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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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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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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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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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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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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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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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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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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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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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Genomic characterization of four novel bacteriophages infecting the clinical pathogen Klebsiella pneumoniae

Estrada Bonilla, B.A. (author), Martins Costa, A.R. (author), van den Berg, D.F. (author), van Rossum, T. (author), Hagedoorn, Stefan (author), Walinga, Hielke (author), Xiao, Minfeng (author), Haas, Pieter Jan (author), Brouns, S.J.J. (author)

Bacteriophages are an invaluable source of novel genetic diversity. Sequencing of phage genomes can reveal new proteins with potential uses as biotechnological and medical tools, and help unravel the diversity of biological mechanisms employed by phages to take over the host during viral infection. Aiming to expand the available collection of...

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

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

Kieper, S.N. (author), Almendros Romero, C. (author), van Eijkeren-Haagsma, A.C. (author), Barendregt, Arjan (author), Heck, Albert J.R. (author), Brouns, S.J.J. (author)

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...

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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An educational guide for nanopore sequencing in the classroom

Salazar, A.N. (author), Luzia de Nobrega, F. (author), Anyansi, C.A. (author), Aparicio Maldonado, C. (author), Martins Costa, A.R. (author), van Eijkeren-Haagsma, A.C. (author), Hiralal, A.A. (author), Mahfouz, A.M.E.T.A. (author), McKenzie, R. (author), van Rossum, T. (author), Brouns, S.J.J. (author), Abeel, T.E.P.M.F. (author)

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...

journal article 2020

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Inactivation of RNA and DNA viruses in water by copper and silver ions and their synergistic effect

Soliman, Mona Youssef Moawad (author), Medema, G.J. (author), Estrada Bonilla, B.A. (author), Brouns, S.J.J. (author), van Halem, D. (author)

Cu and Ag have been used as bactericidal agents since ancient times, yet their antiviral capacity in water remains poorly understood. This study tested the effect of copper (Cu) and silver (Ag) on model RNA and DNA viruses MS2 and PhiX 174 in solution at pH 6–8. Cu caused MS2 inactivation with similar rates at pH 6 and 7 but was inert towards...

journal article 2020

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Mining moon & mars with microbes: Biological approaches to extract iron from Lunar and Martian regolith

Volger, R. (author), Pettersson, G.M. (author), Brouns, S.J.J. (author), Rothschild, L. J. (author), Cowley, A. (author), Lehner, B. (author)

The logistical supply of terrestrial materials to space is costly and puts limitations on exploration mission scenarios. In-situ resource utilization (ISRU) can alleviate logistical requirements and thus enables sustainable exploration of space. In this paper, a novel approach to ISRU, utilizing microorganisms to extract iron from Lunar or...

journal article 2020

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Prophages are associated with extensive CRISPR-Cas auto-immunity

Luzia de Nobrega, F. (author), Walinga, Hielke (author), Dutilh, Bas E. (author), Brouns, S.J.J. (author)

CRISPR-Cas systems require discriminating self from non-self DNA during adaptation and interference. Yet, multiple cases have been reported of bacteria containing self-targeting spacers (STS), i.e. CRISPR spacers targeting protospacers on the same genome. STS has been suggested to reflect potential auto-immunity as an unwanted side effect of...

journal article 2020