Fine-Tuning Tomato Agronomic Properties by Computational Genome Redesign

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

The GB4.0 Platform, an All-In-One Tool for CRISPR/Cas-Based Multiplex Genome Engineering in Plants

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 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

Identification, introgression, and validation of fruit volatile QTLs from a red-fruited wild tomato species

[EN] 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.We thank Erika Moro for valuable help in volatile analysis of the ILs. WB was supported by a fellowship granted by the Universidad de Costa Rica and CSIC-Spain by way of a collaboration agreement between CSIC/UCR. Volatile profiling was performed in the Metabolomic facilities of the IBMCP, CSIC (Spain). This work was supported in part by the Spanish MINECO Project AGL2015-65246-R co-financed by EU FEDER, MINECO Project AGL2011-26957, and the Bilateral agreements of Scientific and Technological Cooperation between the Spanish National Research Council (CSIC) and the Italian National Research Council (CNR). Funding for this project was provided through TRADITOM. TRADITOM has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 634561. Networking activities were supported by COST action Fruit Quality FA 1106.Rambla Nebot, JL.; Medina, A.; Fernández Del Carmen, MA.; Barrantes, W.; Grandillo, S.; Cammareri, M.; López Casado, G.... (2016). Identification, introgression, and validation of fruit volatile QTLs from a red-fruited wild tomato species. Journal of Experimental Botany. 68(3):429-442. https://doi.org/10.1093/jxb/erw455S429442683Alba, J. M., Montserrat, M., & Fernández-Muñoz, R. (2008). Resistance to the two-spotted spider mite (Tetranychus urticae) by acylsucroses of wild tomato (Solanum pimpinellifolium) trichomes studied in a recombinant inbred line population. Experimental and Applied Acarology, 47(1), 35-47. doi:10.1007/s10493-008-9192-4Abegaz, E. G., Tandon, K. S., Scott, J. W., Baldwin, E. A., & Shewfelt, R. L. (2004). Partitioning taste from aromatic flavor notes of fresh tomato (Lycopersicon esculentum, Mill) to develop predictive models as a function of volatile and nonvolatile components. Postharvest Biology and Technology, 34(3), 227-235. doi:10.1016/j.postharvbio.2004.05.023Baldwin, E. A., Goodner, K., & Plotto, A. (2008). Interaction of Volatiles, Sugars, and Acids on Perception of Tomato Aroma and Flavor Descriptors. Journal of Food Science, 73(6), S294-S307. doi:10.1111/j.1750-3841.2008.00825.xBarrantes, W., Fernández-del-Carmen, A., López-Casado, G., González-Sánchez, M. Á., Fernández-Muñoz, R., Granell, A., & Monforte, A. J. (2014). Highly efficient genomics-assisted development of a library of introgression lines of Solanum pimpinellifolium. Molecular Breeding, 34(4), 1817-1831. doi:10.1007/s11032-014-0141-0Buttery, R. G., & Ling, L. C. (1993). Volatile Components of Tomato Fruit and Plant Parts. Bioactive Volatile Compounds from Plants, 23-34. doi:10.1021/bk-1993-0525.ch003Capel, C., Fernández del Carmen, A., Alba, J. M., Lima-Silva, V., Hernández-Gras, F., Salinas, M., … Lozano, R. (2015). Wide-genome QTL mapping of fruit quality traits in a tomato RIL population derived from the wild-relative species Solanum pimpinellifolium L. Theoretical and Applied Genetics, 128(10), 2019-2035. doi:10.1007/s00122-015-2563-4Chen, G., Hackett, R., Walker, D., Taylor, A., Lin, Z., & Grierson, D. (2004). Identification of a Specific Isoform of Tomato Lipoxygenase (TomloxC) Involved in the Generation of Fatty Acid-Derived Flavor Compounds. Plant Physiology, 136(1), 2641-2651. doi:10.1104/pp.104.041608Eisen, M. B., Spellman, P. T., Brown, P. O., & Botstein, D. (1998). Cluster analysis and display of genome-wide expression patterns. Proceedings of the National Academy of Sciences, 95(25), 14863-14868. doi:10.1073/pnas.95.25.14863Fowlkes, E. B., & Mallows, C. L. (1983). A Method for Comparing Two Hierarchical Clusterings. Journal of the American Statistical Association, 78(383), 553. doi:10.2307/2288117Goulet, C., Kamiyoshihara, Y., Lam, N. B., Richard, T., Taylor, M. G., Tieman, D. M., & Klee, H. J. (2015). Divergence in the Enzymatic Activities of a Tomato and Solanum pennellii Alcohol Acyltransferase Impacts Fruit Volatile Ester Composition. Molecular Plant, 8(1), 153-162. doi:10.1016/j.molp.2014.11.007Goulet, C., Mageroy, M. H., Lam, N. B., Floystad, A., Tieman, D. M., & Klee, H. J. (2012). Role of an esterase in flavor volatile variation within the tomato clade. Proceedings of the National Academy of Sciences, 109(46), 19009-19014. doi:10.1073/pnas.1216515109Klee, H. J., & Tieman, D. M. (2013). Genetic challenges of flavor improvement in tomato. Trends in Genetics, 29(4), 257-262. doi:10.1016/j.tig.2012.12.003Kochevenko, A., Araújo, W. L., Maloney, G. S., Tieman, D. M., Do, P. T., Taylor, M. G., … Fernie, A. R. (2012). Catabolism of Branched Chain Amino Acids Supports Respiration but Not Volatile Synthesis in Tomato Fruits. Molecular Plant, 5(2), 366-375. doi:10.1093/mp/ssr108Louveau, T., Leitao, C., Green, S., Hamiaux, C., van der Rest, B., Dechy-Cabaret, O., … Chervin, C. (2010). Predicting the substrate specificity of a glycosyltransferase implicated in the production of phenolic volatiles in tomato fruit. FEBS Journal, 278(2), 390-400. doi:10.1111/j.1742-4658.2010.07962.xMageroy, M. H., Tieman, D. M., Floystad, A., Taylor, M. G., & Klee, H. J. (2011). A Solanum lycopersicum catechol-O-methyltransferase involved in synthesis of the flavor molecule guaiacol. The Plant Journal, 69(6), 1043-1051. doi:10.1111/j.1365-313x.2011.04854.xMathieu, S., Cin, V. D., Fei, Z., Li, H., Bliss, P., Taylor, M. G., … Tieman, D. M. (2008). Flavour compounds in tomato fruits: identification of loci and potential pathways affecting volatile composition. Journal of Experimental Botany, 60(1), 325-337. doi:10.1093/jxb/ern294Matsui, K., Ishii, M., Sasaki, M., Rabinowitch, H. D., & Ben-Oliel, G. (2007). Identification of an Allele Attributable to Formation of Cucumber-like Flavor in Wild Tomato Species (Solanum pennellii) That Was Inactivated during Domestication. Journal of Agricultural and Food Chemistry, 55(10), 4080-4086. doi:10.1021/jf063756bMATSUI, K., MIYAHARA, C., WILKINSON, J., HIATT, B., KNAUF, V., & KAJIWARA, T. (2000). Fatty Acid Hydroperoxide Lyase in Tomato Fruits: Cloning and Properties of a Recombinant Enzyme Expressed inEscherichia coli. Bioscience, Biotechnology, and Biochemistry, 64(6), 1189-1196. doi:10.1271/bbb.64.1189Monforte, A. J., & Tanksley, S. D. (2000). Development of a set of near isogenic and backcross recombinant inbred lines containing most of the Lycopersicon hirsutum genome in a L. esculentum genetic background: A tool for gene mapping and gene discovery. Genome, 43(5), 803-813. doi:10.1139/gen-43-5-803Orzaez, D., Medina, A., Torre, S., Fernández-Moreno, J. P., Rambla, J. L., Fernández-del-Carmen, A., … Granell, A. (2009). A Visual Reporter System for Virus-Induced Gene Silencing in Tomato Fruit Based on Anthocyanin Accumulation. Plant Physiology, 150(3), 1122-1134. doi:10.1104/pp.109.139006Rambla, J. L., Alfaro, C., Medina, A., Zarzo, M., Primo, J., & Granell, A. (2015). Tomato fruit volatile profiles are highly dependent on sample processing and capturing methods. Metabolomics, 11(6), 1708-1720. doi:10.1007/s11306-015-0824-5Rambla, J. L., Tikunov, Y. M., Monforte, A. J., Bovy, A. G., & Granell, A. (2013). The expanded tomato fruit volatile landscape. Journal of Experimental Botany, 65(16), 4613-4623. doi:10.1093/jxb/eru128Saliba-Colombani, V., Causse, M., Langlois, D., Philouze, J., & Buret, M. (2001). Genetic analysis of organoleptic quality in fresh market tomato. 1. Mapping QTLs for physical and chemical traits. Theoretical and Applied Genetics, 102(2-3), 259-272. doi:10.1007/s001220051643Salinas, M., Capel, C., Alba, J. M., Mora, B., Cuartero, J., Fernández-Muñoz, R., … Capel, J. (2012). Genetic mapping of two QTL from the wild tomato Solanum pimpinellifolium L. controlling resistance against two-spotted spider mite (Tetranychus urticae Koch). Theoretical and Applied Genetics, 126(1), 83-92. doi:10.1007/s00122-012-1961-0Sefton, M. A., Skouroumounis, G. K., Elsey, G. M., & Taylor, D. K. (2011). Occurrence, Sensory Impact, Formation, and Fate of Damascenone in Grapes, Wines, and Other Foods and Beverages. Journal of Agricultural and Food Chemistry, 59(18), 9717-9746. doi:10.1021/jf201450qShen, J., Tieman, D., Jones, J. B., Taylor, M. G., Schmelz, E., Huffaker, A., … Klee, H. J. (2014). A 13-lipoxygenase, TomloxC, is essential for synthesis of C5 flavour volatiles in tomato. Journal of Experimental Botany, 65(2), 419-428. doi:10.1093/jxb/ert382Sim, S.-C., Durstewitz, G., Plieske, J., Wieseke, R., Ganal, M. W., Van Deynze, A., … Francis, D. M. (2012). Development of a Large SNP Genotyping Array and Generation of High-Density Genetic Maps in Tomato. PLoS ONE, 7(7), e40563. doi:10.1371/journal.pone.0040563Simkin, A. J., Schwartz, S. H., Auldridge, M., Taylor, M. G., & Klee, H. J. (2004). The tomato carotenoid cleavage dioxygenase 1 genes contribute to the formation of the flavor volatiles β-ionone, pseudoionone, and geranylacetone. The Plant Journal, 40(6), 882-892. doi:10.1111/j.1365-313x.2004.02263.xSkouroumounis GK Massywestropp RA Sefton MA Williams PJ . 1993. beta-Damascenone formation in juices and wines. In: Schreier P Winterhalter P , eds. Progress in flavour precursor studies: analysis, generation, biotechnology. Proceedings of the International Conference, Würzburg, Germany, September 30–October 2, 1992, 275–278.Speirs, J., Lee, E., Holt, K., Yong-Duk, K., Steele Scott, N., Loveys, B., & Schuch, W. (1998). Genetic Manipulation of Alcohol Dehydrogenase Levels in Ripening Tomato Fruit Affects the Balance of Some Flavor Aldehydes and Alcohols. Plant Physiology, 117(3), 1047-1058. doi:10.1104/pp.117.3.1047Tadmor, Y., Fridman, E., Gur, A., Larkov, O., Lastochkin, E., Ravid, U., … Lewinsohn, E. (2002). Identification ofmalodorous, a Wild Species Allele Affecting Tomato Aroma That Was Selected against during Domestication. Journal of Agricultural and Food Chemistry, 50(7), 2005-2009. doi:10.1021/jf011237xTieman, D., Bliss, P., McIntyre, L. M., Blandon-Ubeda, A., Bies, D., Odabasi, A. Z., … Klee, H. J. (2012). The Chemical Interactions Underlying Tomato Flavor Preferences. 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A., Taylor, M. G., Bliss, P., Kirst, M., & Klee, H. J. (2006). Identification of loci affecting flavour volatile emissions in tomato fruits. Journal of Experimental Botany, 57(4), 887-896. doi:10.1093/jxb/erj074Tikunov, Y., Lommen, A., de Vos, C. H. R., Verhoeven, H. A., Bino, R. J., Hall, R. D., & Bovy, A. G. (2005). A Novel Approach for Nontargeted Data Analysis for Metabolomics. Large-Scale Profiling of Tomato Fruit Volatiles. Plant Physiology, 139(3), 1125-1137. doi:10.1104/pp.105.068130Tikunov, Y. M., Molthoff, J., de Vos, R. C. H., Beekwilder, J., van Houwelingen, A., van der Hooft, J. J. J., … Bovy, A. G. (2013). NON-SMOKY GLYCOSYLTRANSFERASE1 Prevents the Release of Smoky Aroma from Tomato Fruit. The Plant Cell, 25(8), 3067-3078. doi:10.1105/tpc.113.114231Van Ooijen JW . 2006. JoinMap® 4. Software for the calculation of genetic linkage maps in experimental populations. Wageningen, The Netherlands: Kyazma BV.Vogel, J. T., Tieman, D. M., Sims, C. A., Odabasi, A. Z., Clark, D. 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GoldenBraid: An Iterative Cloning System for Standardized Assembly of Reusable Genetic Modules

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

Sistema para ensamblado de piezas genéticas

La presente invención describe un sistema de ensamblado in vitro para piezas genéticas. El ensamblado de fragmentos de DNA constituye la base de la ingeniería genética y la biología sintética. Para el diseño de nuevos circuitos genéticos, ambas disciplinas tienden hacia la generación de colecciones de piezas genéticas intercambiables y reciclables (es decir, susceptibles de ser utilizadas en laboratorios distintos y para generar combinaciones genéticas distintas), que puedan ser unidas entre sí mediante el uso de un método estándar de ensamblaje. Es particularmente necesario el desarrollo de métodos que permitan gran eficiencia y versatilidad en el ensamblaje de piezas en los rangos que van entre 5 y 50 piezas individuales, ya que la naturaleza modular de las interacciones genéticas hace que buena parte de la ingeniería se desarrolle en torno a diseños genéticos que abarcan estos rangos de tamaño.Peer reviewedConsejo Superior de Investigaciones Científicas, Universidad Politécnica de ValenciaA1 Solicitud de patente con informe sobre el estado de la técnic

Pilot production of SARS-CoV-2 related proteins in plants: a proof of concept for rapid repurposing of indoors farms into biomanufacturing facilities

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 all 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 its Receptor Binding Domain (RBD) 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 therapeutic responses.This work was supported by H2020 EU projects 760331 Newcotiana and 774078 PharmaFactory. S.S. is recipient of a FPI fellowship BIO2016‐78601‐R from the Spanish Ministry of Science and Competitiveness and R.M. is recipient of a GVA fellowship (ACIF/2019/226).N

A coat-independent superinfection exclusion rapidly imposed in Nicotiana benthamiana cells by tobacco mosaic virus is not prevented by depletion of the movement protein

New evidence is emerging which indicates that population variants in plant virus infections are not uniformly distributed along the plant, but structured in a mosaic-like pattern due to limitation to the superinfection imposed by resident viral clones. The mechanisms that prevent the infection of a challenge virus into a previously infected cell, a phenomenon known as superinfection exclusion (SE) or Homologous Interference, are only partially understood. By taking advantage of a deconstructed tobacco mosaic virus (TMV) system, where the capsid protein (CP) gene is replaced by fluorescent proteins, an exclusion mechanism independent of CP was unveiled. Time-course superinfection experiments provided insights into SE dynamics. Initial infection levels affecting less than 10 % of cells led to full immunization in only 48 h, and measurable immunization levels were detected as early as 6 h post-primary infection. Depletion of a functional movement protein (MP) was also seen to slow down, but not to prevent, the SE mechanism. These observations suggest a CP-independent mechanism based on competition for a host-limiting factor, which operates at very low virus concentration. The possible involvement of host factors in SE has interesting implications as it would enable the host to influence the process. © 2013 Springer Science+Business Media Dordrecht.This work was supported by Projects BIO2010-15384 and IPT-2011-0720-010000 from the Spanish Ministry of Economy and Competitiveness.Peer Reviewe

Pilot production of SARS-CoV-2 related proteins in plants: a proof of concept for rapid repurposing of indoors farms into biomanufacturing facilities

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 all 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 its Receptor Binding Domain (RBD) 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 therapeutic responses.This work was supported by H2020 EU projects 760331 Newcotiana and 774078 PharmaFactory. S.S. is recipient of a FPI fellowship BIO2016‐78601‐R from the Spanish Ministry of Science and Competitiveness and R.M. is recipient of a GVA fellowship (ACIF/2019/226).N

Strong and tunable anti-CRISPR/Cas activities in plants

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.Peer reviewe