From farmers to bioengineers: Sowing genes, harvesting molecules

Twenty-first-century agriculture faces major challenges that urgently need to be answered. In the last decade, new breeding technologies have been developed that can help meet these challenges. These technologies are not only more accurate and efficient, but are also simpler and more accessible, which will facilitate the progressive democratisation of agricultural biotechnology. In this text we discuss future agricultural development in terms of technological democratisation and regulatory relaxation. In this scenario one would expect an increase in the diversity of cultivated varieties and species, the strong development of biofactory crops and, in the long term, the emergence of increasingly fit «smart» crops

DNA assembly standards: Setting the low-level programming code for plant biotechnology

[EN] Synthetic Biology is defined as the application of engineering principles to biology. It aims to increase the speed, ease and predictability with which desirable changes and novel traits can be conferred to living cells. The initial steps in this process aim to simplify the encoding of new instructions in DNA by establishing low-level programming languages for biology. Together with advances in the laboratory that allow multiple DNA molecules to be efficiently assembled together into a desired order in a single step, this approach has simplified the design and assembly of multigene constructs and has even facilitated the automated construction of synthetic chromosomes. These advances and technologies are now being applied to plants, for which there are a growing number of software and wetware tools for the design, construction and delivery of DNA molecules and for the engineering of endogenous genes. Here we review the efforts of the past decade that have established synthetic biology workflows and tools for plants and discuss the constraints and bottlenecks of this emerging field.Marta Vazquez-Vilar is funded by Wageningen University & Research. Diego Orzaez is funded by the Spanish Ministry of Economy and Competitiveness [grant number BIO2016-78601-R]. Nicola Patron is funded by the UK Biotechnological and Biological Sciences Research Council (BBSRC) and Engineering and Physics Research Council (EPSRC)Synthetic Biology for Growth programme [OpenPlant Synthetic Biology Research Centre, grant number BB/L0I4130/1], and by the Earlham DNA Foundry [grant number BB/CCG1720/1].Vázquez-Vilar, M.; Orzáez Calatayud, DV.; Patron, N. (2018). DNA assembly standards: Setting the low-level programming code for plant biotechnology. Plant Science. 273:33-41. https://doi.org/10.1016/j.plantsci.2018.02.024S334127

CRISPR-Cas12a genome editing at the whole-plant level using two compatible RNA virus vectors

[EN] The use of viral vectors that can replicate and move systemically through the host plant to deliver bacterial CRISPR components enables genome editing at the whole-plant level and avoids the requirement for labor-intensive stable transformation. However, this approach usually relies on previously transformed plants that stably express a CRISPR-Cas nuclease. Here, we describe successful DNA-free genome editing of Nicotiana benthamiana using two compatible RNA virus vectors derived from tobacco etch virus (TEV; genus Potyvirus) and potato virus X (PVX; genus Potexvirus), which replicate in the same cells. The TEV and PVX vectors respectively express a Cas12a nuclease and the corresponding guide RNA. This novel two-virus vector system improves the toolbox for transformation-free virus-induced genome editing in plants and will advance efforts to breed more nutritious, resistant, and productive crops.This research was supported by grants BIO2017-83184-R and PID2019-108203RB-I00 from the Ministerio de Ciencia e Innovacion (Spain) through the Agencia Estatal de Investigacion (co-financed by the European Regional Development Fund) and H2020-760331 Newcotiana from the European Commission. M.U. andM.V.-V. are the recipients of fellowships FPU17/05503 from the Ministerio de Ciencia e Innovacion (Spain) and APOSTD/2020/096 from the Generalitat Valenciana (Spain), respectivelyUranga, M.; Vázquez-Vilar, M.; Orzáez Calatayud, DV.; Daròs, J. (2021). CRISPR-Cas12a genome editing at the whole-plant level using two compatible RNA virus vectors. The CRISPR Journal. 4(5):1-9. https://doi.org/10.1089/crispr.2021.0049194

GB_SynP: A Modular dCas9-Regulated Synthetic Promoter Collection for Fine-Tuned Recombinant Gene Expressionin Plants

[EN] Programmable transcriptional factors based on the CRISPR architecture are becoming commonly used in plants for endogenous gene regulation. In plants, a potent CRISPR tool for gene induction is the so-called dCasEV2.1 activation system, which has shown remarkable genome-wide specificity combined with a strong activation capacity. To explore the ability of dCasEV2.1 to act as a transactivator for orthogonal synthetic promoters, a collection of DNA parts was created (GB_SynP) for combinatorial synthetic promoter building. The collection includes (i) minimal promoter parts with the TATA box and 5'UTR regions, (ii) proximal parts containing single or multiple copies of the target sequence for the gRNA, thus functioning as regulatory cis boxes, and (iii) sequence-randomized distal parts that ensure the adequate length of the resulting promoter. A total of 35 promoters were assembled using the GB_SynP collection, showing in all cases minimal background and predictable activation levels depending on the proximal parts used. GB_SynP was also employed in a combinatorial expression analysis of an autoluminescence pathway in Nicotiana benthamiana, showing the value of this tool in extracting important biological information such as the determination of the limiting steps in an enzymatic pathway.This work was funded by Era-CoBiotech SUSPHIRE (PCI2018-092893) and PID2019-108203RB-I00 Plan Nacional I+D, Spanish Ministry of Economy and Competitiveness. E.M.-G. and S.S. acknowledge support by a FPU (FPU18/02019) and a FPI (BIO2016-78601-R) H2020 Research Program: 760331 Newcotiana fellowships, respectively, from the Spanish Ministry of Science, Innovation and Universities. C.C. acknowledges support by a FPI-UPV (PAID-01-20) fellowship from Universitat Politecnica de Valencia. The authors would like to thank Dr. Lynne Yenush for her assistance with the manuscript preparation.Moreno-Giménez, E.; Selma, S.; Calvache-Román, CA.; Orzáez Calatayud, DV. (2022). GB_SynP: A Modular dCas9-Regulated Synthetic Promoter Collection for Fine-Tuned Recombinant Gene Expressionin Plants. ACS Synthetic Biology. 11(9):3037-3048. https://doi.org/10.1021/acssynbio.2c002383037304811

A copper switch for inducing CRISPR/Cas9-based transcriptional activation tightly regulates gene expression in Nicotiana benthamiana

[EN] Background CRISPR-based programmable transcriptional activators (PTAs) are used in plants for rewiring gene networks. Better tuning of their activity in a time and dose-dependent manner should allow precise control of gene expression. Here, we report the optimization of a Copper Inducible system called CI-switch for conditional gene activation in Nicotiana benthamiana. In the presence of copper, the copper-responsive factor CUP2 undergoes a conformational change and binds a DNA motif named copper-binding site (CBS). Results In this study, we tested several activation domains fused to CUP2 and found that the non-viral Gal4 domain results in strong activation of a reporter gene equipped with a minimal promoter, offering advantages over previous designs. To connect copper regulation with downstream programmable elements, several copper-dependent configurations of the strong dCasEV2.1 PTA were assayed, aiming at maximizing activation range, while minimizing undesired background expression. The best configuration involved a dual copper regulation of the two protein components of the PTA, namely dCas9:EDLL and MS2:VPR, and a constitutive RNA pol III-driven expression of the third component, a guide RNA with anchoring sites for the MS2 RNA-binding domain. With these optimizations, the CI/dCasEV2.1 system resulted in copper-dependent activation rates of 2,600-fold and 245-fold for the endogenous N. benthamiana DFR and PAL2 genes, respectively, with negligible expression in the absence of the trigger. Conclusions The tight regulation of copper over CI/dCasEV2.1 makes this system ideal for the conditional production of plant-derived metabolites and recombinant proteins in the field.This work has been funded by EU Horizon 2020 Project Newcotiana Grant 760331, PID2019-108203RB-100 Plan Nacional I + D, Spanish Ministry of Economy and Competitiveness and 'ERACoBioTech' Project SUSPHIRE Grant No. 722361. Vazquez-Vilar, M. is recipient of APOSTD/2020/096 (Generalitat Valenciana and Fondo Social Europeo post-doctoral grant). Garcia-Perez, E. is recipient of ACIF-2020 fellowship (Generalitat Valenciana). Diego-Martin, B. and Moreno-Gimenez, E. are recipients of FPU fellowships. Selma, S. is recipient of FPI fellowship.García-Pérez, E.; Diego-Martín, B.; Quijano-Rubio, A.; Moreno-Giménez, E.; Selma, S.; Orzáez Calatayud, DV.; Vázquez-Vilar, M. (2022). A copper switch for inducing CRISPR/Cas9-based transcriptional activation tightly regulates gene expression in Nicotiana benthamiana. BMC Biotechnology. 22(1):1-13. https://doi.org/10.1186/s12896-022-00741-x11322

Efficient Cas9 multiplex editing using unspaced sgRNA arrays engineering in a Potato virus X vector

[EN] Systems based on the clustered, regularly interspaced, short palindromic repeat (CRISPR) and CRISPR-associated proteins (Cas) have revolutionized genome editing in many organisms, including plants. Most CRISPR-Cas strategies in plants rely on genetic transformation using Agrobacterium tumefaciens to supply the gene editing reagents, such as Cas nucleases or the synthetic guide RNA (sgRNA). While Cas nucleases are constant elements in editing approaches, sgRNAs are target-specific and a screening process is usually required to identify those most effective. Plant virus-derived vectors are an alternative for the fast and efficient delivery of sgRNAs into adult plants, due to the virus capacity for genome amplification and systemic movement, a strategy known as virus-induced genome editing. We engineered Potato virus X (PVX) to build a vector that easily expresses multiple sgRNAs in adult solanaceous plants. Using the PVX-based vector, Nicotiana benthamiana genes were efficiently targeted, producing nearly 80% indels in a transformed line that constitutively expresses Streptococcus pyogenes Cas9. Interestingly, results showed that the PVX vector allows expression of arrays of unspaced sgRNAs, achieving highly efficient multiplex editing in a few days in adult plant tissues. Moreover, virus-free edited progeny can be obtained from plants regenerated from infected tissues or infected plant seeds, which exhibit a high rate of heritable biallelic mutations. In conclusion, this new PVX vector allows easy, fast and efficient expression of sgRNA arrays for multiplex CRISPR-Cas genome editing and will be a useful tool for functional gene analysis and precision breeding across diverse plant species, particularly in Solanaceae crops.This work was supported by grants BIO2017-83184-R and PID2019-108203RB-I00 from Ministerio de Ciencia e Innovacion (Spain) through the Agencia Estatal de Investigacion (co-financed European Regional Development Fund), and H2020-760331 Newcotiana from the European Commission. M.U. and S.S. are the recipients of fellowships FPU17/05503 and BES-2017-0890098, respectively, from Ministerio de Ciencia e Innovacion (Spain)Uranga-Ruiz De Eguino, M.; Aragones, V.; Selma García, S.; Vázquez-Vilar, M.; Orzáez Calatayud, DV.; Daròs, J. (2021). Efficient Cas9 multiplex editing using unspaced sgRNA arrays engineering in a Potato virus X vector. The Plant Journal. 106(2):555-565. https://doi.org/10.1111/tpj.15164555565106

FungalBraid 2.0: expanding the synthetic biology toolbox for the biotechnological exploitation of filamentous fungi

[EN] Fungal synthetic biology is a rapidly expanding field that aims to optimize the biotechnological exploitation of fungi through the generation of standard, readyto-use genetic elements, and universal syntax and rules for contributory use by the fungal research community. Recently, an increasing number of synthetic biology toolkits have been developed and applied to filamentous fungi, which highlights the relevance of these organisms in the biotechnology field. The FungalBraid (FB) modular cloning platform enables interchangeability of DNA parts with the GoldenBraid (GB) platform, which is designed for plants, and other systems that are compatible with the standard Golden Gate cloning and syntax, and uses binary pCAMBIA-derived vectors to allow Agrobacterium tumefaciensmediated transformation of a wide range of fungal species. In this study, we have expanded the original FB catalog by adding 27 new DNA parts that were functionally validated in vivo. Among these are the resistance selection markers for the antibiotics phleomycin and terbinafine, as well as the uridine-auxotrophic marker pyr4. We also used a normalized luciferase reporter system to validate several promoters, such as PpkiA,P7760,Pef1¿, and PafpB constitutive promoters, and PglaA,PamyB, and PxlnA inducible promoters. Additionally, the recently developed dCas9-regulated GB_SynP synthetic promoter collection for orthogonal CRISPR activation (CRISPRa) in plants has been adapted in fungi through the FB system. In general, the expansion of the FB catalog is of great interest to the scientific community since it increases the number of possible modular and interchangeable DNA assemblies, exponentially increasing the possibilities of studying, developing, and exploiting filamentous fungi.This work was supported by PROMETEO/2018/066 from "Conselleria d'Educacio" (Generalitat Valenciana, Comunitat Valenciana, Spain), grant PID2021-125858OB-100, and the Severo Ochoa Excellence Program CEX 2021-001189-S funded by MCIN/AEI/10.13039/501100011033 and by "ERDF A way of making Europe." EM-G was the recipient of a predoctoral grant FPU18/02019 funded by MCIN/AEI/10.13039/501100011033 and by "ESF Investing in your future." SG holds a Juan de la Cierva Incorporacion grant (IJC 2020-042749-I) funded by MCIN/AEI/10.13039/501100011033 and the European Union NextGenerationEU/PRTR.Moreno-Giménez, E.; Gandía, M.; Sáez, Z.; Manzanares, P.; Yenush, L.; Orzáez Calatayud, DV.; Marcos, JF.... (2023). FungalBraid 2.0: expanding the synthetic biology toolbox for the biotechnological exploitation of filamentous fungi. Frontiers in Bioengineering and Biotechnology. 11:1-17. https://doi.org/10.3389/fbioe.2023.12228121171

FungalBraid: A GoldenBraid-based modular cloning platform for the assembly and exchange of DNA elements tailored to fungal synthetic biology

[EN] Current challenges in the study and biotechnological exploitation of filamentous fungi are the optimization of DNA cloning and fungal genetic transformation beyond model fungi, the open exchange of ready-to-use and standardized genetic elements among the research community, and the availability of universal synthetic biology tools and rules. The GoldenBraid (GB) cloning framework is a Golden Gate-based DNA cloning system developed for plant synthetic biology through Agrobacterium tumefaciens-mediated genetic transformation (ATMT). In this study, we develop reagents for the adaptation of GB version 3.0 from plants to filamentous fungi through: (i) the expansion of the GB toolbox with the domestication of fungal-specific genetic elements; (ii) the design of fungal-specific GB structures; and (iii) the ATMT and gene disruption of the plant pathogen Penicillium digitatum as a proof of concept. Genetic elements domesticated into the GB entry vector pUPD2 include promoters, positive and negative selection markers and terminators. Interestingly, some GB elements can be directly exchanged between plants and fungi, as demonstrated with the marker hph for Hyg(R) or the fluorescent protein reporter YFP. The iterative modular assembly of elements generates an endless number of diverse transcriptional units and other higher order combinations in the pDGB3 alpha/pDGB3 Omega destination vectors. Furthermore, the original plant GB syntax was adapted here to incorporate specific GB structures for gene disruption through homologous recombination and dual selection. We therefore have successfully adapted the GB technology for the ATMT of fungi. We propose the name of FungalBraid (FB) for this new branch of the GB technology that provides open, exchangeable and collaborative resources to the fungal research community.This work was funded by grants BIO2015-68790-C2-1-R and BIO2016-78601-R from the "Ministerio de Economia y Competitividad" (MINECO, Spain). SG was recipient of a predoctoral scholarship (FPU13/04584) within the FPU program from "Ministerio de Educacion, Cultura y Deporte" (MECD, Spain). We acknowledge the excellent technical assistance of Tania Campos and the help in the microscopy experiments of Jose M. Coll-Marques (IATA, Valencia, Spain). We also thank Dr. Pilar Moya (Universitat Politecnica de Valencia, Spain) for helpful discussions during the initial stages of this project.Hernanz-Koers, M.; Gandía-Gómez, M.; Garrigues-Cubells, SM.; Manzanares-Mir, PM.; Yenush, L.; Orzáez Calatayud, DV.; Marcos -Lopez, JF. (2018). FungalBraid: A GoldenBraid-based modular cloning platform for the assembly and exchange of DNA elements tailored to fungal synthetic biology. Fungal Genetics and Biology. 116:51-61. https://doi.org/10.1016/j.fgb.2018.04.010S516111

Multigene Engineering by GoldenBraid Cloning: From Plants to Filamentous Fungi and Beyond

This is the peer reviewed version of the following article: Vazquez-Vilar, M., Gandía, M., García-Carpintero, V., Marqués, E., Sarrion-Perdigones, A., Yenush, L., Polaina, J., Manzanares, P., Marcos, J. F., & Orzaez, D. (2020). Multigene engineering by goldenbraid cloning: from plants to filamentous fungi and beyond. Current Protocols in Molecular Biology, 130, e116, doi: 10.1002/cpmb.116, which has been published in final form at https://doi.org/10.1002/cpmb.116. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.[EN] Many synthetic biologists have adopted methods based on Type IIS restriction enzymes and Golden Gate technology in their cloning procedures, as these enable the combinatorial assembly of modular elements in a very efficient way following standard rules. GoldenBraid (GB) is a Golden Gate¿based modular cloning system that, in addition, facilitates the engineering of large multigene constructs and the exchange of DNA parts as result of its iterative cloning scheme. GB was initially developed specifically for plant synthetic biology, and it has been subsequently extended and adapted to other organisms such as Saccharomyces cerevisiae, filamentous fungi, and human cells by incorporating a number of host¿specific features into its basic scheme. Here we describe the general GB cloning procedure and provide detailed protocols for its adaptation to filamentous fungi¿a GB variant known as FungalBraid. The assembly of a cassette for gene disruption by homologous recombination, a fungal¿specific extension of the GB utility, is also shown. Development of FungalBraid was relatively straightforward, as both plants and fungi can be engineered using the same binary plasmids via Agrobacterium¿mediated transformation. We also describe the use of a set of web¿based tools available at the GB website that assist users in all cloning procedures. The availability of plant and fungal versions of GB will facilitate genetic engineering in these industrially relevant organisms.This article is dedicated to the memory of our friend and colleague Dr. Alejandro Sarrion-Perdigones, an early developer of GoldenBraid. We acknowledge the excellent technical assistance provided by Marisol Gascón (IBMCP, Valencia, Spain) with the fluorescent images. This work was funded by Grant BIO2013- 42193 and Grant BIO2016-78601-R, Plan Nacional I+D, Spanish Ministry of Economy and Competitiveness, RTI2018-101115-B-C21 from the Ministerio de Ciencia, Innovación y Universidades (Spain) (MICINN/FEDER Funds), and PROMETEO/ 2018/066 from Conselleria d'Educació (Generalitat Valenciana, Comunitat Valenciana, Spain) and SUSPHIRE PCI2018- 092893-ERA CoBioTech (109) (MCIU/FEDER).Vázquez-Vilar, M.; Gandía, M.; García-Carpintero, V.; Marqués, E.; Sarrion-Perdigones, A.; Yenush, L.; Polaina, J.... (2020). Multigene Engineering by GoldenBraid Cloning: From Plants to Filamentous Fungi and Beyond. Current Protocols in Molecular Biology. 130(1):1-31. https://doi.org/10.1002/cpmb.116S1311301Bernabé‐Orts, J. M., Casas‐Rodrigo, I., Minguet, E. G., Landolfi, V., Garcia‐Carpintero, V., Gianoglio, S., … Orzaez, D. (2019). Assessment of Cas12a‐mediated gene editing efficiency in plants. 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See above.https://gbcloning.upv.es/https://benchling.co

Simultaneous CRISPR/Cas9 Editing of Three PPO Genes Reduces Fruit Flesh Browning in Solanum melongena L.

[EN] Polyphenol oxidases (PPOs) catalyze the oxidization of polyphenols, which in turn causes the browning of the eggplant berry flesh after cutting. This has a negative impact on fruit quality for both industrial transformation and fresh consumption. Ten PPO genes (named SmelPPO1-10) were identified in eggplant thanks to the recent availability of a high-quality genome sequence. A CRISPR/Cas9-based mutagenesis approach was applied to knock-out three target PPO genes (SmelPPO4, SmelPPO5, and SmelPPO6), which showed high transcript levels in the fruit after cutting. An optimized transformation protocol for eggplant cotyledons was used to obtain plants in which Cas9 is directed to a conserved region shared by the three PPO genes. The successful editing of the SmelPPO4, SmelPPO5, and SmelPPO6 loci of in vitro regenerated plantlets was confirmed by Illumina deep sequencing of amplicons of the target sites. Besides, deep sequencing of amplicons of the potential off-target loci identified in silico proved the absence of detectable non-specific mutations. The induced mutations were stably inherited in the T-1 and T-2 progeny and were associated with a reduced PPO activity and browning of the berry flesh after cutting. Our results provide the first example of the use of the CRISPR/Cas9 system in eggplant for biotechnological applications and open the way to the development of eggplant genotypes with low flesh browning which maintain a high polyphenol content in the berries.Research was financially supported by the project CRISPR/Cas9-mediated gene knock-out in eggplant financed by Compagnia San Paolo.Maioli, A.; Gianoglio, S.; Moglia, A.; Acquadro, A.; Valentino, D.; Milani, AM.; Prohens Tomás, J.... (2020). Simultaneous CRISPR/Cas9 Editing of Three PPO Genes Reduces Fruit Flesh Browning in Solanum melongena L. 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