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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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 (2019). - ISSN 1087-0156

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.

Discussion at the international conference crisprcon: Talking about CRISPR-Cas
Oost, J. van der; Fresco, L.O. - \ 2019
Adaptation and application of a two-plasmid inducible CRISPR-Cas9 system in Clostridium beijerinckii
Diallo, M. ; Hocq, Rémi ; Collas, Florent ; Chartier, Gwladys ; Wasels, François ; Wijaya, Hani Surya ; Werten, Marc W.T. ; Wolbert, Emil J.H. ; Kengen, Servé W.M. ; Oost, John van der; Ferreira, Nicolas Lopes ; López-Contreras, A.M. - \ 2019
Methods : a companion to Methods in enzymology (2019). - ISSN 1046-2023 - 10 p.
Clostridium beijerinckii - CRISPR-Cas9 - Genome editing - Nuclease

Recent developments in CRISPR technologies have opened new possibilities for improving genome editing tools dedicated to the Clostridium genus. In this study we adapted a two-plasmid tool based on this technology to enable scarless modification of the genome of two reference strains of Clostridium beijerinckii producing an Acetone/Butanol/Ethanol (ABE) or an Isopropanol/Butanol/Ethanol (IBE) mix of solvents. In the NCIMB 8052 ABE-producing strain, inactivation of the SpoIIE sporulation factor encoding gene resulted in sporulation-deficient mutants, and this phenotype was reverted by complementing the mutant strain with a functional spoIIE gene. Furthermore, the fungal cellulase-encoding celA gene was inserted into the C. beijerinckii NCIMB 8052 chromosome, resulting in mutants with endoglucanase activity. A similar two-plasmid approach was next used to edit the genome of the natural IBE-producing strain C. beijerinckii DSM 6423, which has never been genetically engineered before. Firstly, the catB gene conferring thiamphenicol resistance was deleted to make this strain compatible with our dual-plasmid editing system. As a proof of concept, our dual-plasmid system was then used in C. beijerinckii DSM 6423 ΔcatB to remove the endogenous pNF2 plasmid, which led to a sharp increase of transformation efficiencies.

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.

Hoe verder met CRISPR-Cas?
Oost, John van der; Kok, Esther - \ 2019

Europa zit in haar maag met CRISPR-Cas. De nieuwe technologie heeft de potentie om actuele problemen op te lossen, maar de risico’s zijn nog onbekend en de weerstand is groot. Toetsing op basis van maatschappelijk nut kan mogelijk uitkomst bieden, zo bleek tijdens het Resource-debat op 20 mei.

Multiplex genome editing of microorganisms using CRISPR-Cas
Adiego-Pérez, Belén ; Randazzo, Paola ; Daran, Jean Marc ; Verwaal, René ; Roubos, Johannes A. ; Daran-Lapujade, Pascale ; Oost, John van der - \ 2019
FEMS Microbiology Letters 366 (2019)8. - ISSN 0378-1097
Cas12a - Cas9 - cell factories - CRISPR-Cas - genome editing - multiplex

Microbial production of chemical compounds often requires highly engineered microbial cell factories. During the last years, CRISPR-Cas nucleases have been repurposed as powerful tools for genome editing. Here, we briefly review the most frequently used CRISPR-Cas tools and describe some of their applications. We describe the progress made with respect to CRISPR-based multiplex genome editing of industrial bacteria and eukaryotic microorganisms. We also review the state of the art in terms of gene expression regulation using CRISPRi and CRISPRa. Finally, we summarize the pillars for efficient multiplexed genome editing and present our view on future developments and applications of CRISPR-Cas tools for multiplex genome editing.

Haarfijn gewassen verbeteren
Oost, J. van der - \ 2019
Wageningen : Wageningen University & Research
Wat als tomaten dieper wortelen, zodat ze in droge gebieden kunnen groeien? Kunnen we dan de groeiende wereldbevolking beter voeden? De CRISPR-Cas techniek die deze aanpassingen mogelijk zou kunnen maken is veel nauwkeuriger dan de klassieke veredeling en de aanpassingen zijn vergelijkbaar met spontane natuurlijke mutaties. Maar is deze techniek, waarvan belangrijke principes zijn ontdekt door Wageningse wetenschappers, wel veilig? Wie profiteert ervan? Waar zie jij kansen voor deze techniek?

CRISPR-Cas ribonucleoprotein mediated homology-directed repair for efficient targeted genome editing in microalgae Nannochloropsis oceanica IMET1
Naduthodi, Mihris Ibnu Saleem ; Mohanraju, Prarthana ; Südfeld, Christian ; Adamo, Sarah D'; Barbosa, Maria J. ; Oost, John Van Der - \ 2019
Biotechnology for Biofuels 12 (2019)1. - ISSN 1754-6834
Cas12a - Cas9 - CRISPR - Genome editing - Homologous recombination - Homology-directed repair - Microalgae - Nannochloropsis - Ribonucleoproteins

Background: Microalgae are considered as a sustainable feedstock for the production of biofuels and other value-added compounds. In particular, Nannochloropsis spp. stand out from other microalgal species due to their capabilities to accumulate both triacylglycerol (TAG) and polyunsaturated fatty acids (PUFAs). However, the commercialization of microalgae-derived products is primarily hindered by the high production costs compared to less sustainable alternatives. Efficient genome editing techniques leading to effective metabolic engineering could result in strains with enhanced productivities of interesting metabolites and thereby reduce the production costs. Competent CRISPR-based genome editing techniques have been reported in several microalgal species, and only very recently in Nannochloropsis spp. (2017). All the reported CRISPR-Cas-based systems in Nannochloropsis spp. rely on plasmid-borne constitutive expression of Cas9 and a specific guide, combined with repair of double-stranded breaks (DSB) by non-homologous end joining (NHEJ) for the target gene knockout. Results: In this study, we report for the first time an alternative approach for CRISPR-Cas-mediated genome editing in Nannochloropsis sp.; the Cas ribonucleoproteins (RNP) and an editing template were directly delivered into microalgal cells via electroporation, making Cas expression dispensable and homology-directed repair (HDR) possible with high efficiency. Apart from widely used SpCas9, Cas12a variants from three different bacterium were used for this approach. We observed that FnCas12a from Francisella novicida generated HDR-based targeted mutants with highest efficiency (up to 93% mutants among transformants) while AsCas12a from Acidaminococcus sp. resulted in the lowest efficiency. We initially show that the native homologous recombination (HR) system in N. oceanica IMET1 is not efficient for easy isolation of targeted mutants by HR. Cas9/sgRNA RNP delivery greatly enhanced HR at the target site, generating around 70% of positive mutant lines. Conclusion: We show that the delivery of Cas RNP by electroporation can be an alternative approach to the presently reported plasmid-based Cas9 method for generating mutants of N. oceanica. The co-delivery of Cas-RNPs along with a dsDNA repair template efficiently enhanced HR at the target site, resulting in a remarkable higher percentage of positive mutant lines. Therefore, this approach can be used for efficient generation of targeted mutants in Nannochloropsis sp. In addition, we here report the activity of several Cas12a homologs in N. oceanica IMET1, identifying FnCas12a as the best performer for high efficiency targeted genome editing.

Incorporation of a Synthetic Amino Acid into dCas9 Improves Control of Gene Silencing
Koopal, Balwina ; Kruis, Aleksander J. ; Claassens, Nico J. ; Nobrega, Franklin L. ; Oost, John Van Der - \ 2019
ACS synthetic biology 8 (2019)2. - ISSN 2161-5063 - p. 216 - 222.
Cas9 - CRISPR-Cas - CRISPRi - gene silencing - synthetic amino acid

The CRISPR-Cas9 nuclease has been repurposed as a tool for gene repression (CRISPRi). This catalytically dead Cas9 (dCas9) variant inhibits transcription by blocking either initiation or elongation by the RNA polymerase complex. Conditional control of dCas9-mediated repression has been achieved with inducible promoters that regulate the expression of the dcas9 gene. However, as dCas9-mediated gene silencing is very efficient, even slightly leaky dcas9 expression leads to significant background levels of repression of the target gene. In this study, we report on the development of optimized control of dCas9-mediated silencing through additional regulation at the translation level. We have introduced the TAG stop codon in the dcas9 gene in order to insert a synthetic amino acid, l-biphenylalanine (BipA), at a permissive site in the dCas9 protein. In the absence of BipA, a nonfunctional, truncated dCas9 is produced, but when BipA is present, the TAG codon is translated resulting in a functional, full-length dCas9 protein. This synthetic, BipA-containing dCas9 variant (dCas9-BipA) could still fully repress gene transcription. Comparison of silencing mediated by dCas9 to dCas9-BipA revealed a 14-fold reduction in background repression by the latter system. The here developed proof-of-principle system thus reduces unwanted background levels of gene silencing, allowing for tight and timed control of target gene expression.

Shooting the messenger : RNA-targetting CRISPR-Cas systems
Zhu, Yifan ; Klompe, Sanne E. ; Vlot, Marnix ; Oost, John van der; Staals, Raymond H.J. - \ 2018
Bioscience Reports 38 (2018)3. - ISSN 0144-8463

Since the discovery of CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-associated genes) immune systems, astonishing progress has been made on revealing their mechanistic foundations. Due to the immense potential as genome engineering tools, research has mainly focussed on a subset of Cas nucleases that target DNA. In addition, however, distinct types of RNA-targetting CRISPR-Cas systems have been identified. The focus of this review will be on the interference mechanisms of the RNA targetting type III and type VI CRISPR-Cas systems, their biological relevance and their potential for applications.

Genome editing by natural and engineered CRISPR-associated nucleases
Wu, Wen Y. ; Lebbink, Joyce H.G. ; Kanaar, Roland ; Geijsen, Niels ; Oost, John van der - \ 2018
Nature Chemical Biology 14 (2018)7. - ISSN 1552-4450 - p. 642 - 651.

Over the last decade, research on distinct types of CRISPR systems has revealed many structural and functional variations. Recently, several novel types of single-polypeptide CRISPR-associated systems have been discovered including Cas12a/Cpf1 and Cas13a/C2c2. Despite distant similarities to Cas9, these additional systems have unique structural and functional features, providing new opportunities for genome editing applications. Here, relevant fundamental features of natural and engineered CRISPR-Cas variants are compared. Moreover, practical matters are discussed that are essential for dedicated genome editing applications, including nuclease regulation and delivery, target specificity, as well as host repair diversity.

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.
Progress of CRISPR-Cas based genome editing in Photosynthetic microbes
Naduthodi, M.I.S. ; Barbosa, M.J. ; Oost, J. van der - \ 2018
Biotechnology Journal 13 (2018)9. - ISSN 1860-6768
The carbon footprint caused by unsustainable development and its environmental and economic impact has become a major concern in the past few decades. Photosynthetic microbes such as microalgae and cyanobacteria are capable of accumulating value-added compounds from carbon dioxide, and have been regarded as environmentally friendly alternatives to reduce the usage of fossil fuels, thereby contributing to reducing the carbon footprint. This light-driven generation of green chemicals and biofuels has triggered the research for metabolic engineering of these photosynthetic microbes. CRISPR-Cas systems are successfully implemented across a wide range of prokaryotic and eukaryotic species for efficient genome editing. However, the inception of this genome editing tool in microalgal and cyanobacterial species took off rather slowly due to various complications. In this review, we elaborate on the established CRISPR-Cas based genome editing in various microalgal and cyanobacterial species. The complications associated with CRISPR-Cas based genome editing in these species are addressed along with possible strategies to overcome these issues. It is anticipated that in the near future this will result in improving and expanding the microalgal and cyanobacterial genome engineering toolbox.
Hijacking CRISPR-Cas for high-throughput bacterial metabolic engineering : advances and prospects
Mougiakos, Ioannis ; Bosma, Elleke F. ; Ganguly, Joyshree ; Oost, John van der; Kranenburg, Richard van - \ 2018
Current Opinion in Biotechnology 50 (2018). - ISSN 0958-1669 - p. 146 - 157.
High engineering efficiencies are required for industrial strain development. Due to its user-friendliness and its stringency, CRISPR-Cas-based technologies have strongly increased genome engineering efficiencies in bacteria. This has enabled more rapid metabolic engineering of both the model host Escherichia coli and non-model organisms like Clostridia, Bacilli, Streptomycetes and cyanobacteria, opening new possibilities to use these organisms as improved cell factories. The discovery of novel Cas9-like systems from diverse microbial environments will extend the repertoire of applications and broaden the range of organisms in which it can be used to create novel production hosts. This review analyses the current status of prokaryotic metabolic engineering towards the production of biotechnologically relevant products, based on the exploitation of different CRISPR-related DNA/RNA endonuclease variants.
Complete Genome Sequence of Geobacillus thermodenitrificans T12, A Potential Host for Biotechnological Applications
Daas, Tijn ; Vriesendorp, Bastienne ; Weijer, Tom van de; Oost, John van der; Kranenburg, Richard van - \ 2018
Current Microbiology 75 (2018)1. - ISSN 0343-8651 - p. 49 - 56.
In attempt to obtain a thermophilic host for the conversion of lignocellulose derived substrates into lactic acid, Geobacillus thermodenitrificans T12 was isolated from a compost heap. It was selected from over 500 isolates as a genetically tractable hemicellulolytic lactic acid producer, requiring little nutrients. The strain is able to ferment glucose and xylose simultaneously and can produce lactic acid from xylan, making it a potential host for biotechnological applications. The genome of strain T12 consists of a 3.64 Mb chromosome and two plasmids of 59 and 56 kb. It has a total of 3.676 genes with an average genomic GC content of 48.7%. The T12 genome encodes a denitrification pathway, allowing for anaerobic respiration. The identity and localization of the responsible genes are similar to those of the denitrification pathways found in strain NG80-2. The hemicellulose utilization (HUS) locus was identified based on sequence homology against G. stearothermophilus T-6. It appeared that T12 has all the genes that are present in strain T-6 except for the arabinan degradation cluster. Instead, the HUS locus of strain T12 contains genes for both an inositol and a pectate degradation pathway. Strain T12 has complete pathways for the synthesis of purine and pyrimidine, all 20 amino acids and several vitamins except D-biotin. The host-defense systems present comprise a Type II and a Type III restriction-modification system, as well as a CRISPR-Cas Type II system. It is concluded that G. thermodenitrificans T12 is a potentially interesting candidate for industrial applications.
Structural basis for guide RNA processing and seed-dependent DNA targeting and cleavage by CRISPR-Cas12a
Swarts, Daan C. ; Oost, J. van der; Jinek, Martin - \ 2017
DNA - nuclease - RNA - ribonucleases - gel
Raw imaging data belonging to the manuscript 'Structural basis for guide RNA processing and seed-dependent DNA targeting and cleavage by CRISPR-Cas12a', by Swarts et al.
Improved CRISPR-Cas9 genome editing tool
Louwen, Rogier ; Oost, J. van der - \ 2017
Octrooinummer: WO2017155408, verleend: 2017-09-14.
The invention relates to a Cas-based, preferably Cas9-based nuclease complex, wherein the guide RNA sequence is irreversibly crosslinked to the Cas9 protein. The cross-link may be a covalent binding or a non-covalent binding. Such a complex may be used in delivering constructs to a cell that are capable of gene-editing. Use of this cross-linked complex will result in less off-targeting.
Improved CRISPR-Cpf1 genome editing tool
Oost, J. van der; Baarlen, P. van; Louwen, Rogier - \ 2017
Octrooinummer: WO2017155407, verleend: 2017-09-14.
The invention relates to a Cpf1-based nuclease complex, wherein the guide RNA sequence is irreversibly crosslinked to the Cpf1 protein. The cross-link may be a covalent binding or a non-covalent binding. Such a complex may be used in delivering constructs to a cell that are capable of gene-editing. Use of this cross-linked complex will result in less off-targeting.
FnCpf1: a novel and efficient genome editing tool for Saccharomyces cerevisiae
Swiat, Michal A. ; Dashko, Sofia ; Ridder, Maxime den; Wijsman, Melanie ; Oost, John van der; Daran, Jean Marc ; Daran-Lapujade, Pascale - \ 2017
Nucleic acids research 45 (2017)21. - ISSN 0305-1048 - p. 12585 - 12598.
Cpf1 is a new class II family of CRISPR-Cas RNA-programmable endonucleases with unique features that make it a very attractive alternative or complement to Cas9 for genome engineering. Using constitutively expressed Cpf1 from Francisella novicida, the present study demonstrates that FnCpf1 can mediate RNA-guided DNA cleavage at targeted genomic loci in the popular model and industrial yeast Saccharomyces cerevisiae. FnCpf1 very efficiently and precisely promoted repair DNA recombination with efficiencies up to 100%. Furthermore, FnCpf1 was shown to introduce point mutations with high fidelity. While editing multiple loci with Cas9 is hampered by the need for multiple or complex expression constructs, processing itself a customized CRISPR array FnCpf1 was able to edit four genes simultaneously in yeast with a 100% efficiency. A remarkable observation was the unexpected, strong preference of FnCpf1 to cleave DNA at target sites harbouring 5'-TTTV-3' PAM sequences, a motif reported to be favoured by Cpf1 homologs of Acidaminococcus and Lachnospiraceae. The present study supplies several experimentally tested guidelines for crRNA design, as well as plasmids for FnCpf1 expression and easy construction of crRNA expression cassettes in S. cerevisiae. FnCpf1 proves to be a powerful addition to S. cerevisiae CRISPR toolbox.
Snel en simpel genen repareren: opmars DNA-bewerkingstechniek CRISPR-Cas
Oost, John van der; Groenen, Martien ; Maagd, Ruud de - \ 2017
Characterizing a thermostable Cas9 for bacterial genome editing and silencing
Mougiakos, Ioannis ; Mohanraju, Prarthana ; Bosma, Elleke F. ; Vrouwe, Valentijn ; Finger Bou, Max ; Naduthodi, Mihris I.S. ; Gussak, Alex ; Brinkman, Rudolf B.L. ; Kranenburg, Richard Van; Oost, John Van Der - \ 2017
Nature Communications 8 (2017)1. - ISSN 2041-1723
CRISPR-Cas9-based genome engineering tools have revolutionized fundamental research and biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature of the established Cas9 systems does not allow for applications that require enhanced stability, including engineering at elevated temperatures. Here we identify and characterize ThermoCas9 from the thermophilic bacterium Geobacillus thermodenitrificans T12. We show that in vitro ThermoCas9 is active between 20 and 70 °C, has stringent PAM-preference at lower temperatures, tolerates fewer spacer-protospacer mismatches than SpCas9 and its activity at elevated temperatures depends on the sgRNA-structure. We develop ThermoCas9-based engineering tools for gene deletion and transcriptional silencing at 55 °C in Bacillus smithii and for gene deletion at 37 °C in Pseudomonas putida. Altogether, our findings provide fundamental insights into a thermophilic CRISPR-Cas family member and establish a Cas9-based bacterial genome editing and silencing tool with a broad temperature range.
Efficient Genome Editing of a Facultative Thermophile Using Mesophilic spCas9
Mougiakos, Ioannis ; Bosma, Elleke F. ; Weenink, Koen ; Vossen, Eric ; Goijvaerts, Kirsten ; Oost, John van der; Kranenburg, Richard van - \ 2017
ACS synthetic biology 6 (2017)5. - ISSN 2161-5063 - p. 849 - 861.
Bacillus smithii - bacteria - CRISPR/Cas9 - genome editing - homologous recombination - thermophiles

Well-developed genetic tools for thermophilic microorganisms are scarce, despite their industrial and scientific relevance. Whereas highly efficient CRISPR/Cas9-based genome editing is on the rise in prokaryotes, it has never been employed in a thermophile. Here, we apply Streptococcus pyogenes Cas9 (spCas9)-based genome editing to a moderate thermophile, i.e., Bacillus smithii, including a gene deletion, gene knockout via insertion of premature stop codons, and gene insertion. We show that spCas9 is inactive in vivo above 42 °C, and we employ the wide temperature growth range of B. smithii as an induction system for spCas9 expression. Homologous recombination with plasmid-borne editing templates is performed at 45-55 °C, when spCas9 is inactive. Subsequent transfer to 37 °C allows for counterselection through production of active spCas9, which introduces lethal double-stranded DNA breaks to the nonedited cells. The developed method takes 4 days with 90, 100, and 20% efficiencies for gene deletion, knockout, and insertion, respectively. The major advantage of our system is the limited requirement for genetic parts: only one plasmid, one selectable marker, and a promoter are needed, and the promoter does not need to be inducible or well-characterized. Hence, it can be easily applied for genome editing purposes in both mesophilic and thermophilic nonmodel organisms with a limited genetic toolbox and ability to grow at, or tolerate, temperatures of 37 and at or above 42 °C.

Structural Basis for Guide RNA Processing and Seed-Dependent DNA Targeting by CRISPR-Cas12a
Swarts, Daan C. ; Oost, John van der; Jinek, Martin - \ 2017
Molecular Cell 66 (2017)2. - ISSN 1097-2765 - p. 221 - 233.e4.
Cas12a - Cas9 - Cpf1 - CRISPR RNA - CRISPR-Cas - crRNA processing - nuclease - R-loop - seed sequence - target DNA cleavage

The CRISPR-associated protein Cas12a (Cpf1), which has been repurposed for genome editing, possesses two distinct nuclease activities: endoribonuclease activity for processing its own guide RNAs and RNA-guided DNase activity for target DNA cleavage. To elucidate the molecular basis of both activities, we determined crystal structures of Francisella novicida Cas12a bound to guide RNA and in complex with an R-loop formed by a non-cleavable guide RNA precursor and a full-length target DNA. Corroborated by biochemical experiments, these structures reveal the mechanisms of guide RNA processing and pre-ordering of the seed sequence in the guide RNA that primes Cas12a for target DNA binding. Furthermore, the R-loop complex structure reveals the strand displacement mechanism that facilitates guide-target hybridization and suggests a mechanism for double-stranded DNA cleavage involving a single active site. Together, these insights advance our mechanistic understanding of Cas12a enzymes and may contribute to further development of genome editing technologies.

Multiplex gene editing by CRISPR-Cpf1 using a single crRNA array
Zetsche, Bernd ; Heidenreich, Matthias ; Mohanraju, Prarthana ; Fedorova, Iana ; Kneppers, Jeroen ; Degennaro, Ellen M. ; Winblad, Nerges ; Choudhury, Sourav R. ; Abudayyeh, Omar O. ; Wu, Wen Y. ; Oost, John van der - \ 2017
Nature Biotechnology 35 (2017)1. - ISSN 1087-0156 - p. 31 - 34.

Targeting of multiple genomic loci with Cas9 is limited by the need for multiple or large expression constructs. Here we show that the ability of Cpf1 to process its own CRISPR RNA (crRNA) can be used to simplify multiplexed genome editing. Using a single customized CRISPR array, we edit up to four genes in mammalian cells and three in the mouse brain, simultaneously.

CRISPR-Cas9 gene editing : Delivery aspects and therapeutic potential
Oude Blenke, Erik ; Evers, Martijn J.W. ; Mastrobattista, Enrico ; Oost, John van der - \ 2016
Journal of Controlled Release 244 (2016). - ISSN 0168-3659 - p. 139 - 148.
CRISPR-Cas - CRISPR-Cas9 - Delivery systems - Ex vivo - Gene editing - Genome editing - In vivo - Therapeutic applications

The CRISPR-Cas9 gene editing system has taken the biomedical science field by storm, initiating rumors about future Nobel Prizes and heating up a fierce patent war, but also making significant scientific impact. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), together with CRISPR-associated proteins (Cas) are a part of the prokaryotic adaptive immune system and have successfully been repurposed for genome editing in mammalian cells. The CRISPR-Cas9 system has been used to correct genetic mutations and for replacing entire genes, opening up a world of possibilities for the treatment of genetic diseases. In addition, recently some new CRISPR-Cas systems have been discovered with interesting mechanistic variations. Despite these promising developments, many challenges have to be overcome before the system can be applied therapeutically in human patients and enabling delivery technology is one of the key challenges. Furthermore, the relatively high off-target effect of the system in its current form prevents it from being safely applied directly in the human body. In this review, the transformation of the CRISPR-Cas gene editing systems into a therapeutic modality will be discussed and the currently most realistic . in vivo applications will be highlighted.

Gummen in de genen
Oost, John van der; Schaart, Jan ; Smulders, Rene - \ 2016

De nieuwe technologie Crispr-Cas9 maakt het makkelijker dan ooit om erfelijk materiaal heel gericht te herschrijven. De ontwikkeling van deze gene editing gaat zo snel, dat wetgeving en ethiek moeite hebben het bij te benen. Is dit nu genmodifi catie of klassieke veredeling in de snelkookpan?

Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems
Mohanraju, Prarthana ; Makarova, Kira S. ; Zetsche, Bernd ; Zhang, Feng ; Koonin, Eugene V. ; Oost, John van der - \ 2016
Science 353 (2016)6299. - ISSN 0036-8075 - 14 p.

Adaptive immunity had been long thought of as an exclusive feature of animals. However, the discovery of the CRISPR-Cas defense system, present in almost half of prokaryotic genomes, proves otherwise. Because of the everlasting parasite-host arms race, CRISPR-Cas has rapidly evolved through horizontal transfer of complete loci or individual modules, resulting in extreme structural and functional diversity. CRISPR-Cas systems are divided into two distinct classes that each consist of three types and multiple subtypes. We discuss recent advances in CRISPR-Cas research that reveal elaborate molecular mechanisms and provide for a plausible scenario of CRISPR-Cas evolution. We also briefly describe the latest developments of a wide range of CRISPR-based applications.

Next Generation Prokaryotic Engineering : The CRISPR-Cas Toolkit
Mougiakos, Ioannis ; Bosma, Elleke F. ; Vos, Willem M. de; Kranenburg, Richard van; Oost, John van der - \ 2016
Trends in Biotechnology 34 (2016)7. - ISSN 0167-7799 - p. 575 - 587.
Archaea - Bacteria - Cas9 - CRISPR-Cas - Genome editing - Recombineering

The increasing demand for environmentally friendly production processes of green chemicals and fuels has stimulated research in microbial metabolic engineering. CRISPR-Cas-based tools for genome editing and expression control have enabled fast, easy, and accurate strain development for established production platform organisms, such as Escherichia coli and Saccharomyces cerevisiae. However, the growing interest in alternative production hosts, for which genome editing options are generally limited, requires further developing such engineering tools. In this review, we discuss established and emerging CRISPR-Cas-based tools for genome editing and transcription control of model and non-model prokaryotes, and we analyse the possibilities for further improvement and expansion of these tools for next generation prokaryotic engineering. SpyCas9 has recently been established as an efficient counterselection system in combination with homologous recombination-based strategies for bacterial genome editing.Besides the traditionally used SpyCas9, other CRISPR-Cas systems (both heterologous and native) are currently being evaluated in bacteria for their editing potential.Catalytically inactive variants of CRISPR-Cas systems are used for transcriptional control in bacteria with great potential for fundamental research and applications.

Effects of Argonaute on gene expression in Thermus thermophilus
Swarts, D.C. ; Koehorst, J.J. ; Westra, E.R. ; Schaap, P.J. ; Oost, J. van der - \ 2015
PRJEB8709 - ERP009732 - Thermus thermophilus
To investigate if TtAgo also has the potential to control RNA levels, we analyzed RNA-seq data derived from cultures of four T. thermophilus strain HB27 variants: wild type, TtAgo knockout (Δago), and either strain transformed with a plasmid. Additionally we determined the effect of TtAgo on expression of plasmid-encoded RNA and plasmid DNA levels. Background Eukaryotic Argonaute proteins mediate RNA-guided RNA interference, allowing both regulation of host gene expression and defense against invading mobile genetic elements. Recently, it has become evident that prokaryotic Argonaute homologs mediate DNA-guided DNA interference, and play a role in host defense. Argonaute of the bacterium Thermus thermophilus (TtAgo) targets invading plasmid DNA during and after transformation. Using small interfering DNA guides, TtAgo can cleave single and double stranded DNAs. Although TtAgo additionally has been demonstrated to cleave RNA targets complementary to its DNA guide in vitro, RNA targeting by TtAgo has not been demonstrated in vivo. Methods To investigate if TtAgo also has the potential to control RNA levels, we analyzed RNA-seq data derived from cultures of four T. thermophilus strain HB27 variants: wild type, TtAgo knockout (Δago), and either strain transformed with a plasmid. Additionally we determined the effect of TtAgo on expression of plasmid-encoded RNA and plasmid DNA levels. Results In the absence of exogenous DNA (plasmid), TtAgo presence or absence had no effect on gene expression levels. When plasmid DNA is present, TtAgo reduces plasmid DNA levels 4-fold, and a corresponding reduction of plasmid gene transcript levels was observed. We therefore conclude that TtAgo interferes with plasmid DNA, but not with plasmid RNA. Interestingly, TtAgo presence stimulates expression of specific endogenous genes, but only when exogenous plasmid DNA was present. Specifically, the presence of TtAgo directly or indirectly stimulates expression of CRISPR loci and associated genes, some of which are involved in CRISPR adaptation. This suggests that TtAgo-mediated interference with plasmid DNA stimulates CRISPR adaptation.
Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System
Zetsche, Bernd ; Gootenberg, Jonathan S. ; Abudayyeh, Omar O. ; Slaymaker, Ian M. ; Makarova, Kira S. ; Essletzbichler, Patrick ; Volz, Sara E. ; Joung, Julia ; Oost, John van der; Regev, Aviv ; Koonin, Eugene V. ; Zhang, Feng - \ 2015
Cell 163 (2015)3. - ISSN 0092-8674 - p. 759 - 771.
The microbial adaptive immune system CRISPR mediates defense against foreign genetic elements through two classes of RNA-guided nuclease effectors. Class 1 effectors utilize multi-protein complexes, whereas class 2 effectors rely on single-component effector proteins such as the well-characterized Cas9. Here, we report characterization of Cpf1, a putative class 2 CRISPR effector. We demonstrate that Cpf1 mediates robust DNA interference with features distinct from Cas9. Cpf1 is a single RNA-guided endonuclease lacking tracrRNA, and it utilizes a T-rich protospacer-adjacent motif. Moreover, Cpf1 cleaves DNA via a staggered DNA double-stranded break. Out of 16 Cpf1-family proteins, we identified two candidate enzymes from Acidaminococcus and Lachnospiraceae, with efficient genome-editing activity in human cells. Identifying this mechanism of interference broadens our understanding of CRISPR-Cas systems and advances their genome editing applications.
Analysis of protein-RNA interactions in CRISPR proteins and effector complexes by UV-induced cross-linking and mass spectrometry
Sharma, Kundan ; Hrle, Ajla ; Kramer, Katharina ; Sachsenberg, Timo ; Staals, Raymond H.J. ; Randau, Lennart ; Marchfelder, Anita ; Oost, John van der; Kohlbacher, Oliver ; Conti, Elena ; Urlaub, Henning - \ 2015
Methods : a companion to Methods in enzymology 89 (2015). - ISSN 1046-2023 - p. 138 - 148.
Cas7 - CRISPR-Cas - Mass spectrometry - Protein-RNA interactions - UV cross-linking

Ribonucleoprotein (RNP) complexes play important roles in the cell by mediating basic cellular processes, including gene expression and its regulation. Understanding the molecular details of these processes requires the identification and characterization of protein-RNA interactions. Over the years various approaches have been used to investigate these interactions, including computational analyses to look for RNA binding domains, gel-shift mobility assays on recombinant and mutant proteins as well as co-crystallization and NMR studies for structure elucidation. Here we report a more specialized and direct approach using UV-induced cross-linking coupled with mass spectrometry. This approach permits the identification of cross-linked peptides and RNA moieties and can also pin-point exact RNA contact sites within the protein. The power of this method is illustrated by the application to different single- and multi-subunit RNP complexes belonging to the prokaryotic adaptive immune system, CRISPR-Cas (CRISPR: clustered regularly interspaced short palindromic repeats; Cas: CRISPR associated). In particular, we identified the RNA-binding sites within three Cas7 protein homologs and mapped the cross-linking results to reveal structurally conserved Cas7 - RNA binding interfaces. These results demonstrate the strong potential of UV-induced cross-linking coupled with mass spectrometry analysis to identify RNA interaction sites on the RNA binding proteins.

An updated evolutionary classification of CRISPR-Cas systems
Makarova, K.S. ; Wolf, Y.I. ; Alkhnbashi, O.S. ; Brouns, S.J.J. ; Oost, John Van Der - \ 2015
Nature Reviews Microbiology 13 (2015)11. - ISSN 1740-1526 - p. 722 - 736.

The evolution of CRISPR-cas loci, which encode adaptive immune systems in archaea and bacteria, involves rapid changes, in particular numerous rearrangements of the locus architecture and horizontal transfer of complete loci or individual modules. These dynamics complicate straightforward phylogenetic classification, but here we present an approach combining the analysis of signature protein families and features of the architecture of cas loci that unambiguously partitions most CRISPR-cas loci into distinct classes, types and subtypes. The new classification retains the overall structure of the previous version but is expanded to now encompass two classes, five types and 16 subtypes. The relative stability of the classification suggests that the most prevalent variants of CRISPR-Cas systems are already known. However, the existence of rare, currently unclassifiable variants implies that additional types and subtypes remain to be characterized.

CRISPR sabotage
Oost, John van der; Brouns, S.J.J. - \ 2015
Genome Biology 16 (2015)1. - ISSN 1474-7596

The biological arms race generally involves the rapid co-evolution of anti-virus systems in host organisms and of anti-anti-virus systems in their viral parasites. The CRISPR-Cas system is an example of a prokaryotic immune system in which such co-evolution occurs, as was recently demonstrated by the characterization of a set of viral anti-CRISPR proteins.

Biogenesis pathways of RNA guides in archaeal and bacterial CRISPR-Cas adaptive immunity
Charpentier, Emmanuelle ; Richter, Hagen ; Oost, John van der; White, Malcolm F. - \ 2015
FEMS Microbiology Reviews 39 (2015)3. - ISSN 0168-6445 - p. 428 - 441.
Cas5d - Cas6 - Cas9 - crRNA biogenesis - RNase III - TracrRNA

CRISPR-Cas is an RNA-mediated adaptive immune system that defends bacteria and archaea against mobile genetic elements. Short mature CRISPR RNAs (crRNAs) are key elements in the interference step of the immune pathway. A CRISPR array composed of a series of repeats interspaced by spacer sequences acquired from invading mobile genomes is transcribed as a precursor crRNA (pre-crRNA) molecule. This pre-crRNA undergoes one or two maturation steps to generate the mature crRNAs that guide CRISPR-associated (Cas) protein(s) to cognate invading genomes for their destruction. Different types of CRISPR-Cas systems have evolved distinct crRNA biogenesis pathways that implicate highly sophisticated processing mechanisms. In Types I and III CRISPR-Cas systems, a specific endoribonuclease of the Cas6 family, either standalone or in a complex with other Cas proteins, cleaves the pre-crRNA within the repeat regions. In Type II systems, the trans-acting small RNA (tracrRNA) base pairs with each repeat of the pre-crRNA to form a dual-RNA that is cleaved by the housekeeping RNase III in the presence of the protein Cas9. In this review, we present a detailed comparative analysis of pre-crRNA recognition and cleavage mechanisms involved in the biogenesis of guide crRNAs in the three CRISPR-Cas types.

Effects of Argonaute on Gene Expression in Thermus thermophilus
Swarts, D.C. ; Koehorst, J.J. ; Westra, E.R. ; Schaap, P.J. ; Oost, J. van der - \ 2015
PLoS ONE 10 (2015)4. - ISSN 1932-6203 - 13 p.
BACKGROUND: Eukaryotic Argonaute proteins mediate RNA-guided RNA interference, allowing both regulation of host gene expression and defense against invading mobile genetic elements. Recently, it has become evident that prokaryotic Argonaute homologs mediate DNA-guided DNA interference, and play a role in host defense. Argonaute of the bacterium Thermus thermophilus (TtAgo) targets invading plasmid DNA during and after transformation. Using small interfering DNA guides, TtAgo can cleave single and double stranded DNAs. Although TtAgo additionally has been demonstrated to cleave RNA targets complementary to its DNA guide in vitro, RNA targeting by TtAgo has not been demonstrated in vivo. METHODS: To investigate if TtAgo also has the potential to control RNA levels, we analyzed RNA-seq data derived from cultures of four T. thermophilus strain HB27 variants: wild type, TtAgo knockout (¿ago), and either strain transformed with a plasmid. Additionally we determined the effect of TtAgo on expression of plasmid-encoded RNA and plasmid DNA levels. RESULTS: In the absence of exogenous DNA (plasmid), TtAgo presence or absence had no effect on gene expression levels. When plasmid DNA is present, TtAgo reduces plasmid DNA levels 4-fold, and a corresponding reduction of plasmid gene transcript levels was observed. We therefore conclude that TtAgo interferes with plasmid DNA, but not with plasmid-encoded RNA. Interestingly, TtAgo presence stimulates expression of specific endogenous genes, but only when exogenous plasmid DNA was present. Specifically, the presence of TtAgo directly or indirectly stimulates expression of CRISPR loci and associated genes, some of which are involved in CRISPR adaptation. This suggests that TtAgo-mediated interference with plasmid DNA stimulates CRISPR adaptation
Structural biology. Structures of the CRISPR-Cmr complex reveal mode of RNA target positioning
Taylor, D.W. ; Zhu, Y. ; Staals, R.H.J. ; Kornfeld, J.E. ; Shinkai, A. ; Oost, J. van der; Nogales, E. ; Doudna, J.A. - \ 2015
Science 348 (2015)6234. - ISSN 0036-8075 - p. 581 - 585.
Adaptive immunity in bacteria involves RNA-guided surveillance complexes that use CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) proteins together with CRISPR RNAs (crRNAs) to target invasive nucleic acids for degradation. Whereas type I and type II CRISPR-Cas surveillance complexes target double-stranded DNA, type III complexes target single-stranded RNA. Near-atomic resolution cryo-electron microscopy reconstructions of native type III Cmr (CRISPR RAMP module) complexes in the absence and presence of target RNA reveal a helical protein arrangement that positions the crRNA for substrate binding. Thumblike ß hairpins intercalate between segments of duplexed crRNA:target RNA to facilitate cleavage of the target at 6-nucleotide intervals. The Cmr complex is architecturally similar to the type I CRISPR-Cascade complex, suggesting divergent evolution of these immune systems from a common ancestor.
Bacteriophage exclusion, a new defense system
Barrangou, R. ; Oost, J. van der - \ 2015
The EMBO Journal 34 (2015)2. - ISSN 0261-4189 - p. 134 - 135.
The ability to withstand viral predation is critical for survival of most microbes. Accordingly, a plethora of phage resistance systems has been identified in bacterial genomes (Labrie et al, 2010), including restriction-modification systems (R-M) (Tock & Dryden, 2005), abortive infection (Abi) (Chopin et al, 2005), Argonaute-based interference (Swarts et al, 2014), as well as clustered regularly interspaced short palindromic repeats (CRISPR) and associated protein (Cas) adaptive immune system (CRISPR-Cas) (Barrangou & Marraffini, 2014; Van der Oost et al, 2014). Predictably, the dark matter of bacterial genomes contains a wealth of genetic gold. A study published in this issue of The EMBO Journal by Goldfarb et al (2015) unveils bacteriophage exclusion (BREX) as a novel, widespread bacteriophage resistance system that provides innate immunity against virulent and temperate phage in bacteria.
RNA-targeting by the Type III-A CRISPR-Cas complex of Thermus thermophilus
Staals, R.H.J. ; Zhu, Y. ; Taylor, D.W. ; Kornfeld, J.E. ; Sharma, K. ; Barendregt, A. ; Koehorst, J.J. ; Vlot, M. ; Neupane, N. ; Varossieau, K. ; Sakamoto, K. ; Suzuki, T. ; Schaap, P.J. ; Urlaub, H. ; Heck, A.J.R. ; Nogales, E. ; Doudna, J.A. ; Shinkai, A. ; Oost, J. van der - \ 2014
PRJEB7461 - ERP007191
crRNAs from the Thermus thermophilus CRISPR-Cas Csm complex
RNA Targeting by the Type III-A CRISPR-Cas Csm Complex of Thermus thermophilus
Staals, R.H.J. ; Zhu, Y. ; Taylor, D.W. ; Kornfeld, J.E. ; Sharma, K. ; Barendregt, A. ; Koehorst, J.J. ; Vlot, M. ; Neupane, N. ; Varossieau, K. ; Sakamoto, K. ; Suzuki, T. ; Schaap, P.J. ; Urlaub, H. ; Heck, A.J.R. ; Nogales, E. ; Doudna, J.A. ; Shinkai, A. ; Oost, J. van der - \ 2014
Molecular Cell 56 (2014)4. - ISSN 1097-2765 - p. 518 - 530.
guided surveillance complex - bacterial immune-system - adaptive immunity - mass-spectrometry - crystal-structure - escherichia-coli - haloferax-volcanii - antiviral defense - seed sequence - protein
CRISPR-Cas is a prokaryotic adaptive immune system that provides sequence-specific defense against foreign nucleic acids. Here we report the structure and function of the effector complex of the Type III-A CRISPR-Cas system of Thermus thermophilus: the Csm complex (TtCsm). TtCsm is composed of five different protein subunits (Csm1–Csm5) with an uneven stoichiometry and a single crRNA of variable size (35–53 nt). The TtCsm crRNA content is similar to the Type III-B Cmr complex, indicating that crRNAs are shared among different subtypes. A negative stain EM structure of the TtCsm complex exhibits the characteristic architecture of Type I and Type III CRISPR-associated ribonucleoprotein complexes. crRNA-protein crosslinking studies show extensive contacts between the Csm3 backbone and the bound crRNA. We show that, like TtCmr, TtCsm cleaves complementary target RNAs at multiple sites. Unlike Type I complexes, interference by TtCsm does not proceed via initial base pairing by a seed sequence.
Molecular insights into DNA interference by CRISPR-associated nuclease-helicase Cas3
Gong, B. ; Shin, M. ; Sun, J. ; Jung, C.H. ; Bolt, E.L. ; Oost, J. van der; Kim, J.S. - \ 2014
Proceedings of the National Academy of Sciences of the United States of America 111 (2014)46. - ISSN 0027-8424 - p. 16359 - 16364.
bacterial immune-system - in-vitro reconstitution - escherichia-coli - antiviral defense - adaptive immunity - crystal-structure - structural basis - rna - complex - cascade
Mobile genetic elements in bacteria are neutralized by a system based on clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins. Type I CRISPR-Cas systems use a “Cascade” ribonucleoprotein complex to guide RNA specifically to complementary sequence in invader double-stranded DNA (dsDNA), a process called “interference.” After target recognition by Cascade, formation of an R-loop triggers recruitment of a Cas3 nuclease-helicase, completing the interference process by destroying the invader dsDNA. To elucidate the molecular mechanism of CRISPR interference, we analyzed crystal structures of Cas3 from the bacterium Thermobaculum terrenum, with and without a bound ATP analog. The structures reveal a histidine-aspartate (HD)-type nuclease domain fused to superfamily-2 (SF2) helicase domains and a distinct C-terminal domain. Binding of ATP analog at the interface of the SF2 helicase RecA-like domains rearranges a motif V with implications for the enzyme mechanism. The HD-nucleolytic site contains two metal ions that are positioned at the end of a proposed nucleic acid-binding tunnel running through the SF2 helicase structure. This structural alignment suggests a mechanism for 3' to 5' nucleolytic processing of the displaced strand of invader DNA that is coordinated with ATP-dependent 3' to 5' translocation of Cas3 along DNA. In agreement with biochemical studies, the presented Cas3 structures reveal important mechanistic details on the neutralization of genetic invaders by type I CRISPR-Cas systems.
Crystal structure of the CRISPR RNA–guided surveillance complex from Escherichia coli
Jackson, R.N. ; Golden, S.M. ; Erp, P.B. ; Carter, J. ; Westra, E.R. ; Brouns, S.J.J. ; Oost, J. van der; Terwilliger, T.C. ; Read, R.J. ; Wiedenheft, B. - \ 2014
Science 345 (2014)6203. - ISSN 0036-8075 - p. 1473 - 1479.
bacterial immune-system - processes pre-crrna - thermus-thermophilus - cas systems - interference complex - target recognition - antiviral defense - seed sequence - dna - cascade
Clustered regularly interspaced short palindromic repeats (CRISPRs) are essential components of RNA-guided adaptive immune systems that protect bacteria and archaea from viruses and plasmids. In Escherichia coli, short CRISPR-derived RNAs (crRNAs) assemble into a 405-kilodalton multisubunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Here we present the 3.24 angstrom resolution x-ray crystal structure of Cascade. Eleven proteins and a 61-nucleotide crRNA assemble into a seahorse-shaped architecture that binds double-stranded DNA targets complementary to the crRNA-guide sequence. Conserved sequences on the 3' and 5' ends of the crRNA are anchored by proteins at opposite ends of the complex, whereas the guide sequence is displayed along a helical assembly of six interwoven subunits that present five-nucleotide segments of the crRNA in pseudo–A-form configuration. The structure of Cascade suggests a mechanism for assembly and provides insights into the mechanisms of target recognition.
Unravelling the structural and mechanistic basis of CRISPR-Cas systems
Oost, J. van der; Westra, E.R. ; Jackson, R.N. ; Wiedenheft, B. - \ 2014
Nature Reviews Microbiology 12 (2014)7. - ISSN 1740-1526 - p. 479 - 492.
short palindromic repeats - bacterial immune-system - rna silencing complex - processes pre-crrna - human gut virome - crystal-structure - escherichia-coli - streptococcus-thermophilus - thermus-thermophilus - interference complex
Bacteria and archaea have evolved sophisticated adaptive immune systems, known as CRISPR–Cas (clustered regularly interspaced short palindromic repeats–CRISPR-associated proteins) systems, which target and inactivate invading viruses and plasmids. Immunity is acquired by integrating short fragments of foreign DNA into CRISPR loci, and following transcription and processing of these loci, the CRISPR RNAs (crRNAs) guide the Cas proteins to complementary invading nucleic acid, which results in target interference. In this Review, we summarize the recent structural and biochemical insights that have been gained for the three major types of CRISPR–Cas systems, which together provide a detailed molecular understanding of the unique and conserved mechanisms of RNA-guided adaptive immunity in bacteria and archaea.
The Role of CRISPR-Cas Systems in Virulence of Pathogenic Bacteria
Louwen, R. ; Staals, R.H.J. ; Endtz, H.P. ; Baarlen, P. van; Oost, J. van der - \ 2014
Microbiology and Molecular Biology Reviews 78 (2014)1. - ISSN 1092-2172 - p. 74 - 88.
damage-response framework - short palindromic repeats - guillain-barre-syndrome - swiss army knife - escherichia-coli - campylobacter-jejuni - streptococcus-mutans - yersinia-pestis - salmonella-enterica - mycobacterium-tuberculosis
Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes are present in many bacterial and archaeal genomes. Since the discovery of the typical CRISPR loci in the 1980s, well before their physiological role was revealed, their variable sequences have been used as a complementary typing tool in diagnostic, epidemiologic, and evolutionary analyses of prokaryotic strains. The discovery that CRISPR spacers are often identical to sequence fragments of mobile genetic elements was a major breakthrough that eventually led to the elucidation of CRISPR-Cas as an adaptive immunity system. Key elements of this unique prokaryotic defense system are small CRISPR RNAs that guide nucleases to complementary target nucleic acids of invading viruses and plasmids, generally followed by the degradation of the invader. In addition, several recent studies have pointed at direct links of CRISPR-Cas to regulation of a range of stress-related phenomena. An interesting example concerns a pathogenic bacterium that possesses a CRISPR-associated ribonucleoprotein complex that may play a dual role in defense and/or virulence. In this review, we describe recently reported cases of potential involvement of CRISPR-Cas systems in bacterial stress responses in general and bacterial virulence in particular
Modified cascade ribonucleoproteins and uses thereof
Brouns, S.J.J. ; Oost, J. van der - \ 2013
Octrooinummer: WO2013098244, verleend: 2013-07-04.
A clustered regularly interspaced short palindromic repeat (CRISPR)-associated complex for adaptive antiviral defence(Cascade);the Cascade protein complex comprising at least CRISPR-associated protein subunits Cas7, Cas5 and Cas6 which includes at least one subunit with an additional amino acid sequence possessing nucleic acid or chromatin modifying, visualising, transcription activating or transcription repressing activity. The Cascade complex with additional activity is combined with an RNA molecule to produce a ribonucleoprotein complex. The RNA molecule is selected to have substantial complementarity to a target sequence. Targeted ribonucleoproteins can be used as genetic engineering tools for precise cutting of nucleic acids in homologous recombination, non-homologous end joining, gene modification, gene integration, mutation repair or for their visualisation, transcriptional activation or repression.A pair of ribonucleotides fused to FokI dimers may be used to generate double-strand breakages in the DNA to facilitate these applications in a sequence-specific manner.
crRNA biogenesis
Charpentier, E. ; Oost, J. van der; White, M. - \ 2013
In: CRISPR-Cas Systems / Barrangou, R., Oost, J. van der, Berlin-Heidelberg : Springer Verlag - ISBN 9783642346569 - p. 115 - 144.
Mature crRNAs are key elements in CRISPR-Cas defense against genome invaders. These short RNAs are composed of unique repeat/spacer sequences that guide the Cas protein(s) to the cognate invading nucleic acids for their destruction. The biogenesis of mature crRNAs involves highly precise processing events. Interestingly, different types of CRISPR-Cas systems have evolved distinct crRNA maturation mechanisms. The CRISPR repeat-spacer array is transcribed as a precursor CRISPR RNA molecule (pre-crRNA) that undergoes one or two maturation steps. In type I CRISPR-Cas systems, pre-crRNA is cleaved within the repeat regions by a specific Cas6-like endoribonuclease that at least in some cases is a subunit of a Cascade complex to yield the mature crRNAs. In type III systems, the standalone endoribonuclease Cas6 processes pre-crRNA by cleavage within the repeats, producing an intermediate molecule that is further trimmed to generate the mature crRNAs. Type II systems have evolved a unique crRNA biogenesis pathway, in which a trans-acting small RNA (encoded by the CRISPR-Cas locus) base pairs with each repeat sequence of the pre-crRNA to form a double-stranded RNA template that is cleaved by the housekeeping endoribonuclease III in the presence of protein Cas9 (Csn1). The generated intermediates are then subjected to further maturation by a yet to be revealed mechanism. In this chapter, we present a detailed comparative analysis of pre-crRNA recognition and cleavage mechanisms involved in crRNA biogenesis in the three types of CRISPR-Cas systems
RNA-mediated Adaptive Immunity in Bacteria and Archaea
Barrangou, R. ; Oost, J. van der - \ 2013
In: CRISPR-Cas Systems Berlin-Heidelberg : Springer Verlag - ISBN 9783642346569 - 299 p.
Massive activation of archaeal defense genes during viral infection
Quax, T.E.F. ; Voet, M. ; Sismeiro, O. ; Dillies, M.A. ; Jagla, B. ; Coppée, J.Y. ; Sezonov, G. ; Forterre, P. ; Oost, J. van der; Lavigne, R. ; Prangishvili, D. - \ 2013
Journal of Virology 87 (2013)15. - ISSN 0022-538X - p. 8419 - 8428.
differential expression analysis - turreted icosahedral virus - toxin-antitoxin systems - sulfolobus-solfataricus - cell-division - host interactions - crispr rnas - dna - prokaryotes - complex
Archaeal viruses display unusually high genetic and morphological diversity. Studies of these viruses proved to be instrumental for the expansion of knowledge on viral diversity and evolution. The Sulfolobus islandicus rod-shaped virus 2 (SIRV2) is a model to study virus-host interactions in Archaea. It is a lytic virus that exploits a unique egress mechanism based on the formation of remarkable pyramidal structures on the host cell envelope. Using whole-transcriptome sequencing, we present here a global map defining host and viral gene expression during the infection cycle of SIRV2 in its hyperthermophilic host S. islandicus LAL14/1. This information was used, in combination with a yeast two-hybrid analysis of SIRV2 protein interactions, to advance current understanding of viral gene functions. As a consequence of SIRV2 infection, transcription of more than one-third of S. islandicus genes was differentially regulated. While expression of genes involved in cell division decreased, those genes playing a role in antiviral defense were activated on a large scale. Expression of genes belonging to toxin-antitoxin and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems was specifically pronounced. The observed different degree of activation of various CRISPR-Cas systems highlights the specialized functions they perform. The information on individual gene expression and activation of antiviral defense systems is expected to aid future studies aimed at detailed understanding of the functions and interplay of these systems in vivo
Structure and Activity of the RNA-Targeting Type III-B CRISPR-Cas Complex of Thermus thermophilus
Staals, R.H.J. ; Agari, Y. ; Maki-Yonekura, S. ; Zhu, Y. ; Taylor, D.W. ; Duijn, E. van; Barendregt, A. ; Vlot, M. ; Koehorst, J.J. ; Sakamoto, K. ; Masuda, A. ; Dohmae, N. ; Schaap, P.J. ; Doudna, J.A. ; Heck, A. ; Yonekura, K. ; Oost, J. van der; Shinkai, A. - \ 2013
Molecular Cell 52 (2013)1. - ISSN 1097-2765 - p. 135 - 145.
of-flight instrument - mass-spectrometry - escherichia-coli - silencing complex - antiviral defense - immune-system - protein - interference - transcription - recognition
The CRISPR-Cas system is a prokaryotic host defense system against genetic elements. The Type III-B CRISPR-Cas system of the bacterium Thermus thermophilus, the TtCmr complex, is composed of six different protein subunits (Cmr1-6) and one crRNA with a stoichiometry of Cmr112131445361:crRNA1. The TtCmr complex copurifies with crRNA species of 40 and 46 nt, originating from a distinct subset of CRISPR loci and spacers. The TtCmr complex cleaves the target RNA at multiple sites with 6 nt intervals via a 5' ruler mechanism. Electron microscopy revealed that the structure of TtCmr resembles a "sea worm" and is composed of a Cmr2-3 heterodimer "tail," a helical backbone of Cmr4 subunits capped by Cmr5 subunits, and a curled "head" containing Cmr1 and Cmr6. Despite having a backbone of only four Cmr4 subunits and being both longer and narrower, the overall architecture of TtCmr resembles that of Type I Cascade complexes
A novel link between Campylobacter jejuni bacteriophage defence, virulence and Guillain-Barré syndrome
Louwen, R. ; Horst-Kreft, D. ; Boer, A.G. de; Graaf, L. van der; Knegt, G. de; Hamersma, M. ; Heikema, A.P. ; Timms, A.R. ; Jacobs, B.C. ; Wagenaar, J.A. ; Endtz, H.P. ; Oost, J. van der; Wells, J. ; Nieuwenhuis, E.E.S. ; Vliet, A.H. van; Willemsen, P.T.J. ; Baarlen, P. van; Belkum, A. van - \ 2013
European Journal of Clinical Microbiology and Infectious Diseases 32 (2013)2. - ISSN 0934-9723 - p. 207 - 226.
neisseria-meningitidis - sialic-acid - haemophilus-influenzae - pasteurella-multocida - molecular mimicry - antiganglioside antibodies - pseudomonas-aeruginosa - natural transformation - lipo-oligosaccharide - pathogenic neisseria
Guillain–Barré syndrome (GBS) is a post-infectious disease in which the human peripheral nervous system is affected after infection by specific pathogenic bacteria, including Campylobacter jejuni. GBS is suggested to be provoked by molecular mimicry between sialylated lipooligosaccharide (LOS) structures on the cell envelope of these bacteria and ganglioside epitopes on the human peripheral nerves, resulting in autoimmune-driven nerve destruction. Earlier, the C. jejuni sialyltransferase (Cst-II) was found to be linked to GBS and demonstrated to be involved in the biosynthesis of the ganglioside-like LOS structures. Apart from a role in pathogenicity, we report here that Cst-II-generated ganglioside-like LOS structures confer efficient bacteriophage resistance in C. jejuni. By bioinformatic analysis, it is revealed that the presence of sialyltransferases in C. jejuni and other potential GBS-related pathogens correlated significantly with the apparent degeneration of an alternative anti-virus system: type II Clusters of Regularly Interspaced Short Palindromic Repeat and associated genes (CRISPR-Cas). Molecular analysis of the C. jejuni CRISPR-Cas system confirmed the bioinformatic investigation. CRISPR degeneration and mutations in the cas genes cas2, cas1 and csn1 were found to correlate with Cst-II sialyltransferase presence (p¿
The CRISPRs, They Are A-Changin': How Prokaryotes Generate Adaptive Immunity
Westra, E.R. ; Swarts, D.C. ; Staals, R.H.J. ; Jore, M.M. ; Brouns, S.J.J. ; Oost, J. van der - \ 2012
Annual Review of Genetics 46 (2012). - ISSN 0066-4197 - p. 311 - 339.
short palindromic repeats - restriction-modification systems - regularly spaced repeats - phage abortive infection - thermus-thermophilus hb8 - enterica serovar typhi - ns-mediated repression - escherichia-coli - h-ns - crystal-structure
All organisms need to continuously adapt to changes in their environment. Through horizontal gene transfer, bacteria and archaea can rapidly acquire new traits that may contribute to their survival. However, because new DNA may also cause damage, removal of imported DNA and protection against selfish invading DNA elements are also important. Hence, there should be a delicate balance between DNA uptake and DNA degradation. Here, we describe prokaryotic antiviral defense systems, such as receptor masking or mutagenesis, blocking of phage DNA injection, restriction/modification, and abortive infection. The main focus of this review is on CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated), a prokaryotic adaptive immune system. Since its recent discovery, our biochemical understanding of this defense system has made a major leap forward. Three highly diverse CRISPR/Cas types exist that display major structural and functional differences in their mode of generating resistance against invading nucleic acids. Because several excellent recent reviews cover all CRISPR subtypes, we mainly focus on a detailed description of the type I-E CRISPR/Cas system of the model bacterium Escherichia coli K12
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