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Viral nanoparticles (VNPs) are a new class of virus-based formulations that can be used as building blocks to implement a variety of functions of potential interest in biotechnology and nanomedicine. Viral coat proteins (CP) that exhibit self-assembly properties are particularly appropriate for displaying antigens and antibodies, by generating multivalent VNPs with therapeutic and diagnostic potential. Here, we developed genetically encoded multivalent VNPs derived from two filamentous plant viruses, potato virus X (PVX) and tobacco etch virus (TEV), which were efficiently and inexpensively produced in the biofactory Nicotiana benthamiana plant. PVX and TEV-derived VNPs were decorated with two different nanobodies recognizing two different regions of the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein. The addition of different picornavirus 2A ribosomal skipping peptides between the nanobody and the CP allowed for modulating the degree of VNP decoration. Nanobody-decorated VNPs purified from N. benthamiana tissues successfully recognized the RBD antigen in enzyme-linked immunosorbent assays and showed efficient neutralization activity against pseudoviruses carrying the Spike protein. Interestingly, multivalent PVX and TEV-derived VNPs exhibited a neutralizing activity approximately one order of magnitude higher than the corresponding nanobody in a dimeric format. These properties, combined with the ability to produce VNP cocktails in the same N. benthamiana plant based on synergistic infection of the parent PVX and TEV, make these green nanomaterials an attractive alternative to standard antibodies for multiple applications in diagnosis and therapeutics.

Programmable transcriptional regulators based on CRISPR architecture are promising tools for the induction of plant gene expression. In plants, CRISPR gene activation is effective with respect to modulating development processes, such as the flowering time or customizing biochemical composition. The most widely used method for delivering CRISPR components into the plant is Agrobacterium tumefaciens-mediated genetic transformation, either transient or stable. However, as a result of their versatility and their ability to move, virus-derived systems have emerged as an interesting alternative for supplying the CRISPR components to the plant, in particular guide RNA (gRNA), which represents the variable component in CRISPR strategies. In the present study, we describe a Potato virus X-derived vector that, upon agroinfection in Nicotiana benthamiana, serves as a vehicle for delivery of gRNAs, producing highly specific virus-induced gene activation. The system works in combination with a N. benthamiana transgenic line carrying the remaining complementary CRISPR gene activation components, specifically the dCasEV2.1 cassette, which has been shown previously to mediate strong programmable transcriptional activation in plants. Using an easily scalable, non-invasive spraying method, we show that gRNA-mediated activation programs move locally and systemically, generating a strong activation response in different target genes. Furthermore, by activating three different endogenous MYB transcription factors, we demonstrate that this Potato virus X-based virus-induced gene reprogramming strategy results in program-specific metabolic fingerprints in N. benthamiana leaves characterized by distinctive phenylpropanoid-enriched metabolite profiles.

Transcriptional regulators based on CRISPR architecture expand our ability to reprogramme endogenous gene expression in plants. One of their potential applications is the customization of plant metabolome through the activation of selected enzymes in a given metabolic pathway. Using the previously described multiplexable CRISPR activator dCasEV2.1, we assayed the selective enrichment in Nicotiana benthamiana leaves of four different flavonoids, namely, naringenin, eriodictyol, kaempferol, and quercetin. After careful selection of target genes and guide RNAs combinations, we created successful activation programmes for each of the four metabolites, each programme activating between three and seven genes, and with individual gene activation levels ranging from 4- to 1500-fold. Metabolic analysis of the flavonoid profiles of each multigene activation programme showed a sharp and selective enrichment of the intended metabolites and their glycosylated derivatives. Remarkably, principal component analysis of untargeted metabolic profiles clearly separated samples according to their activation treatment, and hierarchical clustering separated the samples into five groups, corresponding to the expected four highly enriched metabolite groups, plus an un-activated control. These results demonstrate that dCasEV2.1 is a powerful tool for re-routing metabolic fluxes towards the accumulation of metabolites of interest, opening the door for the custom-made design of metabolic contents in plants.

[ES] La tecnología CRISPR-Cas9 se ha convertido en una herramienta esencial en la biotecnología de plantas, cambiando nuestra capacidad para manipular genomas y estudiar redes moleculares gracias a las posibilidades ofrecidas tanto por la edición génica como por la regulación. Los recientes avances en la Biología Sintética de plantas, junto con la aparición del sistema CRISPR/Cas, ha acelerado la mejora de cultivos, dando lugar a cultivos con caracteres mejorados, y la obtención de metabolitos novedosos y de valor añadido, mediante procesos de Molecular Farming. A pesar de la alta eficiencia para la mutagénesis especifica de sitio de CRISPR/Cas9 y que los activadores génicos transcripcionales basados en Cas9 (CRISPRa) reclutan efectivamente a la maquinaria transcripcional en células vegetales, existe la necesidad de desarrollar nuevas herramientas para controlar los fenómenos de actividad inespecífica derivados de CRISPR/Cas9. Por tanto, durante la última década, se ha trabajado en la generación de diferentes alternativas, como proteínas Cas9 de alta fidelidad, inhibidores químicos, y más recientemente, proteínas Anti-CRISPR (Acr). La identificación de las proteínas Acr representó un punto de inflexión para el desarrollo de interruptores de regulación en las aplicaciones basadas en CRISPR/Cas. Estas proteínas, derivadas de fagos, fueron reconocidas como inhibidores naturales de los sistemas CRISPR tipo II, incluyendo a Cas9. Sin embargo, a pesar de haberlas descrito en células bacterianas y de mamífero, todavía no han sido caracterizadas en sistemas vegetales. Este estudio brinda la primera descripción de la actividad de AcrIIA4 (una proteína Anti-CRISPR capaz de desarrollar una actividad inhibitoria mayor a otras proteínas Acr) en el sistema vegetal modelo de Molecular Farming, Nicotiana benthamiana. Nuestros resultados demuestran que esta Anti-CRISPR es capaz de inhibir efectivamente la activación y edición génica mediada por CRISPR/Cas9. AcrIIA4 reprime el CRISPR, [EN] The CRISPR/Cas technology has become an essential tool in plant biotechnology by changing our capacity for genome manipulation and molecular network study thanks to the possibilities offered by both gene edition and regulation. Recent advances in Plant Synthetic Biology added to the emergence of CRISPR/Cas have accelerated crop breeding, leading to crops with improved traits, the obtention of novel-metabolites and better products derived from Molecular Farming approaches. Despite the high efficiency of CRISPR/Cas9 for site directed mutagenesis and that Cas9-based transcriptional activators (CRISPRa) effectively recruit the transcriptional machinery in plant cells, there is a need for developing new tools for controlling the leakiness and off-target CRISPR-derived phenomena. Thus, during the last decade, there has been a constant work on developing different alternatives, as high-fidelity Cas9 proteins, chemical inhibitors, inducible systems for both Cas9 and gRNAs, and lastly, Anti-CRISPR (Acr) proteins. Identification of Acr proteins represented an inflection point for the development of regulation switches in CRISPR/Cas-based applications. These phage-derived proteins were recognized as natural type II CRISPR/Cas systems inhibitors, including Cas9. However, despite being well described in bacterial and mammalian cells, they have not been characterized in plant systems. The present study brings the first description of the activity of AcrIIA4 (an Anti-CRISPR/Cas9 protein that has been proven to develop a higher inhibitory capacity than other Acr proteins) in the Molecular Farming model plant system Nicotiana benthamiana. Our results demonstrate that this Anti-CRISPR is capable of effectively inhibit both CRISPR/Cas9-mediated gene activation and editing. AcrIIA4 represses CRISPRa of both reporter and endogenous genes in a highly efficient, dose-dependent manner. Moreover, the fusion of an auxin degron to AcrIIA4 allowed the auxin-inducible regulation of CRISPRa, [CA] La tecnologia CRISPR-Cas9 s'ha convertit en una ferramenta essencial en la biotecnologia de plantes, canviant la nostra capacitat de manipular genomes i estudiar xarxes moleculars gràcies a les possibilitats ofertes tant per l'edició gènica com per la regulació. Els recents avanços en la Biologia Sintètica de plantes, juntament amb l'aparició del sistema CRISPR / Cas, ha accelerat la millora de cultius, donant lloc a cultius amb caràcters millorats, l'obtenció de metabòlits nous i de valor afegit, mitjançant processos de Molecular Farming.Tot i alta eficiència per a la mutagènesi especifica de lloc de CRISPR / Cas9 i que els activadors gènics transcripcionals basats en Cas9 (CRISPRa) recluten efectivament a la maquinària transcripcional en cèl·lules vegetals, hi ha la necessitat de desenvolupar noves eines per controlar els fenòmens d' activitat inespecífica derivats de CRISPR / CAS9. Per tant, durant l'última dècada, s'ha treballat en la generació de diferents alternatives, com proteïnes Cas9 d'alta fidelitat, inhibidors químics, i més recentment, proteïnes Anti-CRISPR (Acr). La identificació de les proteïnes Acr va representar un punt d'inflexió per al desenvolupament d'interruptors de regulació en les aplicacions basades en CRISPR / Cas. Aquestes proteïnes,derivades de fags, van ser reconegudes com a inhibidors naturals dels sistemes CRISPR tipus II, incloent a Cas9. No obstant això, tot i haver-les descrit en cèl·lules bacterianes i de mamífer, encara no han estat caracteritzades en sistemes vegetals.Aquest estudi ofereix la primera descripció de l'activitat d'AcrIIA4 (una proteïna Anti-CRISPR capaç de desenvolupar una activitat inhibitòria major a altres proteïnes Acr) en el sistema vegetal model de Molecular Farming, Nicotiana benthamiana. Els nostres resultats demostren que aquesta Anti-CRISPR és capaç d'inhibir efectivament l'activació i edició gènica mediada per CRISPR / Cas9. AcrIIA4 reprimeix el CRISPRa tant de gens reporters com endògens d'una maner

[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.

[ES] En los últimos años, la tecnología CRISPR ha revolucionado la ingeniería del genoma en las plantas. El sistema CRISPR / Cas ofrece una alternativa para la generación de rupturas de doble cadena de ADN (DSB) en un lugar específico, allanando el camino para mejorar los rasgos del organismo. La desactivación de genes se basa en los errores del mecanismo de reparación de ADN de unión final no homóloga (NHEJ) tras la producción de DSB hechos a propósito en los genes de interés, lo que da como resultado la interrupción de sus secuencias codificantes. En esta tesis de Master, hemos analizado la generación de estos DSB mediada por CRISPR-Cas12a / Cas9, así como explorar diferentes parámetros que podrían mejorar el rendimiento de Cas12a y Cas9 en N. benthamiana. Entre ellos, se evaluó la influencia de la temperatura en las eficiencia de edición, la capacidad de "multiplexing" de diferentes ortólogos de Cas o la implementación del sistema CRISPR / ErCas12a recientemente descrito. Además, estudiamos la fiabilidad de los algoritmos disponibles para predecir el rendimiento del ARN guía en N. benthamiana observando que los algoritmos disponibles para Cas12a son más precisos que los disponibles para Cas9. En contraste, la reparación de DSB mediada por HR permite su modificación dirigida al introducir una molécula de ADN donante con los cambios previstos para su reparación. Estas son la base de la modificación dirigida de genes y las estrategias de "knock-in". Sin embargo, su eficiencia reducida cuando se aplica en plantas ha limitado su utilidad. Por esta razón, esta tesis de Master también se ha centrado en un sistema para mejorar su rendimiento en N. benthamiana, evaluando el uso de replicones basados ​​en ADN, como geminivirus, para la introducción de la secuencia de ADN utilizada en estas estrategias de edición mediadas por recombinación homóloga., [EN] Over the past years, CRISPR technology has revolutionized genome engineering in plants. The CRISPR/Cas system provides an alternative for the generation of DNA Double Strand Breaks (DSBs) at a specific target locus, paving the way for improving organism traits. Gene knockout relies on the errors of the non-homologous end joining (NHEJ) DNA repair mechanism upon the production of DSBs deliberately on the genes of interest, resulting in the disruption of their coding sequences. In this Master s thesis, we have analyzed CRISPR-Cas12a/Cas9-mediated generation of these DSBs, as well as explored different parameters that could improve Cas12a and Cas9 performance in N. benthamiana. Among them, temperature influence on the editing efficiencies, multiplexing capacity of different Cas orthologues, or the implementation of the recently described CRISPR/ErCas12a system were tested. Additionally, we studied the reliability of the available algorithms to predict guide RNAs performance in N. benthamiana observing that algorithms available for Cas12a are more accurate than those available for Cas9. In contrast, HR-mediated repair of DSBs allows their deliberate modification by introducing a donor DNA molecule with the intended changes for its repairing. These are the basis of gene targeting and gene knock-in strategies. Nevertheless, their reduced efficiency when applied in plants have limited their utility. For this reason, this Master s thesis has also focused on a system to improve their performance in N. benthamiana, evaluating the use of DNA-based replicons, such as geminiviruses, for the delivery of the DNA sequence used on these HR-mediated editing strategies.

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.

[ES] Aunque mucha gente no es consciente de ello, la mejora genética de plantas ha sido aplicada desde los inicios de la agricultura. Desde siempre, se ha establecido una selección artificial, bien sea por la simple elección ejercida por los agricultores de las semillas más productivas o por la realización de cruces artificiales que no se dan en la naturaleza. Durante las últimas décadas, una forma de selección adicional a las ya existentes ha sufrido un crecimiento exponencial. La rápida evolución de las técnicas de la biología molecular ha potenciado a la ingeniería genética hasta límites inimaginables. La ingeniería genética se basa en el uso de la biotecnología para la manipulación dirigida de genes con el propósito de, entre otros, superar los límites reproductivos entre especies diferentes transfiriéndolos de unas a otras, induciendo mutantes con genes silenciados o incluso sobreexpresándolos. Entre todos los aspectos positivos que la ingeniería genética de plantas aporta a la agricultura, merece destacar su capacidad de incrementar los rendimientos de producción, el contenido nutricional de los productos obtenidos o la resistencia a plagas. En general, la edición genética se ha llevado a cabo utilizando nucleasas que permiten inserciones, deleciones y sustituciones específicas de secuencia. Para ello, estas enzimas desencadenan roturas de doble cadena (DSBs) del DNA que pueden ser reparadas, o bien mediante la unión de los extremos no homólogos (NHEJ), o bien mediante la reparación dirigida por homología (HDR), promoviendo así las mutaciones anteriormente mencionadas. Tradicionalmente, los ingenieros genéticos han utilizado meganucleasas, nucleasas de actividad similar a activador de transcripción (TALENs) y nucleasas con dedos de zinc (ZFNs), cada una con sus propias ventajas y desventajas. A pesar del espectacular progreso que se ha conseguido gracias a su uso, una tecnología posteriormente desarrollada que consiste en una nucleasa Cas 9 asociada a CRISPR p, [EN] Although many people are not aware of, plant breeding started at the very beginning of agriculture. A non-naturally occurring selection has always been stablished either by farmer s seed choice or induced plant crosses. During the last decades, an additional way of selecting plants for the desired traits has experienced an exponential growth. The rapid evolution of molecular biology-based techniques has boosted genetic engineering till unexpected limits. Genetic engineering involves the use of biotechnology for direct manipulation of genes aiming, for instance, to transfer them across species boundaries, to knockout, to edit or to overexpress them. Among all possible benefits of plant genetic engineering to agriculture, it should be remarked its ability to increase crop production yields, nutritional content and pest resistance. In general, gene editing has been carried out using specific nucleases that allow targeted insertions, deletions and precise sequence substitutions. To do so, those enzymes trigger double-strand breaks (DSBs) in the DNA that can be repaired either by non-homologous end joining (NHEJ) or by homology-directed repair (HDR), thus promoting the previously mentioned mutations. Traditionally, genetic engineers have used meganucleases, transcription activator like effector nucleases (TALENs) and zinc finger nucleases (ZFNs), each of them presenting their own set of advantages and drawbacks. In spite of the spectacular progress achieved with their application, an afterwards developed technology consisting of a bacterial CRISPR-associated protein 9 nuclease from Streptococcus pyogenes exceeded all their benefits. CRISPR-Cas system has completely revolutionized Genetic Engineering since it is an RNA-based nuclease that relies on base-paring rules between an engineered RNA and the desired target DNA site to be mutated rather than protein-DNA interaction needed for the previous nucleases. However, scientific progress has gone a step further and new

The current CoVid-19 crisis is revealing the strengths and the weaknesses of the world’s capacity to respond to a global health crisis. A critical weakness has resulted from the excessive centralization of the current biomanufacturing capacities, a matter of great concern, if not a source of nationalistic tensions. On the positive side, scientific data and information have been shared at an unprecedented speed fuelled by the preprint phenomena, and this has considerably strengthened our ability to develop new technology-based solutions. In this work, we explore how, in a context of rapid exchange of scientific information, plant biofactories can serve as a rapid and easily adaptable solution for local manufacturing of bioreagents, more specifically recombinant antibodies. For this purpose, we tested our ability to produce, in the framework of an academic lab and in a matter of weeks, milligram amounts of six different recombinant monoclonal antibodies against SARS-CoV-2 in Nicotiana benthamiana. For the design of the antibodies, we took advantage, among other data sources, of the DNA sequence information made rapidly available by other groups in preprint publications. mAbs were engineered as single-chain fragments fused to a human gamma Fc and transiently expressed using a viral vector. In parallel, we also produced the recombinant SARS-CoV-2 N protein and the receptor binding domain (RBD) of the Spike protein in planta and used them to test the binding specificity of the recombinant mAbs. Finally, for two of the antibodies, we assayed a simple scale-up production protocol based on the extraction of apoplastic fluid. Our results indicate that gram amounts of anti-SARS-CoV-2 antibodies could be easily produced in little more than 6 weeks in repurposed greenhouses with little infrastructure requirements using N. benthamiana as production platform. Similar procedures could be easily deployed to produce diagnostic reagents and, eventually, could be adapted for rapid the

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.

[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.

[EN] Synthetic Biology is a novel interdisciplinary field that aims to re-design existing natural biological systems or even to create new ones from an engineering perspective. Modularity, standardization, abstraction, decoupling and orthogonality constitute the mainstays of Synthetic Biology and must be applied to genetic design in a rational and systematic way. Innumerable breakthroughs in sequence-specific nuclease technologies made possible to precisely manipulate plant genomes. These nucleases include meganucleases, zinc-finger nucleases, TALENs or CRISPR/Cas9 and they have become essential tools in Plant Synthetic Biology. The aforementioned technologies allow to generate user-designed organisms by manipulating their genome and expression pattern. Therefore, the natural response of an organism to a specific environment can be improved, taking Synthetic Biology to the next level. One method to efficiently and precisely modify plant genomes involves introducing double-strand breaks produced by sequence-specific endonucleases to stimulate homologous recombination between a donor template and a chromosomal target site. However, precise genome modification remains a challenge due to the low efficiency of homologous recombination, hampering expansion of Plant Synthetic Biology. It was reported that gene-targeting frequency can be further increased by the presence of a high number of repair templates. Bearing this in mind, deconstructed virus strategy, an indispensable tool in Synthetic Biology field, arises as one of the better solution to efficiently deliver both sequence-specific nucleases and repair template. Therefore, it has been postulated that homologous recombination frequency can be enhanced in plant genome using a viral vector strategy. The most powerfulsite-specific nucleases for genome editing are based on CRISPR technology. The easy reprogramming of the system confers a straight-forward, specific and orthogonal strategy to perform targeted genome editin, [ES] La Biología Sintética es un campo innovador de carácter interdisciplinar que tiene como objetivo rediseñar los sistemas biológicos naturales, así como crear otros nuevos desde una perspectiva ingenieril. Modularidad, estandarización, abstracción, desacoplamiento y ortogonalidad constituyen los pilares fundamentales de la Biología Sintética y deben ser considerados en el diseño genético de una manera racional y sistemática. La manipulación precisa del genoma de organismos vegetales ha sido posible gracias a los innumerables avances en las tecnologías basadas en nucleasas específicas de secuencia. Las meganucleasas, nucleasas con dedos de zinc, TALEN o CRISPR/Cas9 se han convertido en herramientas esenciales en Biología Sintética de Plantas que permiten generar organismos a la carta manipulando su genoma y patrón de expresión. De este modo, es posible mejorar la respuesta natural de un organismo frente a un entorno específico impulsando el avance de la Biología Sintética. Un método para modificar de forma eficiente y precisa el genoma de las plantas se basa en la rotura de la doble cadena de ADN por endonucleasas específicas de sitio con el objetivo de estimular la recombinación homóloga entre una secuencia molde y el sitio cromosómico específico. Sin embargo, la modificación precisa del genoma vegetal sigue siendo un desafío debido a la baja eficiencia de la recombinación homóloga, dificultando así la expansión de la Biología Sintética de Plantas. En recientes estudios se ha demostrado que este evento se puede favorecer al incrementarse el número de copias de la secuencia molde. Por este motivo, surge la estrategia basada en el empleo de un virus deconstruido como una de las mejores soluciones para entregar de forma eficiente las endonucleasas y las múltiples copias de la secuencia molde. Por lo tanto, se ha postulado que la frecuencia de recombinación homóloga en el genoma de la planta puede potenciarse usando la estrategia basada en el virus deconstruido. Por

This is a pre-copyedited, author-produced version of an article accepted for publication in Nucleic Acids Research following peer review. The version of record Vázquez-Vilar, M.; Quijano-Rubio, A.; Fernandez Del Carmen, MA.; Sarrion-Perdigones, A.; Ochoa-Fernández, R.; Ziarsolo Areitioaurtena, P.; Blanca Postigo, JM.... (2017). GB3.0: a platform for plant bio-design that connects functional DNA elements with associated biological data. Nucleic Acids Research. 45(4):2196-2209. doi:10.1093/nar/gkw1326 is available online at: http://doi.org/10.1093/nar/gkw1326., [EN] Modular DNA assembly simplifies multigene engineering in Plant Synthetic Biology. Furthermore, the recent adoption of a common syntax to facilitate the exchange of plant DNA parts (phytobricks) is a promising strategy to speed up genetic engineering. Following this lead, here, we present a platform for plant biodesign that incorporates functional descriptions of phytobricks obtained under pre-defined experimental conditions, and systematically registers the resulting information as metadata for documentation. To facilitate the handling of functional descriptions, we developed a new version (v3.0) of the GoldenBraid (GB) webtool that integrates the experimental data and displays it in the form of datasheets. We report the use of the Luciferase/Renilla (Luc/Ren) transient agroinfiltration assay in Nicotiana benthamiana as a standard to estimate relative transcriptional activities conferred by regulatory phytobricks, and show the consistency and reproducibility of this method in the characterization of a synthetic phytobrick based on the CaMV35S promoter. Furthermore, we illustrate the potential for combinatorial optimization and incremental innovation of the GB3.0 platform in two separate examples, (i) the development of a collection of orthogonal transcriptional regulators based on phiC31 integrase and (ii) the design of a small genetic circuit that connects a glucocorticoid switch to a MYB/bHLH transcriptional activation module.

Tesis por compendio, [EN] Synthetic Biology is an emerging interdisciplinary field that aims to apply the engineering principles of modularity, abstraction and standardization to genetic engineering. The nascent branch of Synthetic Biology devoted to plants, Plant Synthetic Biology (PSB), offers new breeding possibilities for crops, potentially leading to enhanced resistance, higher yield, or increased nutritional quality. To this end, the molecular tools in the PSB toolbox need to be adapted accordingly, to become modular, standardized and more precise. Thus, the overall objective of this Thesis was to adapt, expand and refine DNA assembly tools for PSB to enable the incorporation of functional specifications to the description of standard genetic elements (phytobricks) and to facilitate the construction of increasingly complex and precise multigenic devices, including genome editing tools. The starting point of this Thesis was the modular DNA assembly method known as GoldenBraid (GB), based on type IIS restriction enzymes. To further optimize the GB construct-making process and to better catalog the phytobricks collection, a database and a set of software-tools were developed as described in Chapter 1. The final webbased software package, released as GB2.0, was made publicly available at www.gbcloning.upv.es. A detailed description of the functioning of GB2.0, exemplified with the building of a multigene construct for anthocyanin overproduction was also provided in Chapter 1. As the number and complexity of GB constructs increased, the next step forward consisted in the refinement of the standards with the incorporation of experimental information associated to each genetic element (described in Chapter 2). To this end, the GB package was reshaped into an improved version (GB3.0), which is a self-contained, fully traceable assembly system where the experimental data describing the functionality of each DNA element is displayed in the form of a standard datasheet. The utility of the te, [ES] La Biología Sintética es un campo emergente de carácter interdisciplinar que se fundamenta en la aplicación de los principios ingenieriles de modularidad, abstracción y estandarización a la ingeniería genética. Una nueva vertiente de la Biología Sintética aplicada a las plantas, la Biología Sintética Vegetal (BSV), ofrece nuevas posibilidades de mejora de cultivos que podrían llevar a una mejora de la resistencia, a una mayor productividad, o a un aumento de la calidad nutricional. Sin embargo, para alcanzar este fin las herramientas moleculares disponibles en estos momentos para BSV deben ser adaptadas para convertirse en modulares, estándares y más precisas. Por ello se planteó como objetivo general de esta Tesis adaptar, expandir y refinar las herramientas de ensamblaje de DNA de la BSV para permitir la incorporación de especificaciones funcionales en la descripción de elementos genéticos estándar (fitobricks) y facilitar la construcción de estructuras multigénicas cada vez más complejas y precisas, incluyendo herramientas de editado genético. El punto de partida de esta Tesis fue el método de ensamblaje modular de ADN GoldenBraid (GB) basado en enzimas de restricción tipo IIS. Para optimizar el proceso de ensamblaje y catalogar la colección de fitobricks generados se desarrollaron una base de datos y un conjunto de herramientas software, tal y como se describe en el Capítulo 1. El paquete final de software se presentó en formato web como GB2.0, haciéndolo accesible al público a través de www.gbcloning.upv.es. El Capítulo 1 también proporciona una descripción detallada del funcionamiento de GB2.0 ejemplificando su uso con el ensamblaje de una construcción multigénica para la producción de antocianinas. Con el aumento en número y complejidad de las construcciones GB, el siguiente paso necesario fue el refinamiento de los estándar con la incorporación de la información experimental asociada a cada elemento genético (se describe en el Capítulo 2). Para este fin, [CA] La Biologia Sintètica és un camp emergent de caràcter interdisciplinar que es fonamenta amb l'aplicació a la enginyeria genètica dels principis de modularitat, abstracció i estandarització. Una nova vessant de la Biologia Sintètica aplicada a les plantes, la Biologia Sintètica Vegetal (BSV), ofereix noves possibilitats de millora de cultius que podrien portar a una millora de la resistència, a una major productivitat, o a un augment de la qualitat nutricional. Tanmateix, per poder arribar a este fi les eines moleculars disponibles en estos moments per a la BSV han d'adaptar-se per convertir-se en modulars, estàndards i més precises. Per això es plantejà com objectiu general d'aquesta Tesi adaptar, expandir i refinar les eines d'ensamblatge d'ADN de la BSV per permetre la incorporació d'especificacions funcionals en la descripció d'elements genètics estàndards (fitobricks) i facilitar la construcció d'estructures multigèniques cada vegada més complexes i precises, incloent eines d'edidat genètic. El punt de partida d'aquesta Tesi fou el mètode d'ensamblatge d'ADN modular GoldenBraid (GB) basat en enzims de restricció tipo IIS. Per optimitzar el proces d'ensamblatge i catalogar la col.lecció de fitobricks generats es desenvolupà una base de dades i un conjunt d'eines software, tal i com es descriu al Capítol 1. El paquet final de software es presentà en format web com GB2.0, fent-se accessible al públic mitjançant la pàgina web www.gbcloning.upv.es. El Capítol 1 també proporciona una descripció detallada del funcionament de GB2.0, exemplificant el seu ús amb l'ensamblatge d'una construcció multigènica per a la producció d'antocians. Amb l'augment en nombre i complexitat de les construccions GB, el següent pas fou el refinament dels estàndards amb la incorporació de la informació experimental associada a cada element genètic (es descriu en el Capítol 2). Per a aquest fi, el paquet de software de GB es reformulà amb una nova versió anomenada GB3.0. Aquesta versió c

[EN] Background: The efficiency, versatility and multiplexing capacity of RNA-guided genome engineering using the CRISPR/Cas9 technology enables a variety of applications in plants, ranging from gene editing to the construction of transcriptional gene circuits, many of which depend on the technical ability to compose and transfer complex synthetic instructions into the plant cell. The engineering principles of standardization and modularity applied to DNA cloning are impacting plant genetic engineering, by increasing multigene assembly efficiency and by fostering the exchange of well-defined physical DNA parts with precise functional information. Results: Here we describe the adaptation of the RNA-guided Cas9 system to GoldenBraid (GB), a modular DNA con¿ struction framework being increasingly used in Plant Synthetic Biology. In this work, the genetic elements required for CRISPRs-based editing and transcriptional regulation were adapted to GB, and a workflow for gRNAs construction was designed and optimized. New software tools specific for CRISPRs assembly were created and incorporated to the public GB resources site. Conclusions: The functionality and the efficiency of gRNA¿Cas9 GB tools were demonstrated in Nicotiana benthamiana using transient expression assays both for gene targeted mutations and for transcriptional regulation. The availability of gRNA¿Cas9 GB toolbox will facilitate the application of CRISPR/Cas9 technology to plant genome engineering

[EN] Plant synthetic biology aims to apply engineering principles to plant genetic design. One strategic requirement of plant synthetic biology is the adoption of common standardized technologies that facilitate the construction of increasingly complex multigene structures at the DNA level while enabling the exchange of genetic building blocks among plant bioengineers. Here, we describe GoldenBraid 2.0 (GB2.0), a comprehensive technological framework that aims to foster the exchange of standard DNA parts for plant synthetic biology. GB2.0 relies on the use of type IIS restriction enzymes for DNA assembly and proposes a modular cloning schema with positional notation that resembles the grammar of natural languages. Apart from providing an optimized cloning strategy that generates fully exchangeable genetic elements for multigene engineering, the GB2.0 toolkit offers an ever-growing open collection of DNA parts, including a group of functionally tested, premade genetic modules to build frequently used modules like constitutive and inducible expression cassettes, endogenous gene silencing and protein-protein interaction tools, etc. Use of the GB2.0 framework is facilitated by a number of Web resources that include a publicly available database, tutorials, and a software package that provides in silico simulations and laboratory protocols for GB2.0 part domestication and multigene engineering. In short, GB2.0 provides a framework to exchange both information and physical DNA elements among bioengineers to help implement plant synthetic biology projects.