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: keywords==genome editing
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Making the cut(s): how Cas12a cleaves target and non-target DNA
Swarts, Daan C. - \ 2019
Biochemical Society Transactions 47 (2019)5. - ISSN 0300-5127 - p. 1499 - 1510.
cis-cleavage - trans-cleavage - Cas12a - Cpf1 - CRISPR–Cas - genome editing

CRISPR-Cas12a (previously named Cpf1) is a prokaryotic deoxyribonuclease that can be programmed with an RNA guide to target complementary DNA sequences. Upon binding of the target DNA, Cas12a induces a nick in each of the target DNA strands, yielding a double-stranded DNA break. In addition to inducing cis-cleavage of the targeted DNA, target DNA binding induces trans-cleavage of non-target DNA. As such, Cas12a-RNA guide complexes can provide sequence-specific immunity against invading nucleic acids such as bacteriophages and plasmids. Akin to CRISPR-Cas9, Cas12a has been repurposed as a genetic tool for programmable genome editing and transcriptional control in both prokaryotic and eukaryotic cells. In addition, its trans-cleavage activity has been applied for high-sensitivity nucleic acid detection. Despite the demonstrated value of Cas12a for these applications, the exact molecular mechanisms of both cis- and trans-cleavage of DNA were not completely understood. Recent studies have revealed mechanistic details of Cas12a-mediates DNA cleavage: base pairing of the RNA guide and the target DNA induces major conformational changes in Cas12a. These conformational changes render Cas12a in a catalytically activated state in which it acts as deoxyribonuclease. This deoxyribonuclease activity mediates cis-cleavage of the displaced target DNA strand first, and the RNA guide-bound target DNA strand second. As Cas12a remains in the catalytically activated state after cis-cleavage, it subsequently demonstrates trans-cleavage of non-target DNA. Here, I review the mechanistic details of Cas12a-mediated cis- and trans-cleavage of DNA. In addition, I discuss how bacteriophage-derived anti-CRISPR proteins can inhibit Cas12a activity.

Keeping crispr in check: diverse mechanisms of phage-encoded anti-crisprs
Trasanidou, Despoina ; Gerós, Ana Sousa ; Mohanraju, Prarthana ; Nieuwenweg, Anna Cornelia ; Nobrega, Franklin L. ; Staals, Raymond H.J. - \ 2019
FEMS Microbiology Letters 366 (2019)9. - ISSN 0378-1097
crispr-cas - anti-crispr - genome editing - phage
CRISPR-Cas represents the only adaptive immune system of prokaryotes known to date. These immune systems are widespread among bacteria and archaea, and provide protection against invasion of mobile genetic elements, such as bacteriophages and plasmids. As a result of the arms-race between phages and their prokaryotic hosts, phages have evolved inhibitors known as anti-CRISPR (Acr) proteins to evade CRISPR immunity. In the recent years, several Acr proteins have been described in both temperate and virulent phages targeting diverse CRISPR-Cas systems. Here, we describe the strategies of Acr discovery and the multiple molecular mechanisms by which these proteins operate to inhibit CRISPR immunity. We discuss the biological relevance of Acr proteins and speculate on the implications of their activity for the development of improved CRISPR-based research and biotechnological tools.
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.

A comparison of the EU regulatory approach to directed mutagenesis with that of other jurisdictions, consequences for international trade and potential steps forward
Eriksson, Dennis ; Kershen, Drew ; Nepomuceno, Alexandre ; Pogson, Barry J. ; Prieto, Humberto ; Purnhagen, Kai ; Smyth, Stuart ; Wesseler, Justus ; Whelan, Agustina - \ 2019
New Phytologist 222 (2019)4. - ISSN 0028-646X - p. 1673 - 1684.
CJEU - directed mutagenesis - genetically modified organism (GMO) - genome editing - precision breeding

A special regulatory regime applies to products of recombinant nucleic acid modifications. A ruling from the European Court of Justice has interpreted this regulatory regime in a way that it also applies to emerging mutagenesis techniques. Elsewhere regulatory progress is also ongoing. In 2015, Argentina launched a regulatory framework, followed by Chile in 2017 and recently Brazil and Colombia. In March 2018, the USDA announced that it will not regulate genome-edited plants differently if they could have also been developed through traditional breeding. Canada has an altogether different approach with their Plants with Novel Traits regulations. Australia is currently reviewing its Gene Technology Act. This article illustrates the deviation of the European Union's (EU's) approach from the one of most of the other countries studied here. Whereas the EU does not implement a case-by-case approach, this approach is taken by several other jurisdictions. Also, the EU court ruling adheres to a process-based approach while most other countries have a stronger emphasis on the regulation of the resulting product. It is concluded that, unless a functioning identity preservation system for products of directed mutagenesis can be established, the deviation results in a risk of asynchronous approvals and disruptions in international trade.

The impact of genome editing on the introduction of monogenic traits in livestock : Simulation program
Bastiaansen, J.W.M. ; Bovenhuis, H. ; Groenen, M. ; Megens, H.J.W.C. ; Mulder, H.A. - \ 2018
Wageningen University & Research
genome editing - selection emphasis - monogenic trait - selection response - reduced editing efficiency - genomic selection - editing efficiency - polygenic trait - editing procedures - livestock
Background Genome editing technologies provide new tools for genetic improvement and have the potential to become the next game changer in animal and plant breeding. The aim of this study was to investigate how genome editing in combination with genomic selection can accelerate the introduction of a monogenic trait in a livestock population as compared to genomic selection alone. Methods A breeding population was simulated under genomic selection for a polygenic trait. After reaching Bulmer equilibrium, the selection objective was to increase the allele frequency of a monogenic trait, with or without genome editing, in addition to improving the polygenic trait. Scenarios were compared for time to fixation of the desired allele, selection response for the polygenic trait, and level of inbreeding. The costs, in terms of number of editing procedures, were compared to the benefits of having more animals with the desired phenotype of the monogenic trait. Effects of reduced editing efficiency were investigated. Results In a population of 20,000 selection candidates per generation, the total number of edited zygotes needed to reach fixation of the desired allele was 22,118, 7072, or 3912 with, no, moderate, or high selection emphasis on the monogenic trait, respectively. Genome editing resulted in up to four-fold faster fixation of the desired allele when efficiency was 100%, while the loss in long-term selection response for the polygenic trait was up to seven-fold less compared to genomic selection alone. With moderate selection emphasis on the monogenic trait, introduction of genome editing led to a four-fold reduction in the total number of animals showing the undesired phenotype before fixation. However, with a currently realistic editing efficiency of 4%, the number of required editing procedures increased by 72% and loss in selection response increased eight-fold compared to 100% efficiency. With low efficiency, loss in selection response was 29% more compared to genomic selection alone. Conclusions Genome editing strongly decreased the time to fixation for a desired allele compared to genomic selection alone. Reduced editing efficiency had a major impact on the number of editing procedures and on the loss in selection response. In addition to ethical and welfare considerations of genome editing, a careful assessment of its technical costs and benefits is required.
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.

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