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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    We will mail you new results for this query: (q=crispr AND metisnummer==1014585)
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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.
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