Summary
CRISPR/Cas has revolutionized genome engineering in plants. However, the use of anti‐CRISPR proteins as tools to prevent CRISPR/Cas‐mediated gene editing and gene activation in plants has not ...been explored yet. This study describes the characterization of two anti‐CRISPR proteins, AcrIIA4 and AcrVA1, in Nicotiana benthamiana. Our results demonstrate that AcrIIA4 prevents site‐directed mutagenesis in leaves when transiently co‐expressed with CRISPR/Cas9. In a similar way, AcrVA1 is able to prevent CRISPR/Cas12a‐mediated gene editing. Moreover, using a N. benthamiana line constitutively expressing Cas9, we show that the viral delivery of AcrIIA4 using Tobacco etch virus is able to completely abolish the high editing levels obtained when the guide RNA is delivered with a virus, in this case Potato virus X. We also show that AcrIIA4 and AcrVA1 repress CRISPR/dCas‐based transcriptional activation of reporter genes. In the case of AcrIIA4, this repression occurs in a highly efficient, dose‐dependent manner. Furthermore, the fusion of an auxin degron to AcrIIA4 results in auxin‐regulated activation of a downstream reporter gene. The strong anti‐Cas activity of AcrIIA4 and AcrVA1 reported here opens new possibilities for customized control of gene editing and gene expression in plants.
Synthetic Biology requires efficient and versatile DNA assembly systems to facilitate the building of new genetic modules/pathways from basic DNA parts in a standardized way. Here we present ...GoldenBraid (GB), a standardized assembly system based on type IIS restriction enzymes that allows the indefinite growth of reusable gene modules made of standardized DNA pieces. The GB system consists of a set of four destination plasmids (pDGBs) designed to incorporate multipartite assemblies made of standard DNA parts and to combine them binarily to build increasingly complex multigene constructs. The relative position of type IIS restriction sites inside pDGB vectors introduces a double loop ("braid") topology in the cloning strategy that allows the indefinite growth of composite parts through the succession of iterative assembling steps, while the overall simplicity of the system is maintained. We propose the use of GoldenBraid as an assembly standard for Plant Synthetic Biology. For this purpose we have GB-adapted a set of binary plasmids for A. tumefaciens-mediated plant transformation. Fast GB-engineering of several multigene T-DNAs, including two alternative modules made of five reusable devices each, and comprising a total of 19 basic parts are also described.
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
. 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
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
as production platform. Similar procedures could be easily deployed to produce diagnostic reagents and, eventually, could be adapted for rapid therapeutic responses.
Virus-induced gene silencing (VIGS) is a powerful tool for reverse genetics in tomato (Solanum lycopersicum). However, the irregular distribution of the effects of VIGS hampers the identification and ...quantification of nonvisual phenotypes. To overcome this limitation, a visually traceable VIGS system was developed for fruit, comprising two elements: (1) a transgenic tomato line (Del/Ros1) expressing Antirrhinum majus Delila and Rosea1 transcription factors under the control of the fruit-specific E8 promoter, showing a purple-fruited, anthocyanin-rich phenotype; and (2) a modified tobacco rattle virus VIGS vector incorporating partial Rosea1 and Delila sequences, which was shown to restore the red-fruited phenotype upon agroinjection in Del/Ros1 plants. Dissection of silenced areas for subsequent chemometric analysis successfully identified the relevant metabolites underlying gene function for three tomato genes, phytoene desaturase, TomloxC, and SlODO1, used for proof of concept. The C-6 aldehydes derived from lipid 13-hydroperoxidation were found to be the volatile compounds most severely affected by TomloxC silencing, whereas geranial and 6-methyl-5-hepten-2-one were identified as the volatiles most severely reduced by phytoene desaturase silencing in ripening fruit. In a third example, silencing of SlODO1, a tomato homolog of the ODORANT1 gene encoding a myb transcription factor, which regulates benzenoid metabolism in petunia (Petunia hybrida) flowers, resulted in a sharp accumulation of benzaldehyde in tomato fruit. Together, these results indicate that fruit VIGS, enhanced by anthocyanin monitoring, can be a powerful tool for reverse genetics in the study of the metabolic networks operating during fruit ripening.
Volatile organic compounds (VOCs) are major determinants of fruit flavor, a primary objective in tomato breeding. A recombinant inbred line (RIL) population consisting of 169 lines derived from a ...cross between Solanum lycopersicum and a red-fruited wild tomato species Solanum pimpinellifolium accession (SP) was characterized for VOCs in three different seasons. Correlation and hierarchical cluster analyses were performed on the 52 VOCs identified, providing a tool for the putative assignation of individual compounds to metabolic pathways. Quantitative trait locus (QTL) analysis, based on a genetic linkage map comprising 297 single nucleotide polymorphisms (SNPs), revealed 102 QTLs (75% not described previously) corresponding to 39 different VOCs. The SP alleles exerted a positive effect on most of the underlying apocarotenoid volatile QTLs—regarded as desirable for liking tomato—indicating that alleles inherited from SP are a valuable resource for flavor breeding. An introgression line (IL) population developed from the same parental genotypes provided 12 ILs carrying a single SP introgression and covering 85 VOC QTLs, which were characterized at three locations. The results showed that almost half of the QTLs previously identified in the RILs maintained their effect in an IL form, reinforcing the value of these QTLs for flavor/aroma breeding in cultivated tomato.
Antivenoms developed from the plasma of hyperimmunized animals are the only effective treatment available against snakebite envenomation but shortage of supply contributes to the high morbidity and ...mortality toll of this tropical disease. We describe a synthetic biology approach to affordable and cost‐effective antivenom production based on plant‐made recombinant polyclonal antibodies (termed pluribodies). The strategy takes advantage of virus superinfection exclusion to induce the formation of somatic expression mosaics in agroinfiltrated plants, which enables the expression of complex antibody repertoires in a highly reproducible manner. Pluribodies developed using toxin‐binding genetic information captured from peripheral blood lymphocytes of hyperimmunized camels recapitulated the overall binding activity of the immune response. Furthermore, an improved plant‐made antivenom (plantivenom) was formulated using an in vitro selected pluribody against Bothrops asper snake venom toxins and has been shown to neutralize a wide range of toxin activities and provide protection against lethal venom doses in mice.
CRISPR/Cas ability to target several loci simultaneously (multiplexing) is a game-changer in plant breeding. Multiplexing not only accelerates trait pyramiding but also can unveil traits hidden by ...functional redundancy. Furthermore, multiplexing enhances dCas-based programmable gene expression and enables cascade-like gene regulation. However, the design and assembly of multiplex constructs comprising tandemly arrayed guide RNAs (gRNAs) requires scarless cloning and is still troublesome due to the presence of repetitive sequences, thus hampering a more widespread use. Here we present a comprehensive extension of the software-assisted cloning platform GoldenBraid (GB), in which, on top of its multigene cloning software, we integrate new tools for the Type IIS-based easy and rapid assembly of up to six tandemly-arrayed gRNAs with both Cas9 and Cas12a, using the gRNA-tRNA-spaced and the crRNA unspaced approaches, respectively. As stress tests for the new tools, we assembled and used for Agrobacterium-mediated stable transformation a 17 Cas9-gRNAs construct targeting a subset of the Squamosa-Promoter Binding Protein-Like (SPL) gene family in
Nicotiana tabacum
. The 14 selected genes are targets of miR156, thus potentially playing an important role in juvenile-to-adult and vegetative-to-reproductive phase transitions. With the 17 gRNAs construct we generated a collection of Cas9-free
SPL
edited T
1
plants harboring up to 9 biallelic mutations and showing leaf juvenility and more branching. The functionality of GB-assembled dCas9 and dCas12a-based CRISPR/Cas activators and repressors using single and multiplexing gRNAs was validated using a Luciferase reporter with the
Solanum lycopersicum
Mtb promoter or the
Agrobacterium tumefaciens
nopaline synthase promoter in transient expression in
Nicotiana benthamiana
. With the incorporation of the new web-based tools and the accompanying collection of DNA parts, the GB4.0 genome edition turns an all-in-one open platform for plant genome engineering.
Summary
Higher dietary intakes of flavonoids may have a beneficial role in cardiovascular disease prevention. Additionally, supplementation of branched‐chain amino acids (BCAAs) in vegan diets can ...reduce risks associated to their deficiency, particularly in older adults, which can cause loss of skeletal muscle strength and mass. Most plant‐derived foods contain only small amounts of BCAAs, and those plants with high levels of flavonoids are not eaten broadly. Here we describe the generation of metabolically engineered cisgenic tomatoes enriched in both flavonoids and BCAAs. In this approach, coding and regulatory DNA elements, all derived from the tomato genome, were combined to obtain a herbicide‐resistant version of an acetolactate synthase (mSlALS) gene expressed broadly and a MYB12‐like transcription factor (SlMYB12) expressed in a fruit‐specific manner. The mSlALS played a dual role, as a selectable marker as well as being key enzyme in BCAA enrichment. The resulting cisgenic tomatoes were highly enriched in Leucine (21‐fold compared to wild‐type levels), Valine (ninefold) and Isoleucine (threefold) and concomitantly biofortified in several antioxidant flavonoids including kaempferol (64‐fold) and quercetin (45‐fold). Comprehensive metabolomic and transcriptomic analysis of the biofortified cisgenic tomatoes revealed marked differences to wild type and could serve to evaluate the safety of these biofortified fruits for human consumption.
Considering cells as biofactories, we aimed to optimize its internal processes by using the same engineering principles that large industries are implementing nowadays: lean manufacturing. We have ...applied reverse engineering computational methods to transcriptomic, metabolomic and phenomic data obtained from a collection of tomato recombinant inbreed lines to formulate a kinetic and constraint-based model that efficiently describes the cellular metabolism from expression of a minimal core of genes. Based on predicted metabolic profiles, a close association with agronomic and organoleptic properties of the ripe fruit was revealed with high statistical confidence. Inspired in a synthetic biology approach, the model was used for exploring the landscape of all possible local transcriptional changes with the aim of engineering tomato fruits with fine-tuned biotechnological properties. The method was validated by the ability of the proposed genomes, engineered for modified desired agronomic traits, to recapitulate experimental correlations between associated metabolites.
Recombinant antigen production in plants is a safe and economically sound strategy for vaccine development, particularly for oral/mucosal vaccination, but subunit vaccines usually suffer from weak ...immunogenicity and require adjuvants that escort the antigens, target them to relevant sites and/or activate antigen-presenting cells for elicitation of protective immunity. Genetic fusions of antigens with bacterial adjuvants as the B subunit of the cholera toxin have been successful in inducing protective immunity of plant-made vaccines. In addition, several plant compounds, mainly plant defensive molecules as lectins and saponins, have shown strong adjuvant activities. The molecular diversity of the plant kingdom offers a vast source of non-bacterial compounds with adjuvant activity, which can be assayed in emerging plant manufacturing systems for the design of new plant vaccine formulations.
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