Novel phage defense systems featuring diverse enzymatic activities are continually being discovered. Among these, defense systems employing proteolytic enzymes have been identified, revealing a previously unrecognized enzymatic activity in phage defense. These protease-associated defense systems represent an untapped reservoir for new biotechnological tools and may serve as a springboard for the development of proteome editors. This review outlines recent advancements in the discovery and characterization of protease-containing defense systems, proposes methods for further exploration and investigation of protease activity, and considers the prospect of protease defense systems for modulating protein processing and cell fate.

Mass spectrometry-based proteomics focuses on identifying the peptide that generates a tandem mass spectrum. Traditional methods rely on protein databases but are often limited or inapplicable in certain contexts. De novo peptide sequencing, which assigns peptide sequences to spectra without prior information, is valuable for diverse biological applications; however, owing to a lack of accuracy, it remains challenging to apply. Here we introduce InstaNovo, a transformer model that translates fragment ion peaks into peptide sequences. We demonstrate that InstaNovo outperforms state-of-the-art methods and showcase its utility in several applications. We also introduce InstaNovo+, a diffusion model that improves performance through iterative refinement of predicted sequences. Using these models, we achieve improved therapeutic sequencing coverage, discover novel peptides and detect unreported organisms in diverse datasets, thereby expanding the scope and detection rate of proteomics searches. Our models unlock opportunities across domains such as direct protein sequencing, immunopeptidomics and exploration of the dark proteome.

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.

This dissertation provides an experimental and conceptual characterization of Craspase, a CRISPR-controlled protease. The array of functionalities inherent to Craspase — including precise protein cleavage, guided RNA recognition, and self-regulatory capabilities — highlights the complexity that can emerge from the endless bacterium-virus coevolution.

With the discovery of CRISPR-controlled proteases, CRISPR–Cas has moved beyond mere nucleic acid targeting into the territory of targeted protein cleavage. Here, we review the understanding of Craspase, the best-studied member of the growing CRISPR RNA-guided protease family. We recollect the original bioinformatic prediction and early experimental characterizations; evaluate some of the mechanistic structural intricacies and emerging biotechnology; discuss open questions and unexplained mysteries; and indicate future directions for the rapidly moving field of the CRISPR proteases.

In recent years, bacteriophage research has been boosted by a rising interest in using phage therapy to treat antibiotic-resistant bacterial infections. In addition, there is a desire to use phages and their unique proteins for specific biocontrol applications and diagnostics. However, the ability to manipulate phage genomes to understand and control gene functions, or alter phage properties such as host range, has remained challenging due to a lack of universal selectable markers. Here, we discuss the state-of-the-art techniques to engineer and select desired phage genomes using advances in cell-free methodologies and clustered regularly interspaced short palindromic repeats-CRISPR associated protein (CRISPR-Cas) counter-selection approaches.

CRISPR–Cas is a widespread adaptive immune system in bacteria and archaea that protects against viral infection by targeting specific invading nucleic acid sequences. Whereas some CRISPR–Cas systems sense and cleave viral DNA, type III and type VI CRISPR–Cas systems sense RNA that results from viral transcription and perhaps invasion by RNA viruses. The sequence-specific detection of viral RNA evokes a cell-wide response that typically involves global damage to halt the infection. How can one make sense of an immune strategy that encompasses broad, collateral effects rather than specific, targeted destruction? In this Review, we summarize the current understanding of RNA-targeting CRISPR–Cas systems. We detail the composition and properties of type III and type VI systems, outline the cellular defence processes that are instigated upon viral RNA sensing and describe the biological rationale behind the broad RNA-activated immune responses as an effective strategy to combat viral infection.

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.

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.