Craspase Orthologs Cleave a Nonconserved Site in Target Protein Csx30

The Craspase CRISPR-Cas effector consists of the RNA-guided ribonuclease gRAMP and the protease TPR-CHAT, coupling target RNA recognition to protease activation. The natural substrate of Craspase is Csx30, a protein cleaved in two fragments that subsequently activates downstream antiviral pathways. Here, we determined the protease substrate specificity of Craspase from Candidatus “Jettenia caeni” (Jc-Craspase). We find that Jc-Craspase cleaves Jc-Csx30 in a target RNA-dependent fashion in A|S, which is different from the sites found in two other studied Craspases (L|D and M|K for Candidatus “Scalindua brodae” and Desulfonema ishimotonii, respectively). The fact that Craspase cleaves a nonconserved site across orthologs indicates the evolution of specific protein interactions between Craspase and its respective Csx30 target protein. The Craspase family thus represents a panel of proteases with different substrate specificities, which we exploited for the development of a readout for multiplexed RNA detection.BN/Stan Brouns LabBT/Environmental Biotechnolog

TRBP ensures efficient Dicer processing of precursor microRNA in RNA-crowded environments

The RNA-binding protein TRBP is a central component of the Dicer complex. Despite a decade of biochemical and structural studies, the essential functionality of TRBP in microRNA (miRNA) biogenesis remains unknown. Here we show that TRBP is an integral cofactor for time-efficient Dicer processing in RNA-crowded environments. We competed for Dicer processing of pre-miRNA with a large amount of cellular RNA species and found that Dicer-TRBP, but not Dicer alone, remains resilient. To apprehend the mechanism of this substrate selectivity, we use single-molecule fluorescence. The real-time observation reveals that TRBP acts as a gatekeeper, precluding Dicer from engaging with pre-miRNA-like substrates. TRBP acquires the selectivity using the PAZ domain of Dicer, whereas Dicer moderates the RNA-binding affinity of TRBP for fast turnover. This coordinated action between TRBP and Dicer accomplishes an efficient way of discarding pre-miRNA-like substrates.BN/Chirlmin Joo LabBN/Technici en Analiste

Single-molecule pull-down for investigating protein–nucleic acid interactions

The genome and transcriptome are constantly modified by proteins in the cell. Recent advances in single-molecule techniques allow for high spatial and temporal observations of these interactions between proteins and nucleic acids. However, due to the difficulty of obtaining functional protein complexes, it remains challenging to study the interactions between macromolecular protein complexes and nucleic acids. Here, we combined single-molecule fluorescence with various protein complex pull-down techniques to determine the function and stoichiometry of ribonucleoprotein complexes. Through the use of three examples of protein complexes from eukaryotic cells (Drosha, Dicer, and TUT4 protein complexes), we provide step-by-step guidance for using novel single-molecule techniques. Our single-molecule methods provide sub-second and nanometer resolution and can be applied to other nucleoprotein complexes that are essential for cellular processes.Accepted Author ManuscriptBN/Chirlmin Joo LabBN/Technici en Analiste

The gRAMP CRISPR-Cas effector is an RNA endonuclease complexed with a caspase-like peptidase

Type III CRISPR-Cas immunity is widespread in prokaryotes and is generally mediated by multisubunit effector complexes. These complexes recognize complementary viral transcripts and can activate ancillary immune proteins. Here, we describe a type III-E effector from Candidatus “Scalindua brodae” (Sb-gRAMP), which is natively encoded by a single gene with several type III domains fused together. This effector uses CRISPR RNA to guide target RNA recognition and cleaves single-stranded RNA at two defined positions six nucleotides apart. Sb-gRAMP physically combines with the caspase-like TPR-CHAT peptidase to form the CRISPR-guided caspase (Craspase) complex, suggesting a potential mechanism of target RNA-induced protease activity to gain viral immunity.BN/Stan Brouns LabBN/Technici en AnalistenBT/Biocatalysi

Cas4-Cas1 Is a Protospacer Adjacent Motif-Processing Factor Mediating Half-Site Spacer Integration during CRISPR Adaptation

The immunization of bacteria and archaea against invading viruses via CRISPR adaptation is critically reliant on the efficient capture, accurate processing, and integration of CRISPR spacers into the host genome. The adaptation proteins Cas1 and Cas2 are sufficient for successful spacer acquisition in some CRISPR-Cas systems. However, many CRISPR-Cas systems additionally require the Cas4 protein for efficient adaptation. Cas4 has been implied in the selection and processing of spacer precursors, but the detailed mechanistic understanding of how Cas4 contributes to CRISPR adaptation is lacking. Here, we biochemically reconstitute the CRISPR-Cas type I-D adaptation system and show two functionally distinct adaptation complexes: Cas4-Cas1 and Cas1-Cas2. The Cas4-Cas1 complex recognizes and cleaves protospacer adjacent motif (PAM) sequences in 3' overhangs in a sequence-specific manner, while the Cas1-Cas2 complex defines the cleavage of non-PAM sites via host-factor nucleases. Both sub-complexes are capable of mediating half-site integration, facilitating the integration of processed spacers in the correct interference-proficient orientation. We provide a model in which an asymmetric adaptation complex differentially acts on PAM- and non-PAM-containing overhangs, providing cues for the correct orientation of spacer integration.Accepted Author ManuscriptBN/Stan Brouns La

Viral suppressors of RNAI employ a rapid screening mode to discriminate viral RNA from cellular small RNA

RNA interference (RNAi) is an indispensable mechanism for antiviral defense in insects, including mosquitoes that transmit human diseases. To escape this antiviral defense system, viruses encode suppressors of RNAi that prevent elimination of viral RNAs, and thus ensure efficient virus accumulation. Although the first animal Viral Suppressor of RNAi (VSR) was identified more than a decade ago, the molecular basis of RNAi suppression by these viral proteins remains unclear. Here, we developed a single-molecule fluorescence assay to investigate how VSRs inhibit the recognition of viral RNAs by Dcr-2, a key endoribonuclease enzyme in the RNAi pathway. Using VSRs from three insect RNA viruses (Culex Y virus, Drosophila X virus and Drosophila C virus), we reveal bimodal physical interactions between RNA molecules and VSRs. During initial interactions, these VSRs rapidly discriminate short RNA substrates from long dsRNA. VSRs engage nearly irreversible binding with long dsRNAs, thereby shielding it from recognition by Dcr-2. We propose that the length-dependent switch from rapid screening to irreversible binding reflects the main mechanism by which VSRs distinguish viral dsRNA from cellular RNA species such as microRNAs.BN/Chirlmin Joo LabApplied SciencesBN/Technici en Analiste

Craspase is a CRISPR RNA-guided, RNA-activated protease

The CRISPR-Cas type III-E RNA-targeting effector complex gRAMP/Cas7-11 is associated with a caspase-like protein (TPR-CHAT/Csx29) to form Craspase (CRISPR-guided caspase). Here, we use cryo-electron microscopy snapshots of Craspase to explain its target RNA cleavage and protease activation mechanisms. Target-guide pairing extending into the 5' region of the guide RNA displaces a gating loop in gRAMP, which triggers an extensive conformational relay that allosterically aligns the protease catalytic dyad and opens an amino acid side-chain-binding pocket. We further define Csx30 as the endogenous protein substrate that is site-specifically proteolyzed by RNA-activated Craspase. This protease activity is switched off by target RNA cleavage by gRAMP and is not activated by RNA targets containing a matching protospacer flanking sequence. We thus conclude that Craspase is a target RNA-activated protease with self-regulatory capacity.Accepted Author ManuscriptBN/Stan Brouns LabBT/Environmental Biotechnolog

Adaptation by Type V-A and V-B CRISPR-Cas Systems Demonstrates Conserved Protospacer Selection Mechanisms Between Diverse CRISPR-Cas Types

Adaptation of clustered regularly interspaced short palindromic repeats (CRISPR) arrays is a crucial process responsible for the unique, adaptive nature of CRISPR-Cas immune systems. The acquisition of new CRISPR spacers from mobile genetic elements has previously been studied for several types of CRISPR-Cas systems. In this study, we used a high-throughput sequencing approach to characterize CRISPR adaptation of the type V-A system from Francisella novicida and the type V-B system from Alicyclobacillus acidoterrestris. In contrast to other class 2 CRISPR-Cas systems, we found that for the type V-A and V-B systems, the Cas12 nucleases are dispensable for spacer acquisition, with only Cas1 and Cas2 (type V-A) or Cas4/1 and Cas2 (type V-B) being necessary and sufficient. Whereas the catalytic activity of Cas4 is not essential for adaptation, Cas4 activity is required for correct protospacer adjacent motif selection in both systems and for prespacer trimming in type V-A. In addition, we provide evidence for acquisition of RecBCD-produced DNA fragments by both systems, but with spacers derived from foreign DNA being incorporated preferentially over those derived from the host chromosome. Our work shows that several spacer acquisition mechanisms are conserved between diverse CRISPR-Cas systems, but also highlights unexpected nuances between similar systems that generally contribute to a bias of gaining immunity against invading genetic elements. BN/Stan Brouns La

Cas4 Facilitates PAM-Compatible Spacer Selection during CRISPR Adaptation

CRISPR-Cas systems adapt their immunological memory against their invaders by integrating short DNA fragments into clustered regularly interspaced short palindromic repeat (CRISPR) loci. While Cas1 and Cas2 make up the core machinery of the CRISPR integration process, various class I and II CRISPR-Cas systems encode Cas4 proteins for which the role is unknown. Here, we introduced the CRISPR adaptation genes cas1, cas2, and cas4 from the type I-D CRISPR-Cas system of Synechocystis sp. 6803 into Escherichia coli and observed that cas4 is strictly required for the selection of targets with protospacer adjacent motifs (PAMs) conferring I-D CRISPR interference in the native host Synechocystis. We propose a model in which Cas4 assists the CRISPR adaptation complex Cas1-2 by providing DNA substrates tailored for the correct PAM. Introducing functional spacers that target DNA sequences with the correct PAM is key to successful CRISPR interference, providing a better chance of surviving infection by mobile genetic elements. Kieper et al. demonstrate that the ubiquitous protein Cas4 assists Cas1 and Cas2 in the selection of new CRISPR spacers with a PAM licensing efficient CRISPR interference.BN/Stan Brouns LabBN/Technici en Analiste

An educational guide for nanopore sequencing in the classroom

The last decade has witnessed a remarkable increase in our ability to measure genetic information. Advancements of sequencing technologies are challenging the existing methods of data storage and analysis. While methods to cope with the data deluge are progressing, many biologists have lagged behind due to the fast pace of computational advancements and tools available to address their scientific questions. Future generations of biologists must be more computationally aware and capable. This means they should be trained to give them the computational skills to keep pace with technological developments. Here, we propose a model that bridges experimental and bioinformatics concepts using the Oxford Nanopore Technologies (ONT) sequencing platform. We provide both a guide to begin to empower the new generation of educators, scientists, and students in performing long-read assembly of bacterial and bacteriophage genomes and a standalone virtual machine containing all the required software and learning materials for the course.Pattern Recognition and BioinformaticsBN/Stan Brouns LabBN/Technici en Analiste