of the facultative photosynthetic alphaproteobacterium Rhodobacter capsulatus contains several CRISPR-Cas systems of different types, while the related R. sphaeroides does not carry any CRISPR-Cas components. RNAseq analysis revealed increased levels of some of the CRISPR RNAs in presence of singlet oxygen. Northern blots confirmed this result and revealed also effects of other stress conditions on CRISPR RNAs that are part of a class 2, Type VI system. Overexpression of the Cas13a protein from this system resulted in decreased resistance to ampicillin and hydrogen peroxide in R. sphaeroides and in slightly increased resistance to hydrogen peroxide in R. capsulatus. The goal of this project is to elucidate the signaling pathways leading to stress-dependent expression of CRISPR-Cas components in R. capsulatus and the molecular mechanisms that underlie the effect of CRISPR-Cascomponents on stress resistances in both strains. We will test a bigger variety of stress conditions for their effects on expression of CRISPR-Cas components and test strains that overexpress or lack selected CRISPR-Cas components under more growth conditions. The same analyses will be performed in mutant strains that lack certain regulatory factors or known RNases to test their involvement in the responses. A bioinformatic approach should search for RNAs in the R. capsulatus genome, which may be targets of CRISPR RNAs. If such putative targets exist, we will analyze the RNA interaction and possible processing in vitro and turn-over of the RNAs in vivo. For those CRISPR-Cas components that show clear phenotypic effects when deleted or overexpressed, we will perform global transcriptome and proteome studies with the mutant strains to identify all RNAs and proteins with changed expression level. At the start of the project we will concentrate on the class 2 system and the Cas13a protein that is also analyzed in other projects of the priority program and showed clear effects in our preliminary studies, but further CRISPR-Cas components should also be included. Detailed strategies for the elucidation of the CRISPR-Cas mediated pathways will be designed when intermediate results are available.
This project should add to our understanding on CRISPR-Cas functions that are not related to phage defense and should unravel the underlying molecular mechanism and signaling pathways.
Prof. Dr. Anita Marchfelder, Universität Ulm
CRISPR-Cas functions beyond defence in haloarchaea
Homepage Marchfelder Laboratory
Ten years ago CRISPR-Cas was discovered as prokaryotic immune system that is used by bacteria and archaea to defend themselves against viruses. Detailed investigation of this system has since yielded surprising insights into its molecular mechanisms allowing CRISPR-Cas to be harnessed as a tool for molecular biology applications. Bioinformatics analyses are constantly revealing new variants of this system which are currently divided into two classes and six types. New studies are reporting additional functions beyond its initially defined defence function, involving the complete CRISPR-Cas system or one or more of its components. These functions encompass (i) involvement in DNA repair, (ii) regulation of virulence, (iii) regulation of group behaviour and stress tolerance, (iv) functions of individual system components, e.g., in the regulation of gene expression and (v) impacts on ecology and evolution. To date the CRISPR-Cas systems have not been systematically investigated for additional functions, and archaeal CRISPR-Cas systems in particular have not been examined with respect to functions beyond defence. Therefore we will investigate haloarchaeal CRISPR-Cas systems and their potential to perform other tasks beyond defence. We already characterised the defence function of the CRISPR-Cas system of the haloarchaeon H. volcanii in detail. H. volcanii encodes a CRISPR-Cas Type I-B system and we determined the requirements for an active defence reaction (e.g., PAM sequences, parameters for an active crRNA, Cascade composition). In addition, we developed a CRISPRi tool for gene repression in Haloferax. In the frame of this project we want to identify novel functions of haloarchaeal CRISPR-Cas systems. (1) We want to determine additional functions of the Cas1, Cas2 and Cas4 proteins. (2) We will investigate orphan CRISPR-Cas genes. Several haloarchaeal organism contain only CRISPR genes or an incomplete cas gene set. We will analyse whether these genes are still active and what kind of function they have. (3) We want to unravel the regulation of endogenous genes by the CRISPR-Cas system. CRISPR loci can contain spacers matching the own genome. We want to determine whether these spacers can regulate the expression of the endogenous genes. (4) Biofilm formation in bacteria has been reported to be regulated by CRISPR-Cas. We will investigate the regulation of biofilm formation by CRISPR-Cas in haloarchaea.
Prof. Dr. Alice McHardy, Braunschweig
Learning structures in the CRISPR-Cas system using deep learning architectures
Homepage McHardy Laboratory
In recent years, deep neural networks, such as recurrent neural networks (RNN) and convolutional neural networks (CNN), have become a central and remarkably effective modeling tool for classification tasks, such as speech and image recognition, as well for text classification, and outperform classical machine learning approaches, and even humans, in video and image recognition. However, RNNs are still barely tested and applied on genetic datasets. In this application, we present the use of RNNs to model CRISPR regions and their associated genomes, as well as their targets. By visualizing the hidden states of the trained network we will get insights into structural properties, which are shared by CRISPR loci and their associated genomic and target sequences, such as the Protospacer Adjacent Motif (PAM). Due to the fact that nucleotide-level models will be trained unsupervised, the method is capable of detecting yet unknown structural properties of the CRISPR system. We first aim to catalog all CRISPR structures recovered from large collection of metagenomes (’Objective 1’). With this data and together with 2509 already identified CRISPR loci from complete genomes, we will employ RNNs to uncover hidden structures (’Objective 2’). The trained model will also be used to validate putative CRISPR loci, which make up the majority of current CRISPR databases and to refine CRISPR subtype classification (’Objective 3’).
Dr. Lennart Randau, Max-Planck-Institut für terrestrische Mikrobiologie, Marburg
Functional analysis of Shewanella putrefaciens Type I-Fv and Aromatoleum aromaticum Type IV CRISPR-Cas ribonucleoprotein complexes
Homepage Randau Laboratory
Archaea and Bacteria have evolved a variety of strategies to cope with foreign nucleic acids. One of these strategies employs CRISPR-Cas systems, which were originally described to mediate adaptive prokaryotic immunity, providing defense against viral attacks. Cas proteins form ribonucleoprotein complexes with CRISPR RNAs (crRNAs). These RNA molecules contain so-called spacer sequences that identify target nucleic acids via base complementarity. Recently, it emerged that CRISPR-Cas systems can repurpose this RNA guidance mechanism as (i) the spacer content of CRISPR elements is highly dynamic and (ii) a large repertoire of divergent Cas proteins was observed. Our group studies two Class1 CRISPR-Cas ribonucleoprotein complexes with reduced Cas protein content. First, we investigate the Type I-Fv complex of Shewanella putrefaciens, which was shown to target DNA in the absence of commonly observed large and small subunits. The crystal structure of this assembly suggests that unique features of the Cas proteins Cas5fv and Cas7fv compensate for the loss of these subunits. We aim to study the detailed DNA targeting mechanism of this complex and propose to analyze formation of crRNA-DNA interactions using biolayer interferometry and activity assays with Cas protein mutants and target DNA variants. Collaborative efforts aim to investigate the structural and functional involvement of the helicases Cas3 and DinG and the dynamics of DNA scanning. Second, we identified a Type IV CRISPR-Cas system in Aromatoleum aromaticum and produced and purified recombinant Type IV ribonucleoprotein complexes. Type IV CRISPR-Cas does not contain adaptation modules or known DNA nucleases and its cellular function is unknown. We aim to characterize the composition and stoichiometry of the Type IV complexes using gel-filtration and mass-spectrometry approaches.The apparent absence of DNA nucleases suggests that the unknown activity of these CRISPR-Cas systems might not rely on the degradation of target DNA. Therefore, we aim to study the formation and stability of Typ IV complex-mediated crRNA-DNA interactions, so-called R-loops, in vitro and in vivo using footprinting and bisulfite sequencing techniques. The co-occurrence of transposons in the vicinity of Type IV CRISPR-Cas systems indicates that they potentially facilitate crRNA-guided transposition. We aim to test this novel role of CRISPR-Cas systems by following transposon mobility in response to crRNA targeted R-loop formation using next generation sequencing.
Prof. Dr. Ruth Schmitz-Streit / Dr. Anne Kupczok, Christian-Albrechts-Universität Kiel
The Casposon of Methanosarcina mazei Gö1 - function and evolution
Homepage Schmitz-Streit Laboratory
Homepage Kupczok Laboratory
The endonuclease Cas1 is one of the two core proteins of CRISPR/Cas systems. A distinct phylogenetic group of Cas1 proteins is located on newly identified mobile elements termed casposons. Casposons represent the presumed first family of self-synthetizing transposons in prokaryotes. They show a target site preference and highly resemble the eukaryotic DNA transposons of the Polinton/Maverick superfamily. Evidence for recent mobility of casposon was demonstrated by genome comparison of 62different Methanosarcina mazei strains. The casposon identified in the genome of Methanosarcina mazei strain Gö1 belongs to the group of family-2-casposons encoding a predicted casposase(casposon encoded Cas1). In this project we aim to (i) elucidate the transposition mechanism of the newly identified casposon in M. mazei Gö1. We will characterize the casposon, particularly focusing on the casposase and its ability to recognize and bind to the previously identified target sites within the M. mazei genome using in vitro integrase assays, microscale thermophoresis technic and establishing and using a mini casposon. (ii) Casposon mobility will be studied in a long term evolutionary experiment using the wild-type strain as well as a strain overexpressing the casposase challenged with various stresses. This will include the generation and use of a modified caposon containing a selectable marker. (iii) Several new Methanosarcina strains will be isolated and analyzed for casposons. (iv) Bioinformatic analysis of available and newly sequenced Methanosarcina isolates, of available metagenomes and associated metatranscriptomes of a biogas reactor, and of population sequencing of M. mazei with mobilized casposons will give a comprehensive view of the evolution of casposons in Methanosarcina. In particular, we will gain insights into the evolution of the different casposon units, into the casposon insertion sites in the genome, and into the induced genomic rearrangements. Overall combining these experimental and bioinformatic analyses will allow new insights into casposons and their evolution.
Dr. Sabine Schneider, Technical University of Munich, Garching
Elucidation of the molecular mechanism of Cas endonucleases from bacteria and cyanobacteria
Homepage Schneider Laboratory
CRISPR-Cas systems (CRISPR = Clustered Regulatory Interspaced Short Palindromic Repeats, Cas= CRISPR-associated gene) are used by prokaryotes to defend themselves against phages, invading nucleic acids and mobile genetic elements. These adaptive immune systems consist of a genomic CRISPR-locus, which is transcribed in the crRNA (CRISPR-RNA) and one (or a complex) effector nuclease, that cleaves the target nucleic acid in a crRNA-sequence dependent manner. The RNA-guided, sequence-dependent cleavage of double stranded DNA by the Cas9 endonuclease of Streptococcus pyogenes (SpyCas9) is currently exploited for the site-specific genome modification of various organisms and has revolutionised the field of biology. Structural biology played a crucial role to engineer and harness the SpyCas9-system as a universal genome editing tool by deciphering its molecular mechanism. About 100 structures of Cas-proteins and CRISPR-associated proteins have been determined by now. However, prokaryotes and archaea possess numerous, highly diverse Cas-systems, with to date unknown natural functions and mechanisms of action as well as a large number of such systems still remaining to be discovered. Based on bioinformatics analysis of bacterial and archaeal genomes, many novel subtypes of Cas-proteins were predicted and classified in bacteria, archaea and cyanobacteria. Furthermore, recent studies show that an active Class 1 CRISPR-Cas system generates oligoadenylate secondary messengers, resulting in the activation of another endonuclease and RNA-degradation. In addition, current data suggest that CRISPR-Cas systems are not merely prokaryotic defence mechanisms, but also play a role in DNA repair, regulation of gene expression, virulence and horizontal gene transfer. This clearly emphasis that despite the tremendous progress in the characterisation of these intriguing systems which has already been made, a lot of information on their molecular mechanisms and functions in the natural organisms is largely missing.
My research group focuses on the structure-function relationship of nucleic acids and their recognition by protein factors and small molecules. In this proposed project we want to biochemically and structurally characterise Class 2 Cas-proteins from cyanobacteria and bacteria. This will lead to a detailed elucidation of the molecular mechanisms of these bacterial and cyanobacterial Cas-proteins. In order to obtain a full picture of the atomic details and their function in the natural host, we will cooperate with the groups of G. Bange, W. Hess, G. Klug and A. Marchfelder within the framework of this priority program as outlined in the present proposal.
Priv.-Doz. Dr. Dr. Christoph Schoen, Julius-Maximilians-Universität Würzburg
The CRISPR-Cas system in Neisseria meningitidis and its potential role in host cell adhesion
Homepage Schoen Laboratory
Neisseria meningitidis is a commensal pathogen that normally resides in the human nasopharynx but which also constitutes a worldwide leading cause of sepsis and epidemic meningitis. Despite considerable efforts to identify “classical” virulence genes, a clear genetic basis for meningococcal virulence is lacking. Our recent genome-wide association study has suggested an association of the meningococcal type II-C CRISPR-Cas system with carriage lineages, sugesting that this CRISPR-Cas system may function as a “anti-virulence” system. We also revealed the meningococcal type II-C pathway as a streamlined CRISPR-Cas system that restricts horizontal gene transfer in N. meningitidis. In this proposal, we want to combine genetic, cell culture, biochemical and RNA-sequencing approaches to explore the role of the CRISPR-Cas system in the interaction of meningococci with human nasopharyngeal epithelial cells which constitutes a central step in the pathogenesis of meningococcal disease. Our preliminary data indicate that knock-out strains lacking the Cas9 proteinare impaired in the adhesion to human Detroit 562 nasopharyngeal cells in vitro, suggesting that the meningococcal CRISPR-Cas system might regulate the expression of genes involved in host cell interaction. In order to confirm our preliminary data and to obtain novel insights into the interplay between the type II-C CRISPR-Cas system and the interaction between meningococci and human epithelial cells, we thus want to address the following two research questions: First, how does the CRISPR-Cas system impact adhesion of N. meningitidis to human nasopharyngeal epithelial cells? And second, what are the interaction partners of Cas9 in N. meningitidis beyond “classical” CRISPR-Cas system components? In order to analyse the impact of the CRISPR-Cas system on already established factors involved in meningococcal host cell interaction, functional assays will be performed addressing cell adhesion, capsule expression, biofilm formation, bacterial motility and DNA transformation, using a set of (partially already established) CRISPR-Cas system knock-out mutant and complemented strains, respectively, in combination with mutations in selected major and minor meningococcal adhesins. These hypothesis-driven experiments will be complemented by discovery-driven approaches including biochemical along with RNA-sequencing technologies such as conventional RNA-seq and CLIP-seq to detect also novel molecular phenotypes in the CRISPR-Cas mutant strains. Overall, the project aims to identify novel functions of the CRISPR-Cas system beyond its established role in defence against foreign DNA and will contribute to an expanded understanding of the biological roles of the CRISPR-Cas system in human pathogenic bacteria.
Prof. Ralf Seidel, Universität Leipzig
RNA processing and activation of Type III-A CRISPR-Cas systems
Homepage Seidel Laboratory
DNA targeting Type I and Type II CRISPR-Cas systems recognize their nucleic acid targets using complementary base pairing with their crRNAs. crRNA-DNA target pairing is initiated at the protospacer adjacent motif (PAM) from which further hybridization proceeds in a zipper-like fashion. Once the PAM distal end of the target is reached, a conformational change (locking by Type I systems, HNH-domain engagement by Type II systems) triggers cleavage factor recruitment or cleavage itself. Both, PAM recognition together with PAM-distal end verification allows hereby the target verification across its full length.
Effector complexes of Type III systems bind with their crRNA to single-stranded RNA targets and rapidly degrade the bound RNA. In addition, sufficient matching between crRNA and target triggers the activation of the HD domain, an unspecific single-stranded DNAse, of the Cas10 subunit. Type III effector complexes are thought to target emerging transcripts from invader DNA and to simultaneously degrade both the transcript as well as well as the DNA. Compared to Type I and Type II systems, it is, however, rather poorly understood how targets are selected for degradation. Given the structural similarities between Type I and Type III systems we hypothesize that both systems feature similarities in their target recognition mechanism. Here, we want to resolve the mechanism that activates the HD-domain of the Type III-A effector complex (Csm) and understand how the complex maintains the activated state over long times after RNA cleavage. To this end we will employ confocal and wide-field single-molecule fluorescence experiments as well as single-molecule mechanical measurements.
Prof. Dr. Cynthia Sharma, Julius-Maximilians-Universität Würzburg
Mechanisms and functions of endogenous RNA-targeting by CRISPR-Cas9 in Campylobacter jejuni
Homepage Sharma Laboratory
The Type II CRISPR-Cas9 nucleases naturally utilize CRISPR RNAs (crRNAs) and tracrRNA to silence foreign double-stranded DNA. Besides their well-studied role in adaptive immunity, there is emerging evidence that CRISPR-Cas systems have roles beyond defense and impact on phenotypes such as bacterial virulence and group behavior. For example, deletion of Type II components affects virulence in diverse pathogens, such as Francisella novicida, Neisseria meningitidis or Campylobacter jejuni. While recent work has shown that some Cas9 nucleases can also target RNA, RNA recognition has required nuclease modifications or accessory factors. For example, F. novicida Cas9 uses an associated scaRNA and tracrRNA to repress an endogenous lipoprotein mRNA to affect virulence, although the exact mechanism of regulation remains unclear.
Our RNA-seq analysis of multiple strains of the foodborne pathogen C. jejuni revealed a minimal crRNA biogenesis pathway for its abundantly transcribed Type II-C CRISPR-Cas system (Dugar et al, 2013, PLoS Genetics). Moreover, our RIP-seq (co-immunoprecipitation combined with RNA-seq) study revealed that C. jejuni Cas9 (CjCas9) uses its native crRNAs to bind and cleave complementary endogenous mRNAs (Dugar et al., 2018, Mol Cell). Approximately 100 transcripts co-purify with CjCas9 and generally can be subdivided through their base-pairing potential to the four crRNAs of strain NCTC11168. Several of these RNAs underwent cleavage close to the predicted binding site. Mutational analyses revealed RNA targeting was crRNA- and tracrRNA-dependent, and that RNA cleavage required the CjCas9 HNH domain. We further observed that RNA cleavage improved with greater complementarity between the crRNA and the RNA target, and was programmable in vitro. These findings suggest that CjCas9 is a promiscuous nuclease that can coordinately target both DNA and RNA.
Here, we will further investigate the physiological roles and underlying molecular mechanisms of RNA targeting by CjCas9 to understand if it mediates endogenous gene regulation or is just a side effect of immune surveillance. First, RIP-seq of strains expressing different crRNA repertoires will reveal the extent of endogenous RNA targeting and reveal if there are separate crRNA-dependent and -independent regulons. Expression analyses in regulator mutants and under various conditions will show when CRISPR-Cas is regulated to provide insight into its function. RNA-seq and ribosome profiling of CRISPR-Cas mutants will identify global cleavage patterns and may identify targets of post-transcriptional control. We will then use genetics and biochemistry approaches to validate CjCas9 targets and study in detail the molecular mechanisms underlying regulation, including determinants for base-pairing with crRNAs, starting with selected candidate target mRNAs. Finally, we will determine if RNA targeting by CjCas9 regulates virulence. This will provide insight into the roles of CRISPR-Cas in bacterial gene regulation and pathogenicity.
Prof. Dr. Björn Voß, Universität Stuttgart
Metagenomic data mining for the Analysis of CRISPR-Cas systems
Homepage Voß Laboratory
The discovery of a prokaryotic, adaptive immune system, the so called CRISPR-Cas, has revolutionised genetic engineering. The underlying mechanism makes it possible to edit genomic sequences with nucleotide precision, the insertion of genetic material at precisely defined positions, and the accurate deletion of unwanted genomic stretches.
The original function of these systems in bacteria and archaea is the defence against invading DNA, such as viruses and plasmids, and more importantly to confer immunity against these elements. The specificity of CRISPR-Cas is mediated by the so called CRISPR RNAs (crRNAs) that are complementary to the foreign DNA. These crRNAs form a complex with the Cas proteins that recognises the invading DNA and one of the Cas proteins, the Cas endonuclease, cuts the DNA, thereby promoting its degradation. Variants of CRISPR-Cas systems could be identified that are involved in gene regulatory functions in the cell.
The actual challenge in the analysis of CRISPR-Cas systems is to find targets. Because of the fact that CRISPR-Cas is a defence system against foreign DNA, the targeted entities are under a high selective pressure to circumvent this targeting. If they manage to escape, the targeted region has acquired mutations, which hinders their finding based on sequence similarity. If they are unsuccessful to escape, they become extinct. For these reasons, and because viral genomes are underrepresented in existing sequence databases, the search for CRISPR-Cas targets is challenging. Here, metagenomic data have the advantage that they represent a genomic snapshot of a defined habitat at a defined timepoint. Therefore, they likely also contain information about virus-host interactions, as they occur in course of a CRISPR-Cas response to infection. Additionally, the vast majority of prokaryotic species is not culturable, such that metagenomic data also offers the potential to increase the diversity of known CRISPR-Cas systems.
In the proposed project we want to perform a comprehensive analysis of metagenomic data, with the clear aim to identify general features of CRISPR-Cas systems. For example, we will be able to address questions, such as if the defence against invading DNA is the main function of CRISPR-Cas systems, or if other functions, e.g. gene regulation, are more common than expected.
Prof. Dr. Annegret Wilde, Albert-Ludwigs-Universität, Freiburg
Natural functions of CRISPR-Cas systems in Cyanobacteria
Homepage Wilde Laboratory
Sunlight is the universal energy source for life on Earth. Even organisms that do not absorb light for energy production essentially depend on photosynthetically produced oxygen and biomass. Cyanobacteria are very abundant organisms in all aquatic environments, but are often found also in terrestrial habitats. In addition, photosynthetic organisms convert fixed carbon dioxide into different useable carbon-based products what makes them very attractive for biotechnology. However, cyanobacteria can be infected by cyanophages in their natural habitat as well as in production facilities. Similar to many other bacteria and Archaea they have developed defence systems to protect themselves against invasion by foreign nucleic acids. In this programme, we will investigate the interconnection between the CRISPR-based defence system and other cellular functions by testing CRISPR-dependent gene regulation, the role of ribonucleases in this process and by localization and protein-protein interaction studies of different CRISPR associated proteins. In addition, we will test the hypothesis that the cyanobacterial CRISPR-Cas III-B system is able to synthesize second messenger nucleotides and will analyse the mechanism and physiological meaning of such a putative signalling system.
Prof. Dr. Rolf Backofen, Albert-Ludwigs-Universität, Freiburg
Homepage Backofen Laboratory
Bioinformatics analysis has always played a key role in the history of the CRISPR-Cas system. The characteristic repetitive structure of CRISPR loci has been known since 1987 but it was only in 2005 when a computational analysis revealed that spacers could match bacteriophage genomes, leading to the later-confirmed hypothesis that the CRISPR-Cas system acts as an immune system. Bioinformatic approaches have always been instrumental in the detection of new functions and applications, one prominent example being the in-silico detection of endogenous targets of CRISPR-Cas systems which hinted towards endogenous regulatory mechanisms. The discovery of new and unusual CRISPR-Cas elements and functions by computational analyses, however, requires careful definition of the different standard elements of a CRISPR-Cas system. Only a precise and comprehensive classification can avoid the detection of too many false positives, which occur only due to an incomplete detection of all standard elements. Thus, we wish to support the experimental groups of the SPP 2141 in the detection of new functions for CRISPR-Cas systems by setting up a thorough classification of all the major elements of the CRISPRCas system. These classification tools will then be employed in the detection of novel elements and functions in collaboration with the aforementioned experimental groups. This will comprise new and comprehensive detection and classification approaches for: 1) CRISPR arrays and their associated elements such as protospacers and PAM; 2) Cas proteins and complete CRISPR-Cas systems in genomic and metagenomic data; and 3) CRISPR-related sRNAs like tracRNA and scaRNAs. We will use this information to search for CRISPR-Cas systems with unusual composition, such as orphan Cas proteins and CRISPR arrays. However we expect to find many other unusual compositions, especially within metagenomic data, which have not yet been screened for previously in any CRISPR-Cas systems so far. In addition, we will actively assist the experimental groups in the analysis of CRISPR-related NGS data.
Prof. Dr. Henning Urlaub, Max-Planck-Institut für biophysikalische Chemie, Göttingen
Mass spectrometric proteomic and structural proteomics on CRISPR-Cas of archaea and bacteria
Homepage Urlaub Laboratory
Mass spectrometry based proteomics has matured into a central technique to address analytical questions in cell biology and has extended its scope well into structural biology. It can provide answers to the state of protein populations in different cellular conditions both qualitatively and quantitatively. In particular, protein modifications can be assessed quantitatively, as well as protein-protein, protein-nucleic acids, and protein-ligand interactions can be resolved and elucidated at the amino acid level.
This Z-project outlines mass spectrometric services provided to the SPP2141 project required to address questions on proteomics and structural biology regarding applications of CRISPR-Cas systems in archaea and bacteria.
Public outreach module
Dr. Heike Ziegler & Prof. Dr. Wolfgang Nellen, Universität Kassel
CRISPR-Cas -more than defense
Homepage CRISPR whisper
The aim of the project is to bring the research projects of the SPP and results to the general public. As formats we will use a web blog and a roadshow to adress students (high school and university), teachers as well as a broad public -with some affinityto science. The blog will be in shape of a personalized diary (SPP-Diary), but will also contain scienctific information (Scientific-Diary). The roadshow is tend to be a "box of modules", conisting of lectures/discussion, poster, science exhibition, science slam and if desiered a practical public lab cours.