CRISPR-Cas enzymes enable RNA-guided bacterial immunity and are widely used for biotechnological applications including genome editing. In particular, the Class 2 CRISPR-associated enzymes (Cas9, Cas12 and Cas13 families), have been deployed for numerous research, clinical and agricultural applications. However, the immense genetic and biochemical diversity of these proteins in the public domain poses a barrier for researchers seeking to leverage their activities. We present CasPEDIA (http://caspedia.org), the Cas Protein Effector Database of Information and Assessment, a curated encyclopedia that integrates enzymatic classification for hundreds of different Cas enzymes across 27 phylogenetic groups spanning the Cas9, Cas12 and Cas13 families, as well as evolutionarily related IscB and TnpB proteins. All enzymes in CasPEDIA were annotated with a standard workflow based on their primary nuclease activity, target requirements and guide-RNA design constraints. Our functional classification scheme, CasID, is described alongside current phylogenetic classification, allowing users to search related orthologs by enzymatic function and sequence similarity. CasPEDIA is a comprehensive data portal that summarizes and contextualizes enzymatic properties of widely used Cas enzymes, equipping users with valuable resources to foster biotechnological development. CasPEDIA complements phylogenetic Cas nomenclature and enables researchers to leverage the multi-faceted nucleic-acid targeting rules of diverse Class 2 Cas enzymes.

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.

Prokaryotes encode multiple distinct anti-phage defense systems in their genomes. However, the impact of carrying a multitude of defense systems on phage resistance remains unclear, especially in a clinical context. Using a collection of antibiotic-resistant clinical strains of Pseudomonas aeruginosa and a broad panel of phages, we demonstrate that defense systems contribute substantially to defining phage host range and that overall phage resistance scales with the number of defense systems in the bacterial genome. We show that many individual defense systems target specific phage genera and that defense systems with complementary phage specificities co-occur in P. aeruginosa genomes likely to provide benefits in phage-diverse environments. Overall, we show that phage-resistant phenotypes of P. aeruginosa with at least 19 phage defense systems exist in the populations of clinical, antibiotic-resistant P. aeruginosa strains.

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.

Transfer RNAs (tRNAs) in bacteriophage genomes are widespread across bacterial host genera, but their exact function has remained unclear for more than 50 years. Several hypotheses have been proposed, and the most widely accepted one is codon compensation, which suggests that phages encode tRNAs that supplement codons that are less frequently used by the host. Here, we combine several observations and propose a new hypothesis that phage-encoded tRNAs counteract the tRNA-depleting strategies of the host using enzymes such as VapC, PrrC, Colicin D, and Colicin E5 to defend from viral infection. Based on mutational patterns of anticodon loops of tRNAs encoded by phages, we predict that these tRNAs are insensitive to host tRNAses. For phage-encoded tRNAs targeted in the anticodon itself, we observe that phages typically avoid encoding these tRNAs, further supporting the hypothesis that phage tRNAs are selected to be insensitive to host anticodon nucleases. Altogether, our results support the hypothesis that phage-encoded tRNAs have evolved to be insensitive to host anticodon nucleases.

Serratia sp. ATCC 39006 is a Gram-negative bacterium that has been used to study the function of phage defences, such as CRISPR-Cas, and phage counter-defence mechanisms. To expand our phage collection to study the phage-host interaction with Serratia sp. ATCC 39006, we isolated the T4-like myovirus LC53 in Ōtepoti Dunedin, Aotearoa New Zealand. Morphological, phenotypic and genomic characterization revealed that LC53 is virulent and similar to other Serratia, Erwinia and Kosakonia phages belonging to the genus Winklervirus. Using a transposon mutant library, we identified the host ompW gene as essential for phage infection, suggesting that it encodes the phage receptor. The genome of LC53 encodes all the characteristic T4-like core proteins involved in phage DNA replication and generation of viral particles. Furthermore, our bioinformatic analysis suggests that the transcriptional organization of LC53 is similar to that of Escherichia coli phage T4. Importantly, LC53 encodes 18 tRNAs, which likely compensate for differences in GC content between phage and host genomes. Overall, this study describes a newly isolated phage infecting Serratia sp. ATCC 39006 that expands the diversity of phages available to study phage-host interactions.

Bacteriophages (phages) are viruses that specifically attack bacteria. Their use as therapeutics, which constitutes a promising alternative to antibiotics, heavily relies on selecting effective lytic phages against the pathogen of interest. Current selection techniques are laborious and do not allow for direct visualization of phage infection dynamics. Here, we present a method that circumvents these limitations. It can be scaled for high-throughput and permits monitoring of the phage infection in real time via a fluorescence signal readout. This is achieved through the use of a membrane-impermeant nucleic acid dye that stains the DNA of damaged or lysed bacteria and new phage progeny. We have tested the method on Pseudomonas aeruginosa and Klebsiella pneumoniae and show that an increase in fluorescence reflects phage-mediated killing. This is confirmed by other techniques including spot tests, colony plating, flow cytometry and metabolic activity measurements. Furthermore, we illustrate how our method may be used to compare the activity of different phages and to screen the susceptibility of clinical isolates to phage. Altogether, we present a fast, reliable way of selecting phages against Gram-negative bacteria, which may be valuable in optimizing the process of selecting phages for therapeutic use.

Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) has revolutionized genome editing and has great potential for many applications, such as correcting human genetic disorders. To increase the safety of genome editing applications, CRISPR-Cas may benefit from strict control over Cas enzyme activity. Previously, anti-CRISPR proteins and designed oligonucleotides have been proposed to modulate CRISPR-Cas activity. In this study, we report on the potential of guide-complementary DNA oligonucleotides as controlled inhibitors of Cas9 ribonucleoprotein complexes. First, we show that DNA oligonucleotides inhibit Cas9 activity in human cells, reducing both on- A nd off-target cleavage. We then used in vitro assays to better understand how inhibition is achieved and under which conditions. Two factors were found to be important for robust inhibition: The length of the complementary region and the presence of a protospacer adjacent motif-loop on the inhibitor. We conclude that DNA oligonucleotides can be used to effectively inhibit Cas9 activity both ex vivo and in vitro.

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 Klebsiella jumbo myophage ϕKp24 displays an unusually complex arrangement of tail fibers interacting with a host cell. In this study, we combine cryo-electron microscopy methods, protein structure prediction methods, molecular simulations, microbiological and machine learning approaches to explore the capsid, tail, and tail fibers of ϕKp24. We determine the structure of the capsid and tail at 4.1 Å and 3.0 Å resolution. We observe the tail fibers are branched and rearranged dramatically upon cell surface attachment. This complex configuration involves fourteen putative tail fibers with depolymerase activity that provide ϕKp24 with the ability to infect a broad panel of capsular polysaccharide (CPS) types of Klebsiella pneumoniae. Our study provides structural and functional insight into how ϕKp24 adapts to the variable surfaces of capsulated bacterial pathogens, which is useful for the development of phage therapy approaches against pan-drug resistant K. pneumoniae strains.

In the evolutionary arms race against phage, bacteria have assembled a diverse arsenal of antiviral immune strategies. While the recently discovered DISARM (Defense Island System Associated with Restriction-Modification) systems can provide protection against a wide range of phage, the molecular mechanisms that underpin broad antiviral targeting but avoiding autoimmunity remain enigmatic. Here, we report cryo-EM structures of the core DISARM complex, DrmAB, both alone and in complex with an unmethylated phage DNA mimetic. These structures reveal that DrmAB core complex is autoinhibited by a trigger loop (TL) within DrmA and binding to DNA substrates containing a 5′ overhang dislodges the TL, initiating a long-range structural rearrangement for DrmAB activation. Together with structure-guided in vivo studies, our work provides insights into the mechanism of phage DNA recognition and specific activation of this widespread antiviral defense system.

While CRISPR-Cas defence mechanisms have been studied on a population level, their temporal dynamics and variability in individual cells have remained unknown. Using a microfluidic device, time-lapse microscopy and mathematical modelling, we studied invader clearance in Escherichia coli across multiple generations. We observed that CRISPR interference is fast with a narrow distribution of clearance times. In contrast, for invaders with escaping PAM mutations we found large cell-to-cell variability, which originates from primed CRISPR adaptation. Faster growth and cell division and higher levels of Cascade increase the chance of clearance by interference, while slower growth is associated with increased chances of clearance by priming. Our findings suggest that Cascade binding to the mutated invader DNA, rather than spacer integration, is the main source of priming heterogeneity. The highly stochastic nature of primed CRISPR adaptation implies that only subpopulations of bacteria are able to respond quickly to invading threats. We conjecture that CRISPR-Cas dynamics and heterogeneity at the cellular level are crucial to understanding the strategy of bacteria in their competition with other species and phages.

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.

Argonaute proteins use single-stranded RNA or DNA guides to target complementary nucleic acids. This allows eukaryotic Argonaute proteins to mediate RNA interference and long prokaryotic Argonaute proteins to interfere with invading nucleic acids. The function and mechanisms of the phylogenetically distinct short prokaryotic Argonaute proteins remain poorly understood. We demonstrate that short prokaryotic Argonaute and the associated TIR-APAZ (SPARTA) proteins form heterodimeric complexes. Upon guide RNA-mediated target DNA binding, four SPARTA heterodimers form oligomers in which TIR domain-mediated NAD(P)ase activity is unleashed. When expressed in Escherichia coli, SPARTA is activated in the presence of highly transcribed multicopy plasmid DNA, which causes cell death through NAD(P)+ depletion. This results in the removal of plasmid-invaded cells from bacterial cultures. Furthermore, we show that SPARTA can be repurposed for the programmable detection of DNA sequences. In conclusion, our work identifies SPARTA as a prokaryotic immune system that reduces cell viability upon RNA-guided detection of invading DNA.

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.

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.

In-situ resource utilization (ISRU) is increasingly acknowledged as an essential requirement for the construction of sustainable extra-terrestrial colonies. Even with decreasing launch costs, the ultimate goal of establishing colonies must be the usage of resources found at the destination of interest. Typical approaches towards ISRU are often constrained by the mass and energy requirements of transporting processing machineries, such as rovers and massive reactors, and the vast amount of consumables needed. Application of self-reproducing bacteria for the extraction of resources is a promising approach to reduce these pitfalls. In this work, the bacterium Shewanella oneidensis was used to reduce three different types of Lunar and Martian regolith simulants, allowing for the magnetic extraction of iron-rich materials. The combination of bacterial treatment and magnetic extraction resulted in a 5.8-times higher quantity of iron and 43.6% higher iron concentration compared to solely magnetic extraction. The materials were 3D printed into cylinders and the mechanical properties were tested, resulting in a 400% improvement in compressive strength in the bacterially treated samples. This work demonstrates a proof of concept for the on-demand production of construction and replacement parts in space exploration.

The ability to detect specific nucleic acid sequences allows for a wide range of applications such as the identification of pathogens, clinical diagnostics, and genotyping. CRISPR-Cas proteins Cas12a and Cas13a are RNA-guided endonucleases that bind and cleave specific DNA and RNA sequences, respectively. After recognition of a target sequence, both enzymes activate indiscriminate nucleic acid cleavage, which has been exploited for sequence-specific molecular diagnostics of nucleic acids. Here, we present a label-free detection approach that uses a readout based on solution turbidity caused by liquid-liquid phase separation (LLPS). Our approach relies on the fact that the LLPS of oppositely charged polymers requires polymers to be longer than a critical length. This length dependence is predicted by the Voorn-Overbeek model, which we describe in detail and validate experimentally in mixtures of polynucleotides and polycations. We show that the turbidity resulting from LLPS can be used to detect the presence of specific nucleic acid sequences by employing the programmable CRISPR-nucleases Cas12a and Cas13a. Because LLPS of polynucleotides and polycations causes solutions to become turbid, the detection of specific nucleic acid sequences can be observed with the naked eye. We furthermore demonstrate that there is an optimal polynucleotide concentration for detection. Finally, we provide a theoretical prediction that hints towards possible improvements of an LLPS-based detection assay. The deployment of LLPS complements CRISPR-based molecular diagnostic applications and facilitates easy and low-cost nucleotide sequence detection.