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CasPEDIA Database: a functional classification system for class 2 CRISPR-Cas enzymes

Adler, Benjamin A. (author), Trinidad, Marena I. (author), Bellieny-Rabelo, Daniel (author), Zhang, Elaine (author), Karp, Hannah M. (author), Skopintsev, Petr (author), Thornton, Brittney W. (author), Yoon, Peter H. (author), Brouns, S.J.J. (author)

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...

journal article 2024

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Accumulation of defense systems in phage-resistant strains of Pseudomonas aeruginosa

Martins Costa, A.R. (author), van den Berg, D.F. (author), Esser, J.Q. (author), Muralidharan, A. (author), van den Bossche, Halewijn (author), Estrada Bonilla, B.A. (author), van der Steen, B.A. (author), Haas, Pieter Jan (author), Brouns, S.J.J. (author)

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...

journal article 2024

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CRISPR-controlled proteases

van Beljouw, S.P.B. (author), Brouns, S.J.J. (author)

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...

review 2024

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An OmpW-dependent T4-like phage infects Serratia sp. ATCC 39006

Mahler, M.S. (author), Malone Portu, L.M. (author), van den Berg, D.F. (author), Smith, Leah M. (author), Brouns, S.J.J. (author), Fineran, Peter C. (author)

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...

journal article 2023

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Approaches for bacteriophage genome engineering

Mahler, M.S. (author), Martins Costa, A.R. (author), van Beljouw, S.P.B. (author), Fineran, Peter C. (author), Brouns, S.J.J. (author)

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...

review 2023

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Phage tRNAs evade tRNA-targeting host defenses through anticodon loop mutations

van den Berg, D.F. (author), van der Steen, B.A. (author), Martins Costa, A.R. (author), Brouns, S.J.J. (author)

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...

journal article 2023

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Monitoring phage-induced lysis of gram-negatives in real time using a fluorescent DNA dye

Egido, Julia E. (author), Toner-Bartelds, Catherine (author), Martins Costa, A.R. (author), Brouns, S.J.J. (author), Rooijakkers, Suzan H.M. (author), Bardoel, Bart W. (author), Haas, Pieter Jan (author)

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...

journal article 2023

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

Hu, Chunyi (author), van Beljouw, S.P.B. (author), Nam, Ki Hyun (author), Schuler, Gabriel (author), Rodríguez Molina, A. (author), van Eijkeren-Haagsma, A.C. (author), Valk, M. (author), Pabst, Martin (author), Brouns, S.J.J. (author)

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...

journal article 2022

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Short prokaryotic Argonaute systems trigger cell death upon detection of invading DNA

Koopal, Balwina (author), Potocnik, Ana (author), Mutte, Sumanth K. (author), Aparicio Maldonado, C. (author), Lindhoud, Simon (author), Vervoort, Jacques J.M. (author), Brouns, S.J.J. (author), Swarts, Daan C. (author)

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...

journal article 2022

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

Wu, Wen Y. (author), Jackson, Simon A. (author), Almendros Romero, C. (author), van Eijkeren-Haagsma, A.C. (author), Yilmaz, Suzan (author), Gort, Gerrit (author), Van Der Oost, John (author), Brouns, S.J.J. (author), Staals, Raymond H.J. (author)

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...

journal article 2022

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Modulating CRISPR-Cas Genome Editing Using Guide-Complementary DNA Oligonucleotides

Swartjes, Thomas (author), Shang, Peng (author), Van Den Berg, Dennis T.M. (author), Künne, Tim (author), Geijsen, Niels (author), Brouns, S.J.J. (author), Van Der Oost, John (author), Staals, Raymond H.J. (author), Notebaart, Richard A. (author)

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...

journal article 2022

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High-resolution reconstruction of a Jumbo-bacteriophage infecting capsulated bacteria using hyperbranched tail fibers

Ouyang, Ruochen (author), Martins Costa, A.R. (author), Cassidy, C. Keith (author), Otwinowska, Aleksandra (author), Williams, Vera C.J. (author), Latka, Agnieszka (author), Stansfeld, Phill J. (author), Drulis-Kawa, Zuzanna (author), Brouns, S.J.J. (author)

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...

journal article 2022

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RNA-targeting CRISPR–Cas systems

van Beljouw, S.P.B. (author), Sanders, J.A. (author), Rodríguez Molina, A. (author), Brouns, S.J.J. (author)

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...

review 2022

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Single cell variability of CRISPR-Cas interference and adaptation

McKenzie, R. (author), Keizer, Emma M. (author), Vink, J.N.A. (author), van Lopik, Jasper (author), Büke, F. (author), Kalkman, Vera (author), Fleck, Christian (author), Tans, S.J. (author), Brouns, S.J.J. (author)

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...

journal article 2022

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Structural basis for broad anti-phage immunity by DISARM

Bravo, Jack P.K. (author), Aparicio Maldonado, C. (author), Nobrega, Franklin L. (author), Brouns, S.J.J. (author), Taylor, David W. (author)

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...

journal article 2022

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

van Beljouw, S.P.B. (author), van Eijkeren-Haagsma, A.C. (author), Rodriguez Molina, A. (author), van den Berg, D.F. (author), Vink, J.N.A. (author), Brouns, S.J.J. (author)

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...

journal article 2021

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Iron can be microbially extracted from Lunar and Martian regolith simulants and 3D printed into tough structural materials

Castelein, Sofie M. (author), Aarts, Tom F. (author), Schleppi, Juergen (author), Hendrikx, R.W.A. (author), Bottger, A.J. (author), Benz, D. (author), Brouns, S.J.J. (author), Meyer, A.S. (author), Lehner, B. (author)

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...

journal article 2021

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SCOPE enables type III CRISPR-Cas diagnostics using flexible targeting and stringent CARF ribonuclease activation

Steens, Jurre A. (author), Zhu, Y. (author), Taylor, David W. (author), Bravo, Jack P.K. (author), Prinsen, Stijn H.P. (author), Schoen, Cor D. (author), Keijser, Bart J.F. (author), Ossendrijver, Michel (author), Brouns, S.J.J. (author)

Characteristic properties of type III CRISPR-Cas systems include recognition of target RNA and the subsequent induction of a multifaceted immune response. This involves sequence-specific cleavage of the target RNA and production of cyclic oligoadenylate (cOA) molecules. Here we report that an exposed seed region at the 3′ end of the crRNA is...

journal article 2021

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Mechanism for Cas4-assisted directional spacer acquisition in CRISPR–Cas

Hu, Chunyi (author), Almendros Romero, C. (author), Nam, Ki Hyun (author), Sousa Pires da Costa Basto, R.M. (author), Vink, J.N.A. (author), van Eijkeren-Haagsma, A.C. (author), Bagde, Saket R. (author), Brouns, S.J.J. (author), Ke, Ailong (author)

Prokaryotes adapt to challenges from mobile genetic elements by integrating spacers derived from foreign DNA in the CRISPR array¹. Spacer insertion is carried out by the Cas1–Cas2 integrase complex^2–4. A substantial fraction of CRISPR–Cas systems use a Fe–S cluster containing Cas4 nuclease to ensure that spacers are...

journal article 2021

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PAM-repeat associations and spacer selection preferences in single and co-occurring CRISPR-Cas systems

Vink, J.N.A. (author), Baijens, J.H.L. (author), Brouns, S.J.J. (author)

Background: The adaptive CRISPR-Cas immune system stores sequences from past invaders as spacers in CRISPR arrays and thereby provides direct evidence that links invaders to hosts. Mapping CRISPR spacers has revealed many aspects of CRISPR-Cas biology, including target requirements such as the protospacer adjacent motif (PAM). However,...

journal article 2021