Phytophthora palmivora establishes tissue-specific intracellular infection structures in the earliest divergent land plant lineage.
Philip Carella, Anna Gogleva, Marta Tomaselli, Carolin Alfs, and Sebastian Schornack.
PNAS April 17, 2018 115 (16) E3846-E3855
Synergistic binding of bHLH transcription factors to the promoter of the maize NADP-ME gene used in C4 photosynthesis is based on an ancient code found in the ancestral C3 state.
Borba AR, Serra TS, Górska A, Gouveia P, Cordeiro AM, Reyna-Llorens I, Kneřová J, Barros PM, Abreu IA, Oliveira MM, Hibberd JM, Saibo NJM.
Molecular Biology and Evolution, Volume 35, Issue 7, July 2018, Pages 1690–1705.
Electricity generation from digitally printed cyanobacteria.
Sawa, Marin, Andrea Fantuzzi, Paolo Bombelli, Christopher J. Howe, Klaus Hellgardt, and Peter J. Nixon.
Nature Communications 8, no. 1 (2017): 1327.
Insights into Land Plant Evolution Garnered from the Marchantia polymorpha Genome.
Bowman, J.L., Kohchi, T., Yamato, K.T., Jenkins, J., Shu, S., Ishizaki, K., Yamaoka, S., Nishihama, R., Nakamura, Y., Berger, F., Adam, C., Sugamata Aki, S., Althoff, F., Araki, T., Arteaga-Vazquez, M.A., Balasubrmanian, S., Barry, K., Bauer, D., Boehm, C.R., Briginshaw, L., Caballero-Perez, J., Catarino, B., Chen, F., Chiyoda, S., Chovatia, M., Davies, K.M., Delmans, M., Demura, T., Dierschke, T., Dolan, L., Dorantes-Acosta, A.E., Eklund, D.M., Florent, S.N., Flores-Sandoval, E., Fujiyama, A., Fukuzawa, H., Galik, B., Grimanelli, D., Grimwood, J., Grossniklaus, U.
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Human genome editing, 3D-printed replacement organs and artificial photosynthesis – the field of bioengineering offers great promise for tackling the major challenges that face our society. But as a new article out today highlights, these developments provide both opportunities and risks in the short and long term.
Rapid developments in the field of synthetic biology and its associated tools and methods, including more widely available gene editing techniques, have substantially increased our capabilities for bioengineering – the application of principles and techniques from engineering to biological systems, often with the goal of addressing 'real-world' problems.
In a feature article published in the open access journal eLife, an international team of experts led by Dr Bonnie Wintle and Dr Christian R. Boehm from the Centre for the Study of Existential Risk at the University of Cambridge, capture perspectives of industry, innovators, scholars, and the security community in the UK and US on what they view as the major emerging issues in the field. The participants included several OpenPlant researchers and members of the management team.
Dr Wintle says: “The growth of the bio-based economy offers the promise of addressing global environmental and societal challenges, but as our paper shows, it can also present new kinds of challenges and risks. The sector needs to proceed with caution to ensure we can reap the benefits safely and securely.”
The report is intended as a summary and launching point for policy makers across a range of sectors to further explore those issues that may be relevant to them.
Among the issues highlighted by the report as being most relevant over the next five years are:
If technical hurdles can be overcome, such developments might contribute to the future adoption of carbon capture systems, and provide sustainable sources of commodity chemicals and fuel.
Synthetic biology may hold the key to increasing yields on currently farmed land – and hence helping address food security – by enhancing photosynthesis and reducing pre-harvest losses, as well as reducing post-harvest and post-consumer waste.
Gene drives promote the inheritance of preferred genetic traits throughout a species, for example to prevent malaria-transmitting mosquitoes from breeding. However, this technology raises questions about whether it may alter ecosystems, potentially even creating niches where a new disease-carrying species or new disease organism may take hold.
Genome engineering technologies such as CRISPR/Cas9 offer the possibility to improve human lifespans and health. However, their implementation poses major ethical dilemmas. It is feasible that individuals or states with the financial and technological means may elect to provide strategic advantages to future generations.
The areas of synthetic biology in which some defence agencies invest raise the risk of ‘dual-use’. For example, one programme intends to use insects to disseminate engineered plant viruses that confer traits to the target plants they feed on, with the aim of protecting crops from potential plant pathogens – but such technologies could plausibly also be used by others to harm targets.
In the next five to ten years, the authors identified areas of interest including:
While this technology will undoubtedly ease suffering caused by traumatic injuries and a myriad of illnesses, reversing the decay associated with age is still fraught with ethical, social and economic concerns. Healthcare systems would rapidly become overburdened by the cost of replenishing body parts of citizens as they age and could lead new socioeconomic classes, as only those who can pay for such care themselves can extend their healthy years.
The human microbiome is implicated in a large number of human disorders, from Parkinson’s to colon cancer, as well as metabolic conditions such as obesity and type 2 diabetes. Synthetic biology approaches could greatly accelerate the development of more effective microbiota-based therapeutics. However, there is a risk that DNA from genetically engineered microbes may spread to other microbiota in the human microbiome or into the wider environment.
Advancements in automation technology combined with faster and more reliable engineering techniques have resulted in the emergence of robotic 'cloud labs' where digital information is transformed into DNA then expressed in some target organisms. This opens the possibility of new kinds of information security threats, which could include tampering with digital DNA sequences leading to the production of harmful organisms, and sabotaging vaccine and drug production through attacks on critical DNA sequence databases or equipment.
Over the longer term, issues identified include:
Community bio-labs and entrepreneurial startups are customizing and sharing methods and tools for biological experiments and engineering. Combined with open business models and open source technologies, this could herald opportunities for manufacturing therapies tailored to regional diseases that multinational pharmaceutical companies might not find profitable. But this raises concerns around the potential disruption of existing manufacturing markets and raw material supply chains as well as fears about inadequate regulation, less rigorous product quality control and misuse.
Emerging infectious diseases—such as recent Ebola and Zika virus disease outbreaks—and potential biological weapons attacks require scalable, flexible diagnosis and treatment. New technologies could enable the rapid identification and development of vaccine candidates, and plant-based antibody production systems.
The rise of off-patent, generic tools and the lowering of technical barriers for engineering biology has the potential to help those in low-resource settings, benefit from developing a sustainable bioeconomy based on local needs and priorities, particularly where new advances are made open for others to build on.
Dr Jenny Molloy comments: “One theme that emerged repeatedly was that of inequality of access to the technology and its benefits. The rise of open source, off-patent tools could enable widespread sharing of knowledge within the biological engineering field and increase access to benefits for those in developing countries.”
Professor Johnathan Napier from Rothamsted Research adds: “The challenges embodied in the Sustainable Development Goals will require all manner of ideas and innovations to deliver significant outcomes. In agriculture, we are on the cusp of new paradigms for how and what we grow, and where. Demonstrating the fairness and usefulness of such approaches is crucial to ensure public acceptance and also to delivering impact in a meaningful way.”
Dr Christian R. Boehm concludes: “As these technologies emerge and develop, we must ensure public trust and acceptance. People may be willing to accept some of the benefits, such as the shift in ownership away from big business and towards more open science, and the ability to address problems that disproportionately affect the developing world, such as food security and disease. But proceeding without the appropriate safety precautions and societal consensus—whatever the public health benefits—could damage the field for many years to come.”
The research was made possible by the Centre for the Study of Existential Risk, the Synthetic Biology Strategic Research Initiative (both at the University of Cambridge), and the Future of Humanity Institute (University of Oxford). It was based on a workshop co-funded by the Templeton World Charity Foundation and the European Research Council under the European Union’s Horizon 2020 research and innovation programme.
Reference
Wintle, BC, Boehm, CR et al. A transatlantic perspective on 20 emerging issues in biological engineering. eLife; 14 Nov 2017; DOI: 10.7554/eLife.30247
Link to original piece on University News
Hear OpenPlant Coordinator Dr Jenny Molloy discuss the work on BBC Radio 4 'Inside Science'
The text in this work is licensed under a Creative Commons Attribution 4.0 International License. For image use please see separate credits above.
Image Credit: Reaching for the Sky
Susanne Nilsson
Former OpenPlant Fellow Dr Fernan Federici, former OpenPlant PDRA Dr Tim Rudge and colleagues have recently published a pre-print for their low cost and open source multi-fluorescence imaging system for teaching and research in biology and bioengineering, supported by the OpenPlant Fund.
Nuñez, Isaac, Tamara Matute, Roberto Herrera, Juan Keymer, Tim Marzullo, Tim Rudge, and Fernan Federici. "Low cost and open source multi-fluorescence imaging system for teaching and research in biology and bioengineering." bioRxiv (2017): 194324
Examples of images of bacterial colonies and cell-free systems using the microscope. Credit: Federici Lab
The advent of easy-to-use open source microcontrollers, off-the-shelf electronics and customizable manufacturing technologies has facilitated the development of inexpensive scientific devices and laboratory equipment. In this study, we describe an imaging system that integrates low-cost and open-source hardware, software and genetic resources. The multi-fluorescence imaging system consists of readily available 470 nm LEDs, a Raspberry Pi camera and a set of filters made with low cost acrylics. This device allows imaging in scales ranging from single colonies to entire plates.
We developed a set of genetic components (e.g. promoters, coding sequences, terminators) and vectors following the standard framework of Golden Gate, which allowed the fabrication of genetic constructs in a combinatorial, low cost and robust manner. In order to provide simultaneous imaging of multiple wavelength signals, we screened a series of long stokes shift fluorescent proteins that could be combined with cyan/green fluorescent proteins. We found CyOFP1, mBeRFP and sfGFP to be the most compatible set for 3-channel fluorescent imaging. We developed open source Python code to operate the hardware to run time-lapse experiments with automated control of illumination and camera and a Python module to analyze data and extract meaningful biological information.
To demonstrate the potential application of this integral system, we tested its performance on a diverse range of imaging assays often used in disciplines such as microbial ecology, microbiology and synthetic biology. We also assessed its potential for STEM teaching in a high school environment, using it to teach biology, hardware design, optics, and programming. Together, these results demonstrate the successful integration of open source hardware, software, genetic resources and customizable manufacturing to obtain a powerful, low cost and robust system for STEM education, scientific research and bioengineering. All the resources developed here are available under open source license
Dr Mario Juhas and OpenPlant PI Dr Jim Ajioka from the Department of Pathology at the University of Cambridge have contributed to creating reliable and efficient systems for the transfer of synthetic DNA between E. coli and B. subtilis, supported by the OpenPlant Fund.
The majority of the good DNA editing techniques have been developed in Escherichia coli; however, Bacillus subtilis is better host for a plethora of synthetic biology and biotechnology applications.
Using synthetic biology approaches, such as streamlined lambda Red recombineering and Gibson Isothermal Assembly, the team integrated genetic circuits encoding the lysis genes of bacteriophages MS2, ΦX174 and lambda, the thermosensitive repressor and the T7 RNA polymerase into the E. coli chromosome.
In this system the T7 RNA polymerase regulated by the thermosensitive repressor drives the expression of the phage lysis genes. T7 RNA polymerase significantly increases efficiency of cell lysis and transfer of the plasmid and bacterial artificial chromosome-encoded DNA from the lysed E. coli into B. subtilis. The T7 RNA polymerase-driven inducible cell lysis system is therefore suitable for the efficient cell lysis and transfer of the DNA engineered in E. coli to other naturally competent hosts, such as B. subtilis.
The research obtained support from the SynBio Fund and OpenPlant Fund
The full article can be read here.
Image attribution: Debivort at the English language Wikipedia
This file is licensed under the Creative Commons Attribution-Share Alike 3.0 Unported license.
OpenPlant PI Professor Julian Hibberd (Department of Plant Sciences, University of Cambridge) has summarised advances towards efficient plastid transformation in Arabidopsis thaliana as part of a new paper in Plant Physiology
This commentary follows 'Efficient Plastid Transformation in Arabidopsis' reported by Pal Maliga's team in this month's Plant Physiology, which could be regarded as a major advance in plastid transformation. Stable manipulation of the plastid genome of flowering plants was first reported nearly 30 years agoby Svab et al. However, for key species including cereal crops including Arabidopsis (Arabidopsis thaliana), the spectinomycin-based approach using the addition of the aadA gene has proved ineffective.
Hibberd cites Parker et al who showed that ecotypes of Arabidopsis vary in their sensitivity to spectinomycin due to the mutant allele of the nuclear gene ACC2. Its product is targeted to plastids and so provides an alternate to the partially plastid-encoded acetyl-Coenzyme A carboxylase that is inactivated by spectinomycin. Parker et al. proposed that their findings may provide a route to increase efficiency of chloroplast transformation in the Brassicaceae.
Yu et al. (2017) now provide strong support for this conjecture showing that by removing redundancy afforded by ACC2, efficient selection for transplastomic events can be achieved in Arabidopsis.
The next challenge is therefore to identify procedures and ecotypes that facilitate this conversion of transplastomic callus of Arabidopsis into stable and heritable plant material.
Hibberd's full commentary can be read here.
A new publication from the lab of OpenPlant PI Prof. Paul Dupree, University of Cambridge in Biotechnology for Biofuels describes work to improve processing of softwood to biofuels using a synthetic biology strategy to express and assay conifer cell wall synthesis enzymes.
Lyczakowski, J.J., Wicher, K.B., Terrett, O.M., Faria-Blanc, N., Yu, X., Brown, D., Krogh, K.B.R.M., Dupree, P., and Busse-Wicher, M. (2017). Removal of glucuronic acid from xylan is a strategy to improve the conversion of plant biomass to sugars for bioenergy. Biotechnology for Biofuels 10: 224
Background: Plant lignocellulosic biomass can be a source of fermentable sugars for the production of second generation biofuels and biochemicals. The recalcitrance of this plant material is one of the major obstacles in its conversion into sugars. Biomass is primarily composed of secondary cell walls, which is made of cellulose, hemicelluloses and lignin. Xylan, a hemicellulose, binds to the cellulose microfibril and is hypothesised to form an interface between lignin and cellulose. Both softwood and hardwood xylan carry glucuronic acid side branches. As xylan branching may be important for biomass recalcitrance and softwood is an abundant, non-food competing, source of biomass it is important to investigate how conifer xylan is synthesised.
Results: Here, we show using Arabidopsis gux mutant biomass that removal of glucuronosyl substitutions of xylan can allow 30% more glucose and over 700% more xylose to be released during saccharification. Ethanol yields obtained through enzymatic saccharification and fermentation of gux biomass were double those obtained for non-mutant material. Our analysis of additional xylan branching mutants demonstrates that absence of GlcA is unique in conferring the reduced recalcitrance phenotype. As in hardwoods, conifer xylan is branched with GlcA. We use transcriptomic analysis to identify conifer enzymes that might be responsible for addition of GlcA branches onto xylan in industrially important softwood. Using a combination of in vitro and in vivo activity assays, we demonstrate that a white spruce (Picea glauca) gene, PgGUX, encodes an active glucuronosyl transferase. Glucuronic acid introduced by PgGUX reduces the sugar release of Arabidopsis gux mutant biomass to wild-type levels indicating that it can fulfil the same biological function as native glucuronosylation.
Conclusion: Removal of glucuronic acid from xylan results in the largest increase in release of fermentable sugars from Arabidopsis plants that grow to the wild-type size. Additionally, plant material used in this work did not undergo any chemical pretreatment, and thus increased monosaccharide release from gux biomass can be achieved without the use of environmentally hazardous chemical pretreatment procedures. Therefore, the identification of a gymnosperm enzyme, likely to be responsible for softwood xylan glucuronosylation, provides a mutagenesis target for genetically improved forestry trees.
Researchers at the John Innes Centre collaborated with colleagues from the University of Tokyo to understand the regulation of boron transport in Arabidopsis. Nutrient uptake relies on both a regulatory circuit within cells, and a coordinated behaviour across tissues. This work used both computational and molecular biology tools to model the effects of slowing boron uptake, discovering that this peturbation of the system leads to traffic-jam like behaviour of nutrient flow. Read more about it in this JIC news article. Experiments were partly funded through the OpenPlant Fund.
Sotta, N., Duncan, S., Tanaka, M., Takafumi, S., Marée, A.F., Fujiwara, T., Grieneisen, V.A., 2017. Rapid transporter regulation prevents substrate flow traffic jams in boron transport. eLife 2017;6: e27038.
Nutrient uptake by roots often involves substrate-dependent regulated nutrient transporters. For robust uptake, the system requires a regulatory circuit within cells and a collective, coordinated behaviour across the tissue. A paradigm for such systems is boron uptake, known for its directional transport and homeostasis, as boron is essential for plant growth but toxic at high concentrations. In Arabidopsis thaliana Boron up-take occurs via diffusion facilitators (NIPs) and exporters (BORs), each presenting distinct polarity. Intriguingly, although boron soil concentrations are homogenous and stable, both transporters manifest strikingly swift boron-dependent regulation. Through mathematical modelling, we demonstrate that slower regulation of these transporters leads to physiologically detrimental oscillatory behaviour. Cells become periodically exposed to potentially cytotoxic boron levels, and nutrient throughput to the xylem becomes hampered. We conclude that, while maintaining homeostasis, swift transporter regulation within a polarised tissue context is critical to prevent intrinsic traffic-jam like behaviour of nutrient flow.
Researchers at the John Innes Centre, including OpenPlant PI Prof George Lomonossoff, and collaborators, have published research to produce a new polio vaccine in plants, using the HyperTrans transient expression system. The work, funded by the World Health Organisation, was published in Nature Communications. It is hoped that this new polio vaccine will be a move towards global eradication of the disease. The publication was covered by JIC News and the BBC.
Marsian, J., Fox, H., Bahar, M.W., Kotecha, A., Fry, E.E., Stuart, D.I., Macadam, A.J., Rowlands, D.J., & Lomonossoff, G.P. (2017) Plant-made polio type 3 stabilized VLPs—a candidate synthetic polio vaccine. Nature Communications 8, Article number: 245.
Poliovirus (PV) is the causative agent of poliomyelitis, a crippling human disease known since antiquity. PV occurs in two distinct antigenic forms, D and C, of which only the D form elicits a robust neutralizing response. Developing a synthetically produced stabilized virus-like particle (sVLP)-based vaccine with D antigenicity, without the drawbacks of current vaccines, will be a major step towards the final eradication of poliovirus. Such a sVLP would retain the native antigenic conformation and the repetitive structure of the original virus particle, but lack infectious genomic material. In this study, we report the production of synthetically stabilized PV VLPs in plants. Mice carrying the gene for the human PV receptor are protected from wild-type PV when immunized with the plant-made PV sVLPs. Structural analysis of the stabilized mutant at 3.6 Å resolution by cryo-electron microscopy and single-particle reconstruction reveals a structure almost indistinguishable from wild-type PV3.
Researchers in Prof Anne Osbourn's lab at the John Innes Centre, including Prof Osbourn and OpenPlant PDRA Dr Michael Stephenson, have published a new paper detailing their advances in rapidly creating and purifying gram-scale quantities of natural products that were previously not possible to synthesise. This has the potential to reinvigorate drug discovery pipelines by opening up whole regions of chemical diversity for testing and production of potentially medicinally important molecules.
Reed, J., Stephenson, M.J., Miettinen, K., Brouwer, B., Leveau, A., Brett, P., Goss, R.J., Goossens, A., O’Connell, M.A. and Osbourn, A., 2017. A translational synthetic biology platform for rapid access to gram-scale quantities of novel drug-like molecules. Metabolic Engineering. DOI: 10.1016/j.ymben.2017.06.012
Fig 2 from paper: Generation of gram quantities of triterpene using vacuum infiltration a, Vacuum infiltration of N. benthamiana plants. Plants are retained by a bespoke holder, inverted into a bath containing 10 L of A. tumefaciens suspension, and a vacuum applied. Upon release of the vacuum the infiltration process is complete. b, GFP expression in leaves from a vacuum-infiltrated plant 5 days after infiltration (leaves arranged from top left to bottom right in descending order of their height on the plant). The youngest leaves (top left) were formed post-infiltration. c, β-Amyrin purified from vacuum-infiltrated plants following transient expression.
Plants are an excellent source of drug leads. However availability is limited by access to source species, low abundance and recalcitrance to chemical synthesis. Although plant genomics is yielding a wealth of genes for natural product biosynthesis, the translation of this genetic information into small molecules for evaluation as drug leads represents a major bottleneck. For example, the yeast platform for artemisinic acid production is estimated to have taken >150 person years to develop. Here we demonstrate the power of plant transient transfection technology for rapid, scalable biosynthesis and isolation of triterpenes, one of the largest and most structurally diverse families of plant natural products. Using pathway engineering and improved agro-infiltration methodology we are able to generate gram-scale quantities of purified triterpene in just a few weeks. In contrast to heterologous expression in microbes, this system does not depend on re-engineering of the host. We next exploit agro-infection for quick and easy combinatorial biosynthesis without the need for generation of multi-gene constructs, so affording an easy entrée to suites of molecules, some new-to-nature, that are recalcitrant to chemical synthesis. We use this platform to purify a suite of bespoke triterpene analogs and demonstrate differences in anti-proliferative and anti-inflammatory activity in bioassays, providing proof of concept of this system for accessing and evaluating medicinally important bioactives. Together with new genome mining algorithms for plant pathway discovery and advances in plant synthetic biology, this advance provides new routes to synthesize and access previously inaccessible natural products and analogs and has the potential to reinvigorate drug discovery pipelines.
Researchers from the John Innes Centre have published a method for accurate quantification and localization of mRNA in fixed plant samples by detection of individual mRNA molecules. This work was in part supported through the OpenPlant Fund.
Duncan, S., Olsson, T.S.G., Hartley, M., Dean, C., Rosa S., 2017. Single Molecule RNA FISH in Arabidopsis Root Cells. Bio-protocol 7(8): e2240.
Methods that allow the study of gene expression regulation are continually advancing. Here, we present an in situ hybridization protocol capable of detecting individual mRNA molecules in plant root cells, thus permitting the accurate quantification and localization of mRNA within fixed samples (Duncan et al., 2016; Rosa et al., 2016). This single molecule RNA fluorescence in situ hybridization (smFISH) uses multiple single-labelled oligonucleotide probes to bind target RNAs and generate diffraction-limited signals that can be detected using a wide-field fluorescence microscope. We adapted a recent version of this method that uses 48 fluorescently labeled DNA oligonucleotides (20 mers) to hybridize to different portions of each transcript (Raj et al., 2008). This approach is simple to implement and has the advantage that it can be readily applied to any genetic background.
Cambridge researchers including OpenPlant Director Prof Jim Haseloff and OpenPlant PI Dr Nicola Patron (Earlham Institute) have reviewed the state of art and future prospects for Synthetic Botany - the application of synthetic biology to engineering nuclear and chloroplast genomes in plants.
Plants represent the only available platform allowing sustainable bioproduction at the gigatonne scale. Combining modular body plans and developmental plasticity with capacity for photosynthesis and extensive secondary metabolism, plants are highly attractive targets for genetic engineering. However, efforts in this area have been complicated by slow growth rates, physiological complexity, and technical challenges in the handling and manipulation of plants. Furthermore, better experimental and theoretical frameworks are needed to dissect and understand the hierarchies of genetic and physical interactions shaping their multicellular behavior.
Joint first-authors Christian Boehm and Bernardo Pollak and colleagues reviewed the state of the art in genetic engineering of the nuclear and chloroplast genomes in plants, and highlight new approaches to harnessing their potential as custom agronomic systems for large-scale production. In particular, they show how simple plant models like the liverwort Marchantia polymorpha - combined with standard DNA parts and advanced quantitative imaging technqiues - can bridge the complexity gap between microbes and higher plants. Synthetic genetic circuits proven in Marchantia may serve as valuable tools for addressing some of the major challenges in plant metabolic engineering such as the introduction of C4 photosynthesis in C3 crops or the refactoring of nitrogen fixation pathways.
Boehm CR, Pollak B, Purswani N, Patron N & Haseloff J. (2017) Synthetic Biology. CSH Perspect Biol a023887o
The research presented in the following publication was funded in part through the OpenPlant Fund.
Juhas, Mario, and James W. Ajioka. “Integrative bacterial artificial chromosomes for DNA integration into the Bacillus subtilis chromosome.”Journal of microbiological methods 125 (2016): 1-7.
Cambridge Repository Full-Text | Publisher Full-Text
Open Access publication under CC-BY 4.0
Abstract
Bacillus subtilis is a well-characterized model bacterium frequently used for a number of biotechnology and synthetic biology applications. Novel strategies combining the advantages of B. subtilis with the DNA assembly and editing tools of Escherichia coli are crucial for B. subtilis engineering efforts. We combined Gibson Assembly and λ red recombineering in E. coli with RecA-mediated homologous recombination in B. subtilisfor bacterial artificial chromosome-mediated DNA integration into the well-characterized amyE target locus of the B. subtilis chromosome. The engineered integrative bacterial artificial chromosome iBAC(cav) can accept any DNA fragment for integration into B. subtilis chromosome and allows rapid selection of transformants by B. subtilis-specific antibiotic resistance and the yellow fluorescent protein (mVenus) expression. We used the developed iBAC(cav)-mediated system to integrate 10 kb DNA fragment from E. coliK12 MG1655 into B. subtilis chromosome. iBAC(cav)-mediated chromosomal integration approach will facilitate rational design of synthetic biology applications in B. subtilis.
Keywords
The following article was originally published on the John Innes Centre news feed: Using ‘chemical origami’ to generate customisable, high-value chemicals from plants. Anne Osbourn is Co-Director of OpenPlant and this work from her group is highly relevant to the efforts of OpenPlant to create toolkits for plant metabolic engineering, but was funded from other sources.
Following the discovery of a new and very valuable enzyme which folds linear molecules into different shapes, scientists at the John Innes Centre are building a ‘triterpene machine’ which will enable them to custom-build valuable chemical compounds called triterpenes and produce them in large, cost-effective quantities. Working with the pharmaceutical, agricultural and biotechnology industries, they hope to improve existing triterpenes to make better medicines with fewer side effects, or improve the specificity of pesticides. They also hope to make completely new, custom-designed triterpenes to any specification, which could lead to development of new anti-cancer drugs, agrochemicals, industrial chemicals or cosmetics.
In the ancient Japanese art of origami, different ways of folding a single sheet of paper can transform it into an aeroplane, a flower, or a bird. Plants perform origami too – not with paper, but with chemical compounds, taking individual precursor molecules and using enzymes to fold and modify them to create many different variations.
For several years, Professor Anne Osbourn of the John Innes Centre has been studying the ‘chemical origami’ that gives rise to a large group of plant compounds called triterpenes, many of which may have valuable uses in the pharmaceutical, agricultural and biotechnology industries.
Professor Osbourn said:
“Some triterpenes are currently used in drinks as foaming agents, but there are many more exciting possibilities – new medical therapies such as anti-cancer drugs, diabetes medicines and antidepressants, for example; anti-fungal agents in crop protection, or cosmetic ingredients. All of the triterpenes we know about are based on a suite of similar molecular ‘scaffolds’ – we want to understand how these scaffolds are made, ‘folded’ and ‘decorated’ so that we might be able to engineer completely new triterpenes to make new medicines and industrial chemicals, or to improve those we already have.”
In a new research article published this week in the scientific journal Proceedings of the National Academy of Sciences, Professor Osbourn, along with colleagues at the John Innes Centre and collaborators from the USA, describes how she discovered an important part of the triterpene origami process, almost by accident.
By analysing oat plants that had been exposed to a DNA-mutating chemical, the researchers “stumbled across” a handful of mutated versions of an enzyme called SAD1. SAD1 is a triterpene synthase enzyme responsible for a critical step in building triterpenes: in its normal form, it takes a linear precursor molecule called 2,3-oxidosqualene (OS for short), and turns it into a pentacyclic scaffold – a molecule with 5 carbon rings. This is then further modified by other enzymes to produce hundreds of different triterpene compounds.
However, one of the mutated forms, which differed from the normal form by one little change in the enzyme’s structure, produced tetracyclic scaffolds with four carbon rings instead – the scaffold for a completely different set of triterpenes. Incidentally, the same mutation in an equivalent gene from a different plant, Arabidopsis thaliana, gave the same results, suggesting that this ‘molecular switch’ from pentacyclic to tetracyclic triterpene production is conserved between different plant species.
Next, the scientists tried putting the mutant SAD1 gene into yeast, a fast-growing, single-celled organism, to see if it could be used to make large quantities of triterpenes. Here, the team discovered that the SAD1 enzyme favoured dioxidosqualene (DOS) as a substrate rather than OS.
“This was an exciting discovery,” said Professor Osbourn, “because we realised that we could not only modify the enzyme to produce different triterpene scaffolds, but we could also modify the building block to make different more highly oxygenated scaffolds.”
The PNAS article presents just one part of ongoing work by the Osbourn lab to harness the power of genes and enzymes to generate high-value chemicals from plants.
Professor Osbourn said:
“Here at the Norwich Research Park we’re building a ‘Triterpene Machine’; a toolkit of molecular parts we can put into yeast, or a recently developed rapid expression system using tobacco leaves, which we hope will allow us to custom-build valuable triterpenes and produce them in large, cost-effective quantities. Working with the pharmaceutical, agricultural and biotechnology industries, we hope we’ll be able to modify known triterpenes to improve their existing applications – to make better medicines with fewer side effects, or improve the specificity of pesticides, for example. We might even be able to make completely new, custom-designed triterpenes to any specification we want, which could provide us with new anti-cancer drugs, agrochemicals, industrial chemicals or cosmetics. The possibilities are potentially endless!”
This research was funded by the Biotechnology and Biological Sciences Research Council, the John Innes Foundation and a Norwich Research Park Studentship Award.
Image by Ftiercel [Public domain], shared via Wikimedia Commons
OpenPlant PI Professor Rob Field at the John Innes Centre has published work of relevance to those working on algae and microalgae.
O’Neill, G. Saalbach, R.A. Field (2016). Gene Discovery for Synthetic Biology: Exploring the Novel Natural Product Biosynthetic Capacity of Eukaryotic Microalgae. Methods in Enzymology 576, p 99-120.
Eukaryotic microalgae are an incredibly diverse group of organisms whose sole unifying feature is their ability to photosynthesize. They are known for producing a range of potent toxins, which can build up during harmful algal blooms causing damage to ecosystems and fisheries. Genome sequencing is lagging behind in these organisms because of their genetic complexity, but transcriptome sequencing is beginning to make up for this deficit. As more sequence data becomes available, it is apparent that eukaryotic microalgae possess a range of complex natural product biosynthesis capabilities. Some of the genes concerned are responsible for the biosynthesis of known toxins, but there are many more for which we do not know the products. Bioinformatic and analytical techniques have been developed for natural product discovery in bacteria and these approaches can be used to extract information about the products synthesized by algae. Recent analyses suggest that eukaryotic microalgae produce many complex natural products that remain to be discovered.
Image credit: microscopic-view-of-microalgae by Learn 2 Teach, Teach 2 Learn on Flick, licensed under CC-BY-NC 2.0
In September 2015, GARNet and OpenPlant organized a two-day workshop at the John Innes Centre that provided both background information and hands-on training for CRISPR-Cas technology. The report from that meeting is now online, co-authored by Dr Nicola Patron and Dr Colette Matthewman from OpenPlant along with GARNet colleagues.
Parry, G., Patron, N., Bastow, R., & Matthewman, C. (2016). Meeting report: GARNet/OpenPlant CRISPR-Cas workshop. Plant methods, 12(1), 1.
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The article is fully Open Access under a CC-BY 4.0 license so it’s reproduced below!
†Contributed equally
Plant Methods201612:6
DOI: 10.1186/s13007-016-0104-
© Parry et al. 2016
Received: 17 November 201
Accepted: 5 January 2016
Published: 27 January 2016
Targeted genome engineering has been described as a “game-changing technology” for fields as diverse as human genetics and plant biotechnology. One technique used for precise gene editing utilises the CRISPR-Cas system and is an effective method for genetic engineering in a wide variety of plants. However, many researchers remain unaware of both the technical challenges that emerge when using this technique or of its potential benefits. Therefore in September 2015, GARNet and OpenPlant organized a two-day workshop at the John Innes Centre that provided both background information and hands-on training for this important technology.
CRISPR Cas9 Gene Editing Genetic Engineering
Geraint Parry and Nicola Patron contributed equally to this manuscript
Over the past few years, genome engineering (GE), the process of making targeted modifications to the genome, its contexts or its outputs, has been described as a “game-changing technology” for fields as diverse as human genetics and plant biotechnology. The ability to introduce specific changes to genomic loci adds a level of precision not previously available to molecular biologists working in multicellular eukaryotes. Despite overwhelming scientific opinion that Genetically Modified (GM) plants are safe and provide environmental and socioeconomic benefits, they remain broadly unpopular outside of the scientific community [1–3]. This has been blamed both on inaccurate media reporting and public concerns over the ownership of technologies that underpin food production [4–6]. Given these political and public opinions, plant scientists are particularly hopeful about the future use of GE technologies, which are likely to enable precise genetic changes to be made without the ongoing requirement for foreign DNA to be integrated the genome.
However, despite some countries ruling that plants with targeted mutations may not be regulated as GM, there is still much uncertainty [7, 8]. Even as the technologies behind GE are being optimized, the scientific community is engaging with stakeholders to highlight potential positive uses, including how it might be used to develop better crops. This is exemplified by a policy statement from the UK’s Biotechnology and Biological Sciences Research Council (BBSRC) on “New Techniques for Genetic Crop Improvement” that outlines positive uses for GE technologies [9].
The experimental protocols needed to implement these powerful techniques are yet to be embraced by many plant science laboratories. To address this issue GARNet [10] and OpenPlant [11] collaborated to organise a workshop to explain the background of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas technologies for GE in plants and to equip plant scientists with the skills required to implement Cas9-induced targeted mutagenesis. Over 140 researchers registered for the meeting, held at the John Innes Centre (UK), from as far-afield as Ireland and Poland, clearly demonstrating the appetite to apply these technologies to plant systems. The first day was open to all attendees and consisted of conventional ‘seminar-style’ presentations, while day two was a hands-on introduction for 30 researchers. This meeting was made possible by the kind support of Plant Methods.
The meeting was opened by Dr. Jim Haseloff from The University of Cambridge who introduced synthetic biology in plant systems and Dr. Nicola Patron of The Sainsbury Laboratory, Norwich (TSL), the primary organiser, who provided a historical perspective on GE technologies. The specifics of these technologies are discussed in detail as part of this Plant Methodsthematic series. Keynote presentations were given by Professor Holger Puchta (Karlsruhe Institute of Technology) and Professor Bing Yang (Iowa State University) who each provided overviews and success stories from their own laboratories. These were followed by shorter talks from scientists at JIC, TSL and the University of Cambridge who are already working with CRISPR/Cas technologies.
Professor Puchta gave an inspiring talk that provided attendees with the history of his seminal work. He presented earlier work showing that induction of double strand breaks (DSBs) using site-specific endonucleases can enhance the freqeuncy of homologous recombination in plant cells through to his recent work using RNA-guided Cas9 nuclease to induce DSBs [12, 13]. He mentioned that the two most important molecular discoveries of his lifetime had been the Polymerase Chain Reaction (PCR) and GE technologies, the latter he described as having “hit him like a tsunami”. It was exciting to hear about his lab’s recent use of paired nickase variants of Cas9, which cut just one DNA strand, to induce larger endogenous deletions [13, 14]. Professor Puchta was extremely positive about the potential for GE and in his final perspectives noted that “Synthetic nuclease based DSB-induced DNA repair should be applicable for directed mutagenesis in all transformable plants”, and “in the long run synthetic nuclease-based GE will change plant breeding dramatically”. He also thought it possible that plants with targeted mutations might not be regulated in the same way as transgenic GM plants.
Professor Yang echoed this, presenting a letter from the United States Department of Agriculture (USDA) that informed him that the GE rice produced in his laboratory did not fall within its regulatory authority [14, 15]. Professor Yang documented his work on GE in maize and rice, showing that in cultivars where poor transformation efficiency was a significant bottleneck, GE technologies has sped up the process. He also described the induction of a large deletion of 245 kb in rice using RNA-guided Cas9 [15].
Dr. Laurence Tomlinson and Dr. Vladimir Nekrasov, both from TSL, presented their successful applications of RNA-guided Cas9 nuclease to induce targeted mutagenesis in tomatoes. Tomlinson’s work involved GA signaling whilst Nekrasov described the induction of targeted mutations to engineer pathogen-resistance. He took the audience through initial experimental design, through screening of putatively mutated plants to the identification of individuals showing resistance to powdery mildew. It took just 9 months to identify transgene-free, resistant plants with heritable mutations. Nekrasov confirmed that he and his supervisor, Professor Sophien Kamoun, are now investigating options to make their plants available to growers in regions where the pathogen is a significant problem, whilst also undertaking full-genome sequence analysis to determine if the plants contain any additional mutations. University of Cambridge PhD student, Bernando Pollak, introduced the liverwort Marchantia polymorpha, highlighting the ease by which its genome can be manipulated, as well as its potential as an easily engineerable chassis for synthetic biology. Many of the signaling pathways in Marchantialack the redundancy seen in vascular land plants [16] and so it has huge potential as a tool for the study of plant signaling. Additionally, Marchantia is haploid for a large portion of its life cycle and thus the application of programmable nucleases such as RNA-guided Cas9 are even easier to apply. Dr.Oleg Raitskin (TSL) described experiments to further optimize RNA-guided Cas9 nuclease mediated mutagenesis in plants, including the assessment of orthologues and mutants of Cas9 that may expand the number of possible targets in the genome. He also introduced the concepts behind digital droplet PCR and its implementation in the rapid, quantitative assessment of mutations.
The final presentation was delivered by Edward Perello, Chief Business Officer of Desktop Genetics [17], a UK-based software company who develop tools to support the application of CRISPR-associated technologies. Mr. Perello announced that their guide RNA selection software, Guidebook, now supports six plant genomes (Arabidopsis, rice, maize, wheat, barley and Physcomitrella). Plant scientists were encouraged to use this software, which is free for academics, as well as to contact the Desktop Genetics team with feedback and requests for new features and genomes.
For the workshop on the second day, participants were given a detailed introduction to the methods used to induce targeted mutagenesis and gene deletions with RNA-guided Cas9 nuclease. This was a hands-on session designed to give the participants a full understanding of how to undertake three key aspects of the technique: selecting target sequences, constructing plasmid vectors, and screening target loci for induced mutations. The content was tailored for researchers working on any transformable plant species.
As well as discussing targeted mutagenesis, Dr. Patron provided an introduction to Type IIS mediated assembly methods for the facile construction of plasmid vectors. Dr. Patron is an advocate for the adoption of standards in bioengineering. She was the lead author on a recent manuscript that described a broadly agreed common genetic syntax for the exchange of DNA parts for plants [18]. In addition, Dr. Patron has contributed to a toolkit of standard parts for plants and created a series of informative online tutorials that introduces users to the Golden Gate Modular Cloning (MoClo) assembly standard [19, 20]. Participants were instructed in the use of published standard parts (Table 1), compatible with the MoClo binary plasmid backbones to build vectors for multiplexed Cas9-induced mutagenesis. The workshop materials have been provided on the GARNet website [21] but the main points are summarized below.
Designing single guide RNAs (sgRNAs) for use with Streptococcus pyogenes Cas9
![Fig. 1Interaction of a single guide RNA (sgRNA) expressed from a U6 promoter with its cognate genomic target (adapted from Belhaj et al. [23])](https://images.squarespace-cdn.com/content/v1/54a6bdb7e4b08424e69c93a1/1491867915679-K24J2X8DSXIYA871PON6/image-asset.gif)
Fig. 1
Interaction of a single guide RNA (sgRNA) expressed from a U6 promoter with its cognate genomic target (adapted from Belhaj et al. [23])
The target sequence, which is integrated into the single guide RNA (sgRNA), consists of 20 nucleotides (nt). In the genome, target sequences must be located immediately 5′ of an ‘NGG’ sequence, known as the ProtospacerAdjacent Motif (PAM) (Fig. 1). The 6–8nt immediately 5′ of the PAM are called the ‘seed region’ and should be 100 % identical to the target sequence. DSBs may still be induced at targets with one or more mismatches in the 5′ end of the target sequence. The induction of DSBs in sequences that do not exactly match the guide is known as ‘off-target activity’ and may be exploited for simultaneously inducing mutations in closely related sequences although the delivery of multiple sgRNAs that exactly match each target may be more successful.
RNA polymerase III (RNAPol-III) dependent promoters are generally used to transcribe sgRNAs. This is because of their precise transcriptional start site. As the target sequence comprises the 5′ end of the sgRNA, the start of transcription must be preserved. For example, the transcriptional start site of the Arabidopsis U6-26 promoter is a ‘G’ and therefore the transcript will begin with a ‘G’. This nt does not necessarily need to pair with the genomic target. If the desired target sequence does not start with a ‘G’ an additional 5′ non-pairing ‘G’ can be included, extending the target to 21 nts (Fig. 1).
If specific sgRNA identification software is not available for the genome of interest, target sequences can be identified using many DNA analysis software packages by searching for the degenerate sequence ‘N(20)NGG’. Cas9 has been shown to preferentially bind sgRNAs containing purines in the last 4 nucleotides of the spacer sequence whereas pyrimidines are disfavoured [25]. Although unconfirmed in plant systems, users may wish to select targets rich in purines by searching for ‘N(12)R(8)NGG’.
For purposes of creating functional ‘knock-outs’, two or more sgRNAs can be designed to the same gene, thus creating a small deletion. Constructs with multiple sgRNAs, the Cas9 and selection genes as well as other transcriptional units can be easily assembled using the MoClo plasmid system and published standard parts (Table 1) [20, 22, 26].
Once the constructs have been assembled, they are delivered to plant cells using established protocols for the species of interest. Although transient transfection of plasmids and direct delivery of protein-RNA complexes to protoplasts have resulted in targeted mutagenesis [27, 28], regeneration from protoplasts has not yet been established for many plant species. The assembled genes may be integrated as a transgene raft. The resulting transformants can then be analysed for lesions at the target locus. The final part of the workshop was dedicated to simple, rapid techniques for the identification of induced mutations at target loci.
Fig. 2
Detection of induced mutations. a If two single guide RNAs were delivered with the aim of deleting a fragment of DNA, oligonucleoitide primers flanking the targets can be used to PCR amplify the locus. Evidence of an amplicon, smaller that that obtained in a wildtype (WT) control is indicative of a deletion. The absence of an amplicon of equivalent size to the WT may indicate a homozygous deletion. b If the quantity of the deletion amplicon is low or absent, the genomic DNA can be digested with any restriction endonuclease (REN) with one or more recognition sites in the deletion region prior to PCR amplification. This will remove any wild-type sequence enabling the detection of deletions even if at low quantity in the sample. c Double strand breaks (DSBs) are most likely to occur three base pairs before the PAM in the seed-region of the target. Small insertion-deletion events at the target can be detected by digesting a PCR amplicon of the target locus with a REN for which the cognate sequence would be disrupted by imperfect repair of the DSB
Genomic DNA is purified and, if two sgRNAs were used, oligonucleoitide primers flanking the targets sites are used to PCR amplify the locus. Evidence of a deletion can be seen in the form of amplicons smaller than those obtained from a wild type control (Fig. 2a). The absence of the wildtype amplicon may indicate that the deletion was homozygous (Fig. 2a). The sequence of this band may confirm if both sister chromatids were repaired in the same way or if the plant is bialleic. If an amplicon corresponding to the wild-type is also present, the deletion may be heterozygous or, alternatively, the transgenes may be expressed in somatic tissues with cells in the sample showing multiple genotypes. In all cases the seeds will be collected and null-segregent progeny, which have not inherited the transgene, and (unless the deletion was homozygous in the primary transformants) progeny that have inherited the transgene analysed in the same way. The mutation can be classified as heritable and stable when progeny with the same mutant genotype as the parent are recovered and the transgene has been segregated out.
Following PCR amplification, if there is no evidence of smaller band indicating a deletion then two experiments are possible: The first is to digest the purified genomic DNA with a restriction endonuclease with one or more recognition sites between the targets and to PCR amplify the locus with oligonucleotide primers designed to the flanking regions (Fig. 2b). This pre-digestion will remove any wild-type sequence enabling the detection of deletions from just a few cells in the sample. Such plants are highly likely to be chimeric and will need to be progressed to a second generation. The second method allows the detection of small insertion-deletion events at the target rather than a deletion. A DSB is most likely to occur three base pairs before the PAM in the seed-region of the target (Fig. 1). If there is a restriction endonuclease recognition site that would be disrupted by imperfect repair of the DSB, a PCR amplicon of the target locus can be digested with this enzyme. Any amplicon showing resistance to digestion with this enzyme can be sequenced (Fig. 2c). A researcher with sufficient foresight will try to design a target region that contains RE sites that could be used for subsequent screening. Again, the mutation can be classified as heritable and stable when progeny with the same mutant genotype as the parent are recovered and the transgene has been segregated out.
Mutations are detected in at least some cells of at least 5–20 % of primary transformants, with much higher frequencies reported for some species [29]. This rate is dependent on the effectiveness of the specific sgRNAs and species-specific factors including the level of expression of Cas9 and sgRNAs achieved in the tissue to which the transgene is delivered.
One of the main criticisms of programmable nucleases for the induction of targeted mutations is the potential for off-target activity. Although many plant species can be easily backcrossed to ‘clean up’ the genetic background as is done for chemical or radiation-induced mutagenesis, off-targets can only be identified by sequencing either related target sites or the whole genome. Nevertheless, there is little doubt that GE technologies offer immediate opportunities for increasing genetic diversity in crop plants and for understanding the function of plant genes. The take-away message from this workshop was that the technique has enormous potential, but that it can be technically challenging to implement. A post-workshop survey received many positive responses about the breadth of the talks and especially regarding the day two workshop. However, there are still knowledge gaps in the plant science community and therefore GARNet will be organising a further CRISPR-Cas workshop as part of its general meeting, to be held in September 2016 (http://ww.ARNet2016.eebly.om).
Geraint Parry and Nicola Patron contributed equally to this manuscript
The article was prepared with equal contributions by GP and NP. RB and CM were involved in organisation of the workshop. All authors read and approved the final manuscript.
The authors would like to thank the GARNet Advisory Committee for suggestions toward development of the workshop.
The authors declare that they have no competing interests.
Geraint Parry and Ruth Bastow are funded by BBSRC grant BB/M004376/1. Nicola Patron and Colette Matthewman are funded by BBSRC grant BB/L014130/1.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://reativecommons.rg/icenses/y/./), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://reativecommons.rg/ublicdomain/ero/./) applies to the data made available in this article, unless otherwise stated.
OpenPlant PI Nicola Patron and Oleg Raitskin (Earlham Institute, Norwich) have published a review on genome editing with RNA-guided Cas9 nuclease in plants, and the opportunities for multi-gene engineering.
Raitskin, O. and Patron, N.J., 2016. Multi-gene engineering in plants with RNA-guided Cas9 nuclease. Current Opinion in Biotechnology 37, p 69-75.
The use of RNA-guided Cas9 endonuclease for the concurrent engineering of multiple genes has been demonstrated in a number of plant species. Although Cas9 is a large monomeric protein, the single guide RNA (sgRNA) that directs it to a specific DNA target sequence is small and easy to reprogram. It is therefore relatively simple to produce numerous sgRNAs to target multiple endogenous sequences. Several approaches to express multiple sgRNAs and Cas9 in plants for the purpose of simultaneous editing or transcriptional regulation of many genes have recently been reported.
For more information see the full article.
OpenPlant PI Professor Julian Hibberd’s Lab published a significant step towards understanding the efficient form of photosynthesis known as the C4 pathway in The Plant Cell on the 15th of January 2016. In most photosynthetic organisms, ranging from bacteria to land plants, the first step of photosynthesis is catalysed by the enzyme RuBisCO. However, under warm, dry conditions the efficiency of Rubisco is reduced, which can lead to lower crop yields.
Some plants have evolved adaptations to overcome this problem, one of which is known as the C4 photosynthetic pathway, adoption of which allowed fast-growing species such as switchgrass to dominate savannahs and prairies. As C4 photosynthesis requires the co-ordinated action of many genes, Williams and Burgess et al. sought to identify C4 genes that are expressed in mesophyll cells and regulated by the same regulatory elements. Starting with a gene encoding carbonic anhydrase from the C4 species Gynandropsis gynandra they established that its regulation was mediated by a short sequence in the untranslated part of the gene. Furthermore, this sequence was found in additional C4 genes as well as orthologous genes from C3 species, and in each case, regulation appears to act on the translation of RNA to protein.
The work provides evidence that the complex C4 trait is underpinned by the repeated use of simple sequence motifs.
Williams BP, Burgess SJ, Reyna-Llorens I, Knerova J, Aubry S, Stanley S, Hibberd JM. (2016) An untranslated cis-element regulates the accumulation of multiple C4 enzymes in Gynandropsis gynandra mesophyll cells. The Plant Cell.
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