9 research outputs found
GB3.0: a platform for plant bio-design that connects functional DNA elements with associated biological data
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.Spanish Ministry of Economy and Competitiveness [BIO2013-42193-R and BIO2016-78601-R projects to A.G. and D.O.]. Funding for open access charge: Spanish Ministry of Economy and Competitiveness [BIO2013-42193-R and BIO2016-78601-R projects to A.G. and D.O.].Vázquez-Vilar, M.; Quijano-Rubio, A.; Fernández 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. https://doi.org/10.1093/nar/gkw1326S21962209454Dalal, J., Yalamanchili, R., La Hovary, C., Ji, M., Rodriguez-Welsh, M., Aslett, D., … Qu, R. (2015). A novel gateway-compatible binary vector series (PC-GW) for flexible cloning of multiple genes for genetic transformation of plants. Plasmid, 81, 55-62. doi:10.1016/j.plasmid.2015.06.003Cha-aim, K., Fukunaga, T., Hoshida, H., & Akada, R. (2009). Reliable fusion PCR mediated by GC-rich overlap sequences. Gene, 434(1-2), 43-49. doi:10.1016/j.gene.2008.12.014Nour-Eldin, H. H., Geu-Flores, F., & Halkier, B. A. (2010). USER Cloning and USER Fusion: The Ideal Cloning Techniques for Small and Big Laboratories. Methods in Molecular Biology, 185-200. doi:10.1007/978-1-60761-723-5_13Engler, C., Kandzia, R., & Marillonnet, S. (2008). A One Pot, One Step, Precision Cloning Method with High Throughput Capability. PLoS ONE, 3(11), e3647. doi:10.1371/journal.pone.0003647Blake, W. J., Chapman, B. A., Zindal, A., Lee, M. E., Lippow, S. M., & Baynes, B. M. (2010). Pairwise selection assembly for sequence-independent construction of long-length DNA. Nucleic Acids Research, 38(8), 2594-2602. doi:10.1093/nar/gkq123De Paoli, H. C., Tuskan, G. A., & Yang, X. (2016). An innovative platform for quick and flexible joining of assorted DNA fragments. Scientific Reports, 6(1). doi:10.1038/srep19278Engler, C., Gruetzner, R., Kandzia, R., & Marillonnet, S. (2009). Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes. PLoS ONE, 4(5), e5553. doi:10.1371/journal.pone.0005553Weber, E., Engler, C., Gruetzner, R., Werner, S., & Marillonnet, S. (2011). A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLoS ONE, 6(2), e16765. doi:10.1371/journal.pone.0016765Sarrion-Perdigones, A., Falconi, E. E., Zandalinas, S. I., Juárez, P., Fernández-del-Carmen, A., Granell, A., & Orzaez, D. (2011). GoldenBraid: An Iterative Cloning System for Standardized Assembly of Reusable Genetic Modules. PLoS ONE, 6(7), e21622. doi:10.1371/journal.pone.0021622Patron, N. J., Orzaez, D., Marillonnet, S., Warzecha, H., Matthewman, C., Youles, M., … Rogers, C. (2015). Standards for plant synthetic biology: a common syntax for exchange ofDNAparts. New Phytologist, 208(1), 13-19. doi:10.1111/nph.13532Liu, W., & Stewart, C. N. (2015). Plant synthetic biology. Trends in Plant Science, 20(5), 309-317. doi:10.1016/j.tplants.2015.02.004Wang, Y.-H., Wei, K. Y., & Smolke, C. D. (2013). Synthetic Biology: Advancing the Design of Diverse Genetic Systems. Annual Review of Chemical and Biomolecular Engineering, 4(1), 69-102. doi:10.1146/annurev-chembioeng-061312-103351Engler, C., Youles, M., Gruetzner, R., Ehnert, T.-M., Werner, S., Jones, J. D. G., … Marillonnet, S. (2014). A Golden Gate Modular Cloning Toolbox for Plants. ACS Synthetic Biology, 3(11), 839-843. doi:10.1021/sb4001504Canton, B., Labno, A., & Endy, D. (2008). Refinement and standardization of synthetic biological parts and devices. Nature Biotechnology, 26(7), 787-793. doi:10.1038/nbt1413Sarrion-Perdigones, A., Vazquez-Vilar, M., Palaci, J., Castelijns, B., Forment, J., Ziarsolo, P., … Orzaez, D. (2013). GoldenBraid 2.0: A Comprehensive DNA Assembly Framework for Plant Synthetic Biology. PLANT PHYSIOLOGY, 162(3), 1618-1631. doi:10.1104/pp.113.217661Orzaez, 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.109.139006Yoo, S.-D., Cho, Y.-H., & Sheen, J. (2007). Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nature Protocols, 2(7), 1565-1572. doi:10.1038/nprot.2007.199Schneider, C. A., Rasband, W. S., & Eliceiri, K. W. (2012). NIH Image to ImageJ: 25 years of image analysis. Nature Methods, 9(7), 671-675. doi:10.1038/nmeth.2089Gutiérrez, S., Yvon, M., Thébaud, G., Monsion, B., Michalakis, Y., & Blanc, S. (2010). Dynamics of the Multiplicity of Cellular Infection in a Plant Virus. PLoS Pathogens, 6(9), e1001113. doi:10.1371/journal.ppat.1001113Vazquez-Vilar, M., Sarrion-Perdigones, A., Ziarsolo, P., Blanca, J., Granell, A., & Orzaez, D. (2015). Software-Assisted Stacking of Gene Modules Using GoldenBraid 2.0 DNA-Assembly Framework. Plant Functional Genomics, 399-420. doi:10.1007/978-1-4939-2444-8_20Vazquez-Vilar, M., Bernabé-Orts, J. M., Fernandez-del-Carmen, A., Ziarsolo, P., Blanca, J., Granell, A., & Orzaez, D. (2016). A modular toolbox for gRNA–Cas9 genome engineering in plants based on the GoldenBraid standard. Plant Methods, 12(1). doi:10.1186/s13007-016-0101-2Quinn, J. Y., Cox, R. S., Adler, A., Beal, J., Bhatia, S., Cai, Y., … Sauro, H. M. (2015). SBOL Visual: A Graphical Language for Genetic Designs. PLOS Biology, 13(12), e1002310. doi:10.1371/journal.pbio.1002310Thorpe, H. M., Wilson, S. E., & Smith, M. C. M. (2000). Control of directionality in the site-specific recombination system of the Streptomyces phage phiC31. Molecular Microbiology, 38(2), 232-241. doi:10.1046/j.1365-2958.2000.02142.xKhaleel, T., Younger, E., McEwan, A. R., Varghese, A. S., & Smith, M. C. M. (2011). A phage protein that binds φC31 integrase to switch its directionality. Molecular Microbiology, 80(6), 1450-1463. doi:10.1111/j.1365-2958.2011.07696.xOrt, D. R., Merchant, S. S., Alric, J., Barkan, A., Blankenship, R. E., Bock, R., … Zhu, X. G. (2015). Redesigning photosynthesis to sustainably meet global food and bioenergy demand. Proceedings of the National Academy of Sciences, 112(28), 8529-8536. doi:10.1073/pnas.1424031112Smolke, C. D. (2009). Building outside of the box: iGEM and the BioBricks Foundation. Nature Biotechnology, 27(12), 1099-1102. doi:10.1038/nbt1209-1099Cambray, G., Mutalik, V. K., & Arkin, A. P. (2011). Toward rational design of bacterial genomes. Current Opinion in Microbiology, 14(5), 624-630. doi:10.1016/j.mib.2011.08.001Mutalik, V. K., Guimaraes, J. C., Cambray, G., Mai, Q.-A., Christoffersen, M. J., Martin, L., … Arkin, A. P. (2013). Quantitative estimation of activity and quality for collections of functional genetic elements. Nature Methods, 10(4), 347-353. doi:10.1038/nmeth.2403Cambray, G., Guimaraes, J. C., Mutalik, V. K., Lam, C., Mai, Q.-A., Thimmaiah, T., … Endy, D. (2013). Measurement and modeling of intrinsic transcription terminators. Nucleic Acids Research, 41(9), 5139-5148. doi:10.1093/nar/gkt163Mutalik, V. K., Guimaraes, J. C., Cambray, G., Lam, C., Christoffersen, M. J., Mai, Q.-A., … Endy, D. (2013). Precise and reliable gene expression via standard transcription and translation initiation elements. Nature Methods, 10(4), 354-360. doi:10.1038/nmeth.2404Bartley, B., Beal, J., Clancy, K., Misirli, G., Roehner, N., Oberortner, E., … Sauro, H. (2015). Synthetic Biology Open Language (SBOL) Version 2.0.0. Journal of Integrative Bioinformatics, 12(2). doi:10.1515/jib-2015-272Angov, E. (2011). Codon usage: Nature’s roadmap to expression and folding of proteins. Biotechnology Journal, 6(6), 650-659. doi:10.1002/biot.201000332Hillson, N. J., Rosengarten, R. D., & Keasling, J. D. (2011). j5 DNA Assembly Design Automation Software. ACS Synthetic Biology, 1(1), 14-21. doi:10.1021/sb2000116Brazma, A., Hingamp, P., Quackenbush, J., Sherlock, G., Spellman, P., Stoeckert, C., … Vingron, M. (2001). Minimum information about a microarray experiment (MIAME)—toward standards for microarray data. Nature Genetics, 29(4), 365-371. doi:10.1038/ng1201-365Taylor, C. F., Paton, N. W., Lilley, K. S., Binz, P.-A., Julian, R. K., Jones, A. R., … Hermjakob, H. (2007). The minimum information about a proteomics experiment (MIAPE). Nature Biotechnology, 25(8), 887-893. doi:10.1038/nbt1329Yang, Y., Li, R., & Qi, M. (2000). In vivo analysis of plant promoters and transcription factors by agroinfiltration of tobacco leaves. The Plant Journal, 22(6), 543-551. doi:10.1046/j.1365-313x.2000.00760.xKapila, J., De Rycke, R., Van Montagu, M., & Angenon, G. (1997). An Agrobacterium-mediated transient gene expression system for intact leaves. Plant Science, 122(1), 101-108. doi:10.1016/s0168-9452(96)04541-4Schaumberg, K. A., Antunes, M. S., Kassaw, T. K., Xu, W., Zalewski, C. S., Medford, J. I., & Prasad, A. (2015). Quantitative characterization of genetic parts and circuits for plant synthetic biology. Nature Methods, 13(1), 94-100. doi:10.1038/nmeth.3659Dafny-Yelin, M., & Tzfira, T. (2007). Delivery of Multiple Transgenes to Plant Cells. Plant Physiology, 145(4), 1118-1128. doi:10.1104/pp.107.106104TZFIRA, T. (2004). Agrobacterium T-DNA integration: molecules and models. Trends in Genetics, 20(8), 375-383. doi:10.1016/j.tig.2004.06.004De Buck, S., De Wilde, C., Van Montagu, M., & Depicker, A. (2000). Determination of the T-DNA Transfer and the T-DNA Integration Frequencies upon Cocultivation ofArabidopsis thalianaRoot Explants. Molecular Plant-Microbe Interactions, 13(6), 658-665. doi:10.1094/mpmi.2000.13.6.658Konermann, S., Brigham, M. D., Trevino, A. E., Joung, J., Abudayyeh, O. O., Barcena, C., … Zhang, F. (2014). Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature, 517(7536), 583-588. doi:10.1038/nature14136Gilbert, L. A., Horlbeck, M. A., Adamson, B., Villalta, J. E., Chen, Y., Whitehead, E. H., … Weissman, J. S. (2014). Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation. Cell, 159(3), 647-661. doi:10.1016/j.cell.2014.09.029Moore, I., Samalova, M., & Kurup, S. (2006). Transactivated and chemically inducible gene expression in plants. The Plant Journal, 45(4), 651-683. doi:10.1111/j.1365-313x.2006.02660.xButelli, E., Titta, L., Giorgio, M., Mock, H.-P., Matros, A., Peterek, S., … Martin, C. (2008). Enrichment of tomato fruit with health-promoting anthocyanins by expression of select transcription factors. Nature Biotechnology, 26(11), 1301-1308. doi:10.1038/nbt.1506Müller, K., Siegel, D., Rodriguez Jahnke, F., Gerrer, K., Wend, S., Decker, E. L., … Zurbriggen, M. D. (2014). A red light-controlled synthetic gene expression switch for plant systems. Mol. BioSyst., 10(7), 1679-1688. doi:10.1039/c3mb70579jDavuluri, G. R., van Tuinen, A., Fraser, P. D., Manfredonia, A., Newman, R., Burgess, D., … Bowler, C. (2005). Fruit-specific RNAi-mediated suppression of DET1 enhances carotenoid and flavonoid content in tomatoes. Nature Biotechnology, 23(7), 890-895. doi:10.1038/nbt1108Zhang, Y., Butelli, E., Alseekh, S., Tohge, T., Rallapalli, G., Luo, J., … Martin, C. (2015). Multi-level engineering facilitates the production of phenylpropanoid compounds in tomato. Nature Communications, 6(1). doi:10.1038/ncomms9635Ori, N., Juarez, M. T., Jackson, D., Yamaguchi, J., Banowetz, G. M., & Hake, S. (1999). Leaf Senescence Is Delayed in Tobacco Plants Expressing the Maize Homeobox Gene knotted1 under the Control of a Senescence-Activated Promoter. The Plant Cell, 11(6), 1073-1080. doi:10.1105/tpc.11.6.1073Azuma, M., Morimoto, R., Hirose, M., Morita, Y., Hoshino, A., Iida, S., … Shiratake, K. (2015). A petal‐specific
In
MYB
1
promoter from Japanese morning glory: a useful tool for molecular breeding of floricultural crops. Plant Biotechnology Journal, 14(1), 354-363. doi:10.1111/pbi.12389Paine, J. A., Shipton, C. A., Chaggar, S., Howells, R. M., Kennedy, M. J., Vernon, G., … Drake, R. (2005). Improving the nutritional value of Golden Rice through increased pro-vitamin A content. Nature Biotechnology, 23(4), 482-487. doi:10.1038/nbt1082Houmard, N. M., Mainville, J. L., Bonin, C. P., Huang, S., Luethy, M. H., & Malvar, T. M. (2007). High-lysine corn generated by endosperm-specific suppression of lysine catabolism using RNAi. Plant Biotechnology Journal, 5(5), 605-614. doi:10.1111/j.1467-7652.2007.00265.xPadidam, M. (2003). Chemically regulated gene expression in plants. Current Opinion in Plant Biology, 6(2), 169-177. doi:10.1016/s1369-5266(03)00005-0Kinkema, M., Geijskes, R. J., Shand, K., Coleman, H. D., De Lucca, P. C., Palupe, A., … Sainz, M. B. (2013). An improved chemically inducible gene switch that functions in the monocotyledonous plant sugar cane. Plant Molecular Biology, 84(4-5), 443-454. doi:10.1007/s11103-013-0140-
The GB4.0 Platform, an All-In-One Tool for CRISPR/Cas-Based Multiplex Genome Engineering in Plants
[EN] 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.This work had been funded by EU Horizon 2020 Project Newcotiana Grant 760331 and PID2019-108203RB-100 Plan Nacional I+D, Spanish Ministry of Economy and Competitiveness. MV-V was recipient of aGeneralitat Valenciana and Fondo Social Europeo post-doctoral grant. JB-O and SS were recipients of FPI fellowships. CP was recipient of a Santiago Grisolia fellowship (Generalitat Valenciana).Vazquez-Vilar, M.; Garcia-Carpintero, V.; Selma, S.; Bernabé-Orts, JM.; Sánchez-Vicente, J.; Salazar-Sarasua, B.; Ressa, A.... (2021). The GB4.0 Platform, an All-In-One Tool for CRISPR/Cas-Based Multiplex Genome Engineering in Plants. Frontiers in Plant Science. 12:1-14. https://doi.org/10.3389/fpls.2021.6899371141
A modular toolbox for gRNA-Cas9 genome engineering in plants based on the GoldenBraid standard
[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
engineeringThis work has been funded by Grant BIO2013-42193-R from Plan Nacional I + D of the Spanish Ministry of Economy and Competitiveness. Vazquez-Vilar M. is a recipient of a Junta de Ampliacion de Estudios fellowship. Bernabe-Orts J.M. is a recipient of a FPI fellowship. We want to thank Nicola J. Patron and Mark Youles for kindly providing humanCas9 and U6-26 clones. We also want to thank Eugenio Gomez for providing Arabidopsis thaliana genomic DNA and Concha Domingo for providing rice genomic DNA. We also want to thank the COST Action FA1006 for the support in the development of the software tools.Vázquez-Vilar, M.; Bernabé-Orts, JM.; Fernández Del Carmen, MA.; Ziarsolo Areitioaurtena, P.; Blanca Postigo, JM.; Granell Richart, A.; Orzáez Calatayud, DV. (2016). A modular toolbox for gRNA-Cas9 genome engineering in plants based on the GoldenBraid standard. Plant Methods. 12. https://doi.org/10.1186/s13007-016-0101-2S12Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013;8(11):2281–308. doi: 10.1038/nprot.2013.143 .Yang X. Applications of CRISPR-Cas9 mediated genome engineering. Mil Med Res. 2015;2:11. doi: 10.1186/s40779-015-0038-1 .Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell. 2013;153(4):910–8. doi: 10.1016/j.cell.2013.04.025 .Bortesi L, Fischer R. The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnol Adv. 2015;33(1):41–52. doi: 10.1016/j.biotechadv.2014.12.006 .Belhaj K, Chaparro-Garcia A, Kamoun S, Patron NJ, Nekrasov V. Editing plant genomes with CRISPR/Cas9. Curr Opin Biotechnol. 2015;32:76–84. doi: 10.1016/j.copbio.2014.11.007 .Shan Q, Wang Y, Li J, Zhang Y, Chen K, Liang Z, et al. Targeted genome modification of crop plants using a CRISPR-Cas system. Nat Biotechnol. 2013;31(8):686–8. doi: 10.1038/nbt.2650 .Gao J, Wang G, Ma S, Xie X, Wu X, Zhang X, et al. CRISPR/Cas9-mediated targeted mutagenesis in Nicotiana tabacum. Plant Mol Biol. 2015;87(1–2):99–110. doi: 10.1007/s11103-014-0263-0 .Fauser F, Schiml S, Puchta H. Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana. Plant J. 2014;79(2):348–59. doi: 10.1111/tpj.12554 .Schiml S, Fauser F, Puchta H. The CRISPR/Cas system can be used as nuclease for in planta gene targeting and as paired nickases for directed mutagenesis in Arabidopsis resulting in heritable progeny. Plant J. 2014;80(6):1139–50. doi: 10.1111/tpj.12704 .Piatek A, Ali Z, Baazim H, Li L, Abulfaraj A, Al-Shareef S, et al. RNA-guided transcriptional regulation in planta via synthetic dCas9-based transcription factors. Plant Biotechnol J. 2015;13(4):578–89. doi: 10.1111/pbi.12284 .Beerli RR, Barbas CF 3rd. Engineering polydactyl zinc-finger transcription factors. Nat Biotechnol. 2002;20(2):135–41. doi: 10.1038/nbt0202-135 .Bogdanove AJ, Voytas DF. TAL effectors: customizable proteins for DNA targeting. Science. 2011;333(6051):1843–6. doi: 10.1126/science.1204094 .Nielsen AA, Voigt CA. Multi-input CRISPR/Cas genetic circuits that interface host regulatory networks. Mol Syst Biol. 2014;10:763. doi: 10.15252/msb.20145735 .Eeckhaut T, Lakshmanan PS, Deryckere D, Van Bockstaele E, Van Huylenbroeck J. Progress in plant protoplast research. Planta. 2013. doi: 10.1007/s00425-013-1936-7 .Mikami M, Toki S, Endo M. Comparison of CRISPR/Cas9 expression constructs for efficient targeted mutagenesis in rice. Plant Mol Biol. 2015. doi: 10.1007/s11103-015-0342-x .Patron NJ, Orzaez D, Marillonnet S, Warzecha H, Matthewman C, Youles M, et al. Standards for plant synthetic biology: a common syntax for exchange of DNA parts. New Phytol. 2015. doi: 10.1111/nph.13532 .Liu W, Stewart CN Jr. Plant synthetic biology. Trends Plant Sci. 2015;20(5):309–17. doi: 10.1016/j.tplants.2015.02.004 .Sarrion-Perdigones A, Vazquez-Vilar M, Palaci J, Castelijns B, Forment J, Ziarsolo P, et al. GoldenBraid 2.0: a comprehensive DNA assembly framework for plant synthetic biology. Plant Physiol. 2013;162(3):1618–31. doi: 10.1104/pp.113.217661 .Vazquez-Vilar M, Sarrion-Perdigones A, Ziarsolo P, Blanca J, Granell A, Orzaez D. Software-assisted stacking of gene modules using GoldenBraid 2.0 DNA-assembly framework. Methods Mol Biol. 2015;1284:399–420. doi: 10.1007/978-1-4939-2444-8_20 .Duportet X, Wroblewska L, Guye P, Li Y, Eyquem J, Rieders J, et al. A platform for rapid prototyping of synthetic gene networks in mammalian cells. Nucleic Acids Res. 2014;42(21):13440–51. doi: 10.1093/nar/gku1082 .Guo Y, Dong J, Zhou T, Auxillos J, Li T, Zhang W, et al. YeastFab: the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiae. Nucleic Acids Res. 2015;43(13):e88. doi: 10.1093/nar/gkv464 .Engler C, Gruetzner R, Kandzia R, Marillonnet S. Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS ONE. 2009;4(5):e5553. doi: 10.1371/journal.pone.0005553 .Sarrion-Perdigones A, Falconi EE, Zandalinas SI, Juarez P, Fernandez-del-Carmen A, Granell A, et al. GoldenBraid: an iterative cloning system for standardized assembly of reusable genetic modules. PLoS ONE. 2011;6(7):e21622. doi: 10.1371/journal.pone.0021622 .Lei Y, Lu L, Liu HY, Li S, Xing F, Chen LL. CRISPR-P: a web tool for synthetic single-guide RNA design of CRISPR-system in plants. Mol Plant. 2014;7(9):1494–6. doi: 10.1093/mp/ssu044 .Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, et al. RNA-guided human genome engineering via Cas9. Science. 2013;339(6121):823–6. doi: 10.1126/science.1232033 .Li JF, Norville JE, Aach J, McCormack M, Zhang D, Bush J, et al. Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat Biotechnol. 2013;31(8):688–91. doi: 10.1038/nbt.2654 .Bikard D, Jiang W, Samai P, Hochschild A, Zhang F, Marraffini LA. Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Res. 2013;41(15):7429–37. doi: 10.1093/nar/gkt520 .Xie K, Minkenberg B, Yang Y. Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system. Proc Natl Acad Sci USA. 2015;112(11):3570–5. doi: 10.1073/pnas.1420294112 .Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S. A modular cloning system for standardized assembly of multigene constructs. PLoS ONE. 2011;6(2):e16765. doi: 10.1371/journal.pone.0016765 .Sakuma T, Nishikawa A, Kume S, Chayama K, Yamamoto T. Multiplex genome engineering in human cells using all-in-one CRISPR/Cas9 vector system. Sci Rep. 2014;4:5400. doi: 10.1038/srep05400 .Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, et al. A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol Plant. 2015. doi: 10.1016/j.molp.2015.04.007 .Lowder LG, Zhang D, Baltes NJ, Paul JW 3rd, Tang X, Zheng X, et al. A CRISPR/Cas9 toolbox for multiplexed plant genome editing and transcriptional regulation. Plant Physiol. 2015;169(2):971–85. doi: 10.1104/pp.15.00636 .Nekrasov V, Staskawicz B, Weigel D, Jones JD, Kamoun S. Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nat Biotechnol. 2013;31(8):691–3. doi: 10.1038/nbt.2655 .Upadhyay SK, Kumar J, Alok A, Tuli R. RNA-guided genome editing for target gene mutations in wheat. G3. 2013;3(12):2233–8. doi: 10.1534/g3.113.008847 .Senis E, Fatouros C, Grosse S, Wiedtke E, Niopek D, Mueller AK, et al. CRISPR/Cas9-mediated genome engineering: an adeno-associated viral (AAV) vector toolbox. Biotechnol J. 2014;9(11):1402–12. doi: 10.1002/biot.201400046 .Port F, Chen HM, Lee T, Bullock SL. Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila. Proc Natl Acad Sci USA. 2014;111(29):E2967–76. doi: 10.1073/pnas.1405500111 .Xing HL, Dong L, Wang ZP, Zhang HY, Han CY, Liu B, et al. A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol. 2014;14:327. doi: 10.1186/s12870-014-0327-y
Cloning and characterization of theYarrowia lipolytica squalene synthase (SQS1) gene and functional complementation of theSaccharomyces cerevisiae erg9 mutation
Comparative analysis of the Squamosa Promoter Binding-Like (SPL) gene family in Nicotiana benthamiana and Nicotiana tabacum
[EN] SQUAMOSA PROMOTER BINDING-LIKE (SPL) proteins constitute a large family of transcription factors known to play key roles in growth and developmental processes, including juvenile-to-adult and vegetative-to-reproductive phase transitions. This makes SPLs interesting targets for precision breeding in plants of the Nicotiana genus used as e.g. recombinant biofactories. We report the identification of 49 SPL genes in Nicotiana tabacum cv. K326 and 43 SPL genes in Nicotiana benthamiana LAB strain, which were classified into eight phylogenetic groups according to the SPL classification in Arabidopsis. Exon-intron gene structure and DNA-binding domains were highly conserved between homeologues and orthologues. Thirty of the NbSPL genes and 33 of the NtSPL genes were found to be possible targets of microRNA 156. The expression of SPL genes in leaves was analysed by RNA-seq at three different stages, revealing that genes not under miR156 control were in general constitutively expressed at high levels, whereas miR156-regulated genes showed lower expression, often developmentally regulated. We selected the N. benthamiana SPL13_1a gene as target for a CRISPR/Cas9 knock-out experiment. We show here that a full knock-out in this single gene leads to a significant delay in flowering time, a trait that could be exploited to increase biomass for recombinant protein production.This work was supported by grant H2020 - 760331 Newcotiana from the European Commission. C.D and M.V.V are the recipients of fellowships GRISOLIAP/2019/013 and APOSTD/2020/096 from the Generalitat Valenciana (Spain) , respectively.De Paola, C.; García-Carpintero, V.; Vázquez-Vilar, M.; Kaminski, K.; Fernandez-Del-Carmen, A.; Sierro, N.; Ivanov, NV.... (2023). Comparative analysis of the Squamosa Promoter Binding-Like (SPL) gene family in Nicotiana benthamiana and Nicotiana tabacum. Plant Science (Online). 335. https://doi.org/10.1016/j.plantsci.2023.11179733
Evaluation of Unintended Effects in the Composition of Tomatoes Expressing a Human Immunoglobulin A against Rotavirus
The
production of neutralizing immunoglobulin A (IgA) in edible
fruits as a means of oral passive immunization is a promising strategy
for the inexpensive treatment of mucosal diseases. This approach is
based on the assumption that the edible status remains unaltered in
the immunoglobulin-expressing fruit, and therefore extensive purification
is not required for mucosal delivery. However, unintended effects
associated with IgA expression such as toxic secondary metabolites
and protein allergens cannot be dismissed a priori and need to be
investigated. This paper describes a collection of independent transgenic
tomato lines expressing a neutralizing human IgA against rotavirus,
a mucosal pathogen producing severe diarrhea episodes. This collection
was used to evaluate possible unintended effects associated with recombinant
IgA expression. A comparative analysis of protein and secondary metabolite
profiles using wild type lines and other commercial varieties failed
to find unsafe features significantly associated with IgA expression.
Preliminary, the data indicate that formulations derived from IgA
tomatoes are as safe for consumption as equivalent formulations derived
from wild type tomatoes
MOESM1 of A modular toolbox for gRNA–Cas9 genome engineering in plants based on the GoldenBraid standard
Additional file 1: Figure S1. Cloning efficiency of representative Level ≥1 GBelements. Figure S2. Schema of the dead Cas9 transcriptional units tested on the repression and activation experiments
Benchmarking plant diversity of Palaearctic grasslands and other open habitats
Abstract
Aims: Understanding fine-grain diversity patterns across large spatial extents is fundamental for macroecological research and biodiversity conservation. Using the GrassPlot database, we provide benchmarks of fine-grain richness values of Palaearctic open habitats for vascular plants, bryophytes, lichens and complete vegetation (i.e., the sum of the former three groups).
Location: Palaearctic biogeographic realm.
Methods: We used 126,524 plots of eight standard grain sizes from the GrassPlot database: 0.0001, 0.001, 0.01, 0.1, 1, 10, 100 and 1,000 m² and calculated the mean richness and standard deviations, as well as maximum, minimum, median, and first and third quartiles for each combination of grain size, taxonomic group, biome, region, vegetation type and phytosociological class.
Results: Patterns of plant diversity in vegetation types and biomes differ across grain sizes and taxonomic groups. Overall, secondary (mostly semi-natural) grasslands and natural grasslands are the richest vegetation type. The open-access file ”GrassPlot Diversity Benchmarks” and the web tool “GrassPlot Diversity Explorer” are now available online (https://edgg.org/databases/GrasslandDiversityExplorer) and provide more insights into species richness patterns in the Palaearctic open habitats.
Conclusions: The GrassPlot Diversity Benchmarks provide high-quality data on species richness in open habitat types across the Palaearctic. These benchmark data can be used in vegetation ecology, macroecology, biodiversity conservation and data quality checking. While the amount of data in the underlying GrassPlot database and their spatial coverage are smaller than in other extensive vegetation-plot databases, species recordings in GrassPlot are on average more complete, making it a valuable complementary data source in macroecology