Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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Direct Visualization of Native CRISPR Target Search in Live Bacteria Reveals Cascade DNA Surveillance Mechanism
Vink, Jochem N.A. ; Martens, Koen J.A. ; Vlot, Marnix ; McKenzie, Rebecca E. ; Almendros, Cristóbal ; Estrada Bonilla, Boris ; Brocken, Daan J.W. ; Hohlbein, Johannes ; Brouns, Stan J.J. - \ 2020
Molecular Cell 77 (2020)1. - ISSN 1097-2765 - p. 39 - 50.e10.
arms race - Cascade - CRISPR-Cas - PALM - PAM - photo-activation localization microscopy - single molecule - single-particle tracking - target search

Vink et al. tracked single CRISPR RNA-surveillance complexes (Cascade) in the native host cell and determined the influence of Cascade copy numbers, PAM scanning speed, and the presence of CRISPR arrays and transcription on their ability to find and clear invading mobile genetic elements from the cell.

Harnessing type I CRISPR–Cas systems for genome engineering in human cells
Cameron, Peter ; Coons, Mary M. ; Klompe, Sanne E. ; Lied, Alexandra M. ; Smith, Stephen C. ; Vidal, Bastien ; Donohoue, Paul D. ; Rotstein, Tomer ; Kohrs, Bryan W. ; Nyer, David B. ; Kennedy, Rachel ; Banh, Lynda M. ; Williams, Carolyn ; Toh, Mckenzi S. ; Irby, Matthew J. ; Edwards, Leslie S. ; Lin, Chun Han ; Owen, Arthur L.G. ; Künne, Tim ; Oost, John van der; Brouns, Stan J.J. ; Slorach, Euan M. ; Fuller, Chris K. ; Gradia, Scott ; Kanner, Steven B. ; May, Andrew P. ; Sternberg, Samuel H. - \ 2019
Nature Biotechnology 37 (2019). - ISSN 1087-0156 - p. 1471 - 1477.

Type I CRISPR–Cas systems are the most abundant adaptive immune systems in bacteria and archaea1,2. Target interference relies on a multi-subunit, RNA-guided complex called Cascade3,4, which recruits a trans-acting helicase-nuclease, Cas3, for target degradation5–7. Type I systems have rarely been used for eukaryotic genome engineering applications owing to the relative difficulty of heterologous expression of the multicomponent Cascade complex. Here, we fuse Cascade to the dimerization-dependent, non-specific FokI nuclease domain8–11 and achieve RNA-guided gene editing in multiple human cell lines with high specificity and efficiencies of up to ~50%. FokI–Cascade can be reconstituted via an optimized two-component expression system encoding the CRISPR-associated (Cas) proteins on a single polycistronic vector and the guide RNA (gRNA) on a separate plasmid. Expression of the full Cascade–Cas3 complex in human cells resulted in targeted deletions of up to ~200 kb in length. Our work demonstrates that highly abundant, previously untapped type I CRISPR–Cas systems can be harnessed for genome engineering applications in eukaryotic cells.

Visualisation of dCas9 target search in vivo using an open-microscopy framework
Martens, Koen J.A. ; Beljouw, Sam P.B. van; Els, Simon van der; Vink, Jochem N.A. ; Baas, Sander ; Vogelaar, George A. ; Brouns, Stan J.J. ; Baarlen, Peter van; Kleerebezem, Michiel ; Hohlbein, Johannes - \ 2019
Nature Communications 10 (2019)1. - ISSN 2041-1723

CRISPR-Cas9 is widely used in genomic editing, but the kinetics of target search and its relation to the cellular concentration of Cas9 have remained elusive. Effective target search requires constant screening of the protospacer adjacent motif (PAM) and a 30 ms upper limit for screening was recently found. To further quantify the rapid switching between DNA-bound and freely-diffusing states of dCas9, we developed an open-microscopy framework, the miCube, and introduce Monte-Carlo diffusion distribution analysis (MC-DDA). Our analysis reveals that dCas9 is screening PAMs 40% of the time in Gram-positive Lactoccous lactis, averaging 17 ± 4 ms per binding event. Using heterogeneous dCas9 expression, we determine the number of cellular target-containing plasmids and derive the copy number dependent Cas9 cleavage. Furthermore, we show that dCas9 is not irreversibly bound to target sites but can still interfere with plasmid replication. Taken together, our quantitative data facilitates further optimization of the CRISPR-Cas toolbox.

Conserved motifs in the CRISPR leader sequence control spacer acquisition levels in Type I-D CRISPR-Cas systems
Kieper, Sebastian N. ; Almendros, Cristóbal ; Brouns, Stan J.J. - \ 2019
FEMS Microbiology Letters 366 (2019)11. - ISSN 0378-1097
CRISPR adaptation - CRISPR leader - spacer acquisition - type I-D CRISPR-Cas system

Integrating short DNA fragments at the correct leader-repeat junction is key to successful CRISPR-Cas memory formation. The Cas1-2 proteins are responsible to carry out this process. However, the CRISPR adaptation process additionally requires a DNA element adjacent to the CRISPR array, called leader, to facilitate efficient localization of the correct integration site. In this work, we introduced the core CRISPR adaptation genes cas1 and cas2 from the Type I-D CRISPR-Cas system of Synechocystis sp. 6803 into Escherichia coli and assessed spacer integration efficiency. Truncation of the leader resulted in a significant reduction of spacer acquisition levels and revealed the importance of different conserved regions for CRISPR adaptation rates. We found three conserved sequence motifs in the leader of I-D CRISPR arrays that each affected spacer acquisition rates, including an integrase anchoring site. Our findings support the model in which the leader sequence is an integral part of type I-D adaptation in Synechocystis sp. acting as a localization signal for the adaptation complex to drive CRISPR adaptation at the first repeat of the CRISPR array.

Cas4-Cas1 fusions drive efficient PAM selection and control CRISPR adaptation
Almendros, Cristóbal ; Nobrega, Franklin L. ; McKenzie, Rebecca E. ; Brouns, Stan J.J. - \ 2019
Nucleic acids research 47 (2019)10. - ISSN 0305-1048 - p. 5223 - 5230.

Microbes have the unique ability to acquire immunological memories from mobile genetic invaders to protect themselves from predation. To confer CRISPR resistance, new spacers need to be compatible with a targeting requirement in the invader's DNA called the protospacer adjacent motif (PAM). Many CRISPR systems encode Cas4 proteins to ensure new spacers are integrated that meet this targeting prerequisite. Here we report that a gene fusion between cas4 and cas1 from the Geobacter sulfurreducens I-U CRISPR-Cas system is capable of introducing functional spacers carrying interference proficient TTN PAM sequences at much higher frequencies than unfused Cas4 adaptation modules. Mutations of Cas4-domain catalytic residues resulted in dramatically decreased naïve and primed spacer acquisition, and a loss of PAM selectivity showing that the Cas4 domain controls Cas1 activity. We propose the fusion gene evolved to drive the acquisition of only PAM-compatible spacers to optimize CRISPR interference.

Systematic analysis of Type I-E Escherichia coli CRISPR-Cas PAM sequences ability to promote interference and primed adaptation
Musharova, Olga ; Sitnik, Vasily ; Vlot, Marnix ; Savitskaya, Ekaterina ; Datsenko, Kirill A. ; Krivoy, Andrey ; Fedorov, Ivan ; Semenova, Ekaterina ; Brouns, Stan J.J. ; Severinov, Konstantin - \ 2019
Molecular Microbiology 111 (2019)6. - ISSN 0950-382X - p. 1558 - 1570.

CRISPR interference occurs when a protospacer recognized by the CRISPR RNA is destroyed by Cas effectors. In Type I CRISPR-Cas systems, protospacer recognition can lead to «primed adaptation» – acquisition of new spacers from in cis located sequences. Type I CRISPR-Cas systems require the presence of a trinucleotide protospacer adjacent motif (PAM) for efficient interference. Here, we investigated the ability of each of 64 possible trinucleotides located at the PAM position to induce CRISPR interference and primed adaptation by the Escherichia coli Type I-E CRISPR-Cas system. We observed clear separation of PAM variants into three groups: those unable to cause interference, those that support rapid interference and those that lead to reduced interference that occurs over extended periods of time. PAM variants unable to support interference also did not support primed adaptation; those that supported rapid interference led to no or low levels of adaptation, while those that caused attenuated levels of interference consistently led to highest levels of adaptation. The results suggest that primed adaptation is fueled by the products of CRISPR interference. Extended over time interference with targets containing «attenuated» PAM variants provides a continuous source of new spacers leading to high overall level of spacer acquisition.

Addiction systems antagonize bacterial adaptive immunity
Sluijs, Lisa van; Houte, Stineke van; Oost, John van der; Brouns, Stan J.J. ; Buckling, Angus ; Westra, Edze R. - \ 2019
FEMS Microbiology Letters 366 (2019)5. - ISSN 0378-1097
adaptive immunity - bacteria - CRISPR - plasmid - TA - toxin

CRISPR-Cas systems provide adaptive immunity against mobile genetic elements, but employment of this resistance mechanism is often reported with a fitness cost for the host. Whether or not CRISPR-Cas systems are important barriers for the horizontal spread of conjugative plasmids, which play a crucial role in the spread of antibiotic resistance, will depend on the fitness costs of employing CRISPR-based defences and the benefits of resisting conjugative plasmids. To estimate these costs and benefits we measured bacterial fitness associated with plasmid immunity using Escherichia coli and the conjugative plasmid pOX38-Cm. We find that CRISPR-mediated immunity fails to confer a fitness benefit in the absence of antibiotics, despite the large fitness cost associated with carrying the plasmid in this context. Similar to many other conjugative plasmids, pOX38-Cm carries a CcdAB toxin-anti-toxin (TA) addiction system. These addiction systems encode long-lived toxins and short-lived anti-toxins, resulting in toxic effects following the loss of the TA genes from the bacterial host. Our data suggest that the lack of a fitness benefit associated with CRISPR-mediated defence is due to expression of the TA system before plasmid detection and degradation. As most antibiotic resistance plasmids encode TA systems this could have important consequences for the role of CRISPR-Cas systems in limiting the spread of antibiotic resistance.

CRISPR-Cas Systems Reduced to a Minimum
Almendros, Cristóbal ; Kieper, Sebastian N. ; Brouns, Stan J.J. - \ 2019
Molecular Cell 73 (2019)4. - ISSN 1097-2765 - p. 641 - 642.

In two recent studies in Molecular Cell, Wright et al. (2019) report complete spacer integration by a Cas1 mini-integrase and Edraki et al. (2019) describe accurate genome editing by a small Cas9 ortholog with less stringent PAM requirements.

Using CAPTURE to detect spacer acquisition in native CRISPR arrays
McKenzie, Rebecca E. ; Almendros, Cristóbal ; Vink, Jochem N.A. ; Brouns, Stan J.J. - \ 2019
Nature protocols (2019)14. - ISSN 1754-2189 - p. 976 - 990.

CRISPR–Cas systems are able to acquire immunological memories (spacers) from bacteriophages and plasmids in order to survive infection; however, this often occurs at low frequency within a population, which can make it difficult to detect. Here we describe CAPTURE (CRISPR adaptation PCR technique using reamplification and electrophoresis), a versatile and adaptable protocol to detect spacer-acquisition events by electrophoresis imaging with high-enough sensitivity to identify spacer acquisition in 1 in 10 5 cells. Our method harnesses two simple PCR steps, separated by automated electrophoresis and extraction of size-selected DNA amplicons, thus allowing the removal of unexpanded arrays from the sample pool and enabling 1,000-times more sensitive detection of new spacers than alternative PCR protocols. CAPTURE is a straightforward method that requires only 1 d to enable the detection of spacer acquisition in all native CRISPR systems and facilitate studies aimed both at unraveling the mechanism of spacer integration and more sensitive tracing of integration events in natural ecosystems.

Molecular and Evolutionary Determinants of Bacteriophage Host Range
Jonge, Patrick A. de; Nobrega, Franklin L. ; Brouns, Stan J.J. ; Dutilh, Bas E. - \ 2019
Trends in Microbiology 27 (2019)1. - ISSN 0966-842X - p. 51 - 63.
bacteriophage - broad-host-range phages - phage therapy - specificity - virus–host interaction

The host range of a bacteriophage is the taxonomic diversity of hosts it can successfully infect. Host range, one of the central traits to understand in phages, is determined by a range of molecular interactions between phage and host throughout the infection cycle. While many well studied model phages seem to exhibit a narrow host range, recent ecological and metagenomics studies indicate that phages may have specificities that range from narrow to broad. There is a growing body of studies on the molecular mechanisms that enable phages to infect multiple hosts. These mechanisms, and their evolution, are of considerable importance to understanding phage ecology and the various clinical, industrial, and biotechnological applications of phage. Here we review knowledge of the molecular mechanisms that determine host range, provide a framework defining broad host range in an evolutionary context, and highlight areas for additional research.

Role of nucleotide identity in effective CRISPR target escape mutations
Künne, Tim ; Zhu, Yifan ; Silva, Fausia da; Konstantinides, Nico ; McKenzie, Rebecca E. ; Jackson, Ryan N. ; Brouns, Stan J.J. - \ 2018
Nucleic acids research 46 (2018)19. - ISSN 0305-1048 - p. 10395 - 10404.

Prokaryotes use primed CRISPR adaptation to update their memory bank of spacers against invading genetic elements that have escaped CRISPR interference through mutations in their protospacer target site. We previously observed a trend that nucleotide-dependent mismatches between crRNA and the protospacer strongly influence the efficiency of primed CRISPR adaptation. Here we show that guanine-substitutions in the target strand of the protospacer are highly detrimental to CRISPR interference and interference-dependent priming, while cytosine-substitutions are more readily tolerated. Furthermore, we show that this effect is based on strongly decreased binding affinity of the effector complex Cascade for guanine-mismatched targets, while cytosine-mismatched targets only minimally affect target DNA binding. Structural modeling of Cascade-bound targets with mismatches shows that steric clashes of mismatched guanines lead to unfavorable conformations of the RNA-DNA duplex. This effect has strong implications for the natural selection of target site mutations that lead to effective escape from type I CRISPR-Cas systems.

Targeting mechanisms of tailed bacteriophages
Nobrega, Franklin L. ; Vlot, Marnix ; Jonge, Patrick A. de; Dreesens, Lisa L. ; Beaumont, Hubertus J.E. ; Lavigne, Rob ; Dutilh, Bas E. ; Brouns, Stan J.J. - \ 2018
Nature Reviews Microbiology 16 (2018). - ISSN 1740-1526 - p. 760 - 773.

Phages differ substantially in the bacterial hosts that they infect. Their host range is determined by the specific structures that they use to target bacterial cells. Tailed phages use a broad range of receptor-binding proteins, such as tail fibres, tail spikes and the central tail spike, to target their cognate bacterial cell surface receptors. Recent technical advances and new structure–function insights have begun to unravel the molecular mechanisms and temporal dynamics that govern these interactions. Here, we review the current understanding of the targeting machinery and mechanisms of tailed phages. These new insights and approaches pave the way for the application of phages in medicine and biotechnology and enable deeper understanding of their ecology and evolution.

Complete genome sequences of two T4-like Escherichia coli bacteriophages
Costa, Ana R. ; Brouns, Stan J.J. ; Nobrega, Franklin L. - \ 2018
Genome Announcements 6 (2018)26. - ISSN 2169-8287

Bacteriophages and their proteins have potential applications in biotechnology for the detection and control of bacterial diseases. Here, we describe the sequencing and genome annotations of two strictly virulent Escherichia coli bacteriophages that may be explored for biocontrol strategies and to expand the understanding of phage-host interactions.

Repetitive DNA Reeling by the Cascade-Cas3 Complex in Nucleotide Unwinding Steps
Loeff, Luuk ; Brouns, Stan J.J. ; Joo, Chirlmin - \ 2018
Molecular Cell 70 (2018)3. - ISSN 1097-2765 - p. 385 - 394.
adaptive - Cas3 - cascade - CRISPR - FRET - helicase - immunity - interference - single-molecule

CRISPR-Cas provides RNA-guided adaptive immunity against invading genetic elements. Interference in type I systems relies on the RNA-guided Cascade complex for target DNA recognition and the Cas3 helicase/nuclease protein for target degradation. Even though the biochemistry of CRISPR interference has been largely covered, the biophysics of DNA unwinding and coupling of the helicase and nuclease domains of Cas3 remains elusive. Here, we employed single-molecule Förster resonance energy transfer (FRET) to probe the helicase activity with high spatiotemporal resolution. We show that Cas3 remains tightly associated with the target-bound Cascade complex while reeling the DNA using a spring-loaded mechanism. This spring-loaded reeling occurs in distinct bursts of 3 bp, which underlie three successive 1-nt unwinding events. Reeling is highly repetitive, allowing Cas3 to repeatedly present its inefficient nuclease domain with single-strand DNA (ssDNA) substrate. Our study reveals that the discontinuous helicase properties of Cas3 and its tight interaction with Cascade ensure controlled degradation of target DNA only. Loeff et al. report on a single-molecule fluorescence analysis of the E. coli CRISPR-Cas3 protein. The Cas3 protein uses a spring-loaded unwinding mechanism, reeling the target DNA 3 bp at a time. Facilitated by slipping, Cas3 repeatedly presents its intrinsically inefficient nuclease domain with DNA substrate, which may contribute to promoting a robust immune response.

Bacteriophage DNA glucosylation impairs target DNA binding by type I and II but not by type V CRISPR–Cas effector complexes
Vlot, Marnix ; Houkes, Joep ; Lochs, Silke J.A. ; Swarts, Daan C. ; Zheng, Peiyuan ; Kunne, Tim ; Mohanraju, Prarthana ; Anders, Carolin ; Jinek, Martin ; Oost, John Van Der; Dickman, Mark J. ; Brouns, Stan J.J. - \ 2018
Nucleic acids research 46 (2018)2. - ISSN 0305-1048 - p. 873 - 885.
Prokaryotes encode various host defense systems that provide protection against mobile genetic elements. Restriction–modification (R–M) and CRISPR–Cas systems mediate host defense by sequence specific targeting of invasive DNA. T-even bacteriophages employ covalent modifications of nucleobases to avoid binding and therefore cleavage of their DNA by restriction endonucleases. Here, we describe that DNA glucosylation of bacteriophage genomes affects interference of some but not all CRISPR–Cas systems. We show that glucosyl modification of 5-hydroxymethylated cytosines in the DNA of bacteriophage T4 interferes with type I-E and type II-A CRISPR–Cas systems by lowering the affinity of the Cascade and Cas9–crRNA complexes for their target DNA. On the contrary, the type V-A nuclease Cas12a (also known as Cpf1) is not impaired in binding and cleavage of glucosylated target DNA, likely due to a more open structural architecture of the protein. Our results suggest that CRISPR–Cas systems have contributed to the selective pressure on phages to develop more generic solutions to escape sequence specific host defense systems.
Cas4 Facilitates PAM-Compatible Spacer Selection during CRISPR Adaptation
Kieper, Sebastian N. ; Almendros, Cristóbal ; Behler, Juliane ; McKenzie, Rebecca E. ; Nobrega, Franklin L. ; Haagsma, Anna C. ; Vink, Jochem N.A. ; Hess, Wolfgang R. ; Brouns, Stan J.J. - \ 2018
Cell Reports 22 (2018)13. - ISSN 2211-1247 - p. 3377 - 3384.
Cas4 - CRISPR adaptation - spacer acquisition - type I-D CRISPR-Cas system
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.
Complete genome sequence of the Escherichia coli phage Ayreon
Vlot, Marnix ; Nobrega, Franklin L. ; Wong, Che F.A. ; Liu, Yue ; Brouns, Stan J.J. - \ 2018
Genome Announcements 6 (2018)2. - ISSN 2169-8287
We report the whole-genome sequence of a new Escherichia coli temperate phage, Ayreon, comprising a linear double-stranded DNA (dsDNA) genome of 44,708 bp.
DNA modification
Vlot, M. ; Brouns, S.J.J. - \ 2017
Octrooinummer: WO2017055514, gepubliceerd: 2017-04-06.
The invention relates to the field of genetic engineering, and to the modification of nucleic acids and organismal genomes in particular. Specifically, the invention concerns the chemical modification of nucleic acids for the improved transformation of cells. Accordingly, the invention provides for modified nucleic acids, methods of modifying nucleic acids, methods of transforming cells and methods for the sequence-directed site-specific genetic modification of cells. The invention additionally encompasses cells transformed with modified nucleic acids and expression constructs for delivery and expression of modified nucleic acids. The nucleic acids, expression constructs, cells and methods of the invention find application in many areas of biotechnology, including, for example, genome editing.
CRISPR-Cas : Adapting to change
Jackson, Simon A. ; McKenzie, Rebecca E. ; Fagerlund, Robert D. ; Kieper, Sebastian N. ; Fineran, Peter C. ; Brouns, Stan J.J. - \ 2017
Science 356 (2017)6333. - ISSN 0036-8075
Bacteria and archaea are engaged in a constant arms race to defend against the ever-present threats of viruses and invasion by mobile genetic elements. The most flexible weapons in the prokaryotic defense arsenal are the CRISPR-Cas adaptive immune systems. These systems are capable of selective identification and neutralization of foreign DNA and/or RNA. CRISPR-Cas systems rely on stored genetic memories to facilitate target recognition. Thus, to keep pace with a changing pool of hostile invaders, the CRISPR memory banks must be regularly updated with new information through a process termed CRISPR adaptation. In this Review, we outline the recent advances in our understanding of the molecular mechanisms governing CRISPR adaptation. Specifically, the conserved protein machinery Cas1-Cas2 is the cornerstone of adaptive immunity in a range of diverse CRISPR-Cas systems.
CRISPR Cas3 Plasmid degradation assays
Kunne, T.A. ; Kieper, Sebastian ; Bannenberg, J.W. ; Vogel, A.I.M. ; Miellet, Willem ; Klein, Misha ; Depken, Martin ; Suarez Diez, M. ; Brouns, S.J.J. - \ 2016
A plasmid degradation assay with CRISPR Cas3 was performed on different plasmids to investigate the respective degradation patterns.
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