[{"status":"public","_id":"17233","doi":"10.21769/BioProtoc.5029","date_updated":"2025-03-06T10:28:18Z","volume":14,"date_created":"2024-07-14T22:01:11Z","article_type":"original","language":[{"iso":"eng"}],"oa":1,"user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","issue":"13","acknowledgement":"This work was supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (project 772103-BRIDGING to E.M.B.).","oa_version":"Published Version","month":"07","has_accepted_license":"1","date_published":"2024-07-05T00:00:00Z","quality_controlled":"1","department":[{"_id":"MiSi"}],"publisher":"Bio-Protocol","abstract":[{"text":"CRISPR-Cas9 technology has become an essential tool for plant genome editing. Recent advancements have significantly improved the ability to target multiple genes simultaneously within the same genetic background through various strategies. Additionally, there has been significant progress in developing methods for inducible or tissue-specific editing. These advancements offer numerous possibilities for tailored genome modifications. Building upon existing research, we have developed an optimized and modular strategy allowing the targeting of several genes simultaneously in combination with the synchronized expression of the Cas9 endonuclease in the egg cell. This system allows significant editing efficiency while avoiding mosaicism. In addition, the versatile system we propose allows adaptation to inducible and/or tissue-specific edition according to the promoter chosen to drive the expression of the Cas9 gene. Here, we describe a step-by-step protocol for generating the binary vector necessary for establishing Arabidopsis edited lines using a versatile cloning strategy that combines Gateway® and Golden Gate technologies. We describe a versatile system that allows the cloning of as many guides as needed to target DNA, which can be multiplexed into a polycistronic gene and combined in the same construct with sequences for the expression of the Cas9 endonuclease. The expression of Cas9 is controlled by selecting from among a collection of promoters, including constitutive, inducible, ubiquitous, or tissue-specific promoters. Only one vector containing the polycistronic gene (tRNA-sgRNA) needs to be constructed. For that, sgRNA (composed of protospacers chosen to target the gene of interest and sgRNA scaffold) is cloned in tandem with the pre-tRNA sequence. Then, a single recombination reaction is required to assemble the promoter, the zCas9 coding sequence, and the tRNA-gRNA polycistronic gene. Each element is cloned in an entry vector and finally assembled according to the Multisite Gateway® Technology. Here, we detail the process to express zCas9 under the control of egg cell promoter fused to enhancer sequence (EC1.2en-EC1.1p) and to simultaneously target two multiple C2 domains and transmembrane region protein genes (MCTP3 and MCTP4, respectively at3g57880 and at1g51570), using one or two sgRNA per gene.","lang":"eng"}],"citation":{"mla":"LI, ZIQIANG, et al. “Versatile Cloning Strategy for Efficient Multigene Editing in Arabidopsis.” <i>Bio-Protocol</i>, vol. 14, no. 13, e5029, Bio-Protocol, 2024, doi:<a href=\"https://doi.org/10.21769/BioProtoc.5029\">10.21769/BioProtoc.5029</a>.","short":"Z. LI, J. Huard, E.M. Bayer, V. Wattelet-Boyer, Bio-Protocol 14 (2024).","ieee":"Z. LI, J. Huard, E. M. Bayer, and V. Wattelet-Boyer, “Versatile cloning strategy for efficient multigene editing in Arabidopsis,” <i>Bio-protocol</i>, vol. 14, no. 13. Bio-Protocol, 2024.","apa":"LI, Z., Huard, J., Bayer, E. M., &#38; Wattelet-Boyer, V. (2024). Versatile cloning strategy for efficient multigene editing in Arabidopsis. <i>Bio-Protocol</i>. Bio-Protocol. <a href=\"https://doi.org/10.21769/BioProtoc.5029\">https://doi.org/10.21769/BioProtoc.5029</a>","chicago":"LI, ZIQIANG, Jennifer Huard, Emmanuelle M. Bayer, and Valérie Wattelet-Boyer. “Versatile Cloning Strategy for Efficient Multigene Editing in Arabidopsis.” <i>Bio-Protocol</i>. Bio-Protocol, 2024. <a href=\"https://doi.org/10.21769/BioProtoc.5029\">https://doi.org/10.21769/BioProtoc.5029</a>.","ama":"LI Z, Huard J, Bayer EM, Wattelet-Boyer V. Versatile cloning strategy for efficient multigene editing in Arabidopsis. <i>Bio-protocol</i>. 2024;14(13). doi:<a href=\"https://doi.org/10.21769/BioProtoc.5029\">10.21769/BioProtoc.5029</a>","ista":"LI Z, Huard J, Bayer EM, Wattelet-Boyer V. 2024. Versatile cloning strategy for efficient multigene editing in Arabidopsis. Bio-protocol. 14(13), e5029."},"title":"Versatile cloning strategy for efficient multigene editing in Arabidopsis","type":"journal_article","publication_status":"published","year":"2024","intvolume":"        14","article_number":"e5029","file_date_updated":"2024-07-16T06:16:11Z","scopus_import":"1","publication":"Bio-protocol","external_id":{"pmid":["39007160"]},"article_processing_charge":"Yes","license":"https://creativecommons.org/licenses/by/4.0/","file":[{"content_type":"application/pdf","relation":"main_file","file_size":2896048,"success":1,"checksum":"c8671c0ad483da6407cb16cc3fef1990","file_id":"17242","access_level":"open_access","date_created":"2024-07-16T06:16:11Z","date_updated":"2024-07-16T06:16:11Z","file_name":"2024_BioProtocol_Li.pdf","creator":"dernst"}],"ddc":["570"],"publication_identifier":{"eissn":["2331-8325"]},"tmp":{"legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode","name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)","image":"/images/cc_by.png","short":"CC BY (4.0)"},"author":[{"full_name":"Li, Ziqiang","id":"922e68bb-1727-11ee-857c-966e8cc1b6c3","last_name":"Li","first_name":"Ziqiang"},{"full_name":"Huard, Jennifer","first_name":"Jennifer","last_name":"Huard"},{"first_name":"Emmanuelle M.","last_name":"Bayer","full_name":"Bayer, Emmanuelle M."},{"first_name":"Valérie","last_name":"Wattelet-Boyer","full_name":"Wattelet-Boyer, Valérie"}],"day":"05","pmid":1},{"year":"2018","publication_status":"published","intvolume":"         8","corr_author":"1","file_date_updated":"2020-07-14T12:46:29Z","publication":"Bio-protocol","article_processing_charge":"No","pubrep_id":"970","file":[{"file_size":11352389,"checksum":"6644ba698206eda32b0abf09128e63e3","content_type":"application/pdf","relation":"main_file","date_updated":"2020-07-14T12:46:29Z","date_created":"2018-12-12T10:17:43Z","file_name":"IST-2018-970-v1+1_2018_Lanxin_Real-time_analysis.pdf","creator":"system","file_id":"5299","access_level":"open_access"}],"ddc":["576","581"],"project":[{"name":"International IST Doctoral Program","_id":"2564DBCA-B435-11E9-9278-68D0E5697425","call_identifier":"H2020","grant_number":"665385"}],"publication_identifier":{"eissn":["2331-8325"]},"ec_funded":1,"tmp":{"legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode","name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)","image":"/images/cc_by.png","short":"CC BY (4.0)"},"publist_id":"7381","author":[{"full_name":"Li, Lanxin","id":"367EF8FA-F248-11E8-B48F-1D18A9856A87","first_name":"Lanxin","orcid":"0000-0002-5607-272X","last_name":"Li"},{"id":"2B819732-F248-11E8-B48F-1D18A9856A87","full_name":"Krens, Gabriel","first_name":"Gabriel","orcid":"0000-0003-4761-5996","last_name":"Krens"},{"last_name":"Fendrych","orcid":"0000-0002-9767-8699","first_name":"Matyas","id":"43905548-F248-11E8-B48F-1D18A9856A87","full_name":"Fendrych, Matyas"},{"first_name":"Jirí","orcid":"0000-0002-8302-7596","last_name":"Friml","id":"4159519E-F248-11E8-B48F-1D18A9856A87","full_name":"Friml, Jirí"}],"related_material":{"record":[{"status":"public","id":"10083","relation":"dissertation_contains"}]},"day":"05","status":"public","_id":"442","doi":"10.21769/BioProtoc.2685","date_updated":"2026-05-26T22:31:19Z","volume":8,"date_created":"2018-12-11T11:46:30Z","article_type":"original","language":[{"iso":"eng"}],"user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","oa":1,"issue":"1","oa_version":"Published Version","acknowledgement":"This protocol was adapted from Fendrych et al., 2016. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie Grant Agreement No. 665385, and Austrian Science Fund (FWF) [M 2128-B21]. ","month":"01","has_accepted_license":"1","date_published":"2018-01-05T00:00:00Z","quality_controlled":"1","department":[{"_id":"JiFr"},{"_id":"Bio"}],"publisher":"Bio-protocol","abstract":[{"lang":"eng","text":"The rapid auxin-triggered growth of the Arabidopsis hypocotyls involves the nuclear TIR1/AFB-Aux/IAA signaling and is accompanied by acidification of the apoplast and cell walls (Fendrych et al., 2016). Here, we describe in detail the method for analysis of the elongation and the TIR1/AFB-Aux/IAA-dependent auxin response in hypocotyl segments as well as the determination of relative values of the cell wall pH."}],"citation":{"chicago":"Li, Lanxin, Gabriel Krens, Matyas Fendrych, and Jiří Friml. “Real-Time Analysis of Auxin Response, Cell Wall PH and Elongation in Arabidopsis Thaliana Hypocotyls.” <i>Bio-Protocol</i>. Bio-protocol, 2018. <a href=\"https://doi.org/10.21769/BioProtoc.2685\">https://doi.org/10.21769/BioProtoc.2685</a>.","short":"L. Li, G. Krens, M. Fendrych, J. Friml, Bio-Protocol 8 (2018).","mla":"Li, Lanxin, et al. “Real-Time Analysis of Auxin Response, Cell Wall PH and Elongation in Arabidopsis Thaliana Hypocotyls.” <i>Bio-Protocol</i>, vol. 8, no. 1, Bio-protocol, 2018, doi:<a href=\"https://doi.org/10.21769/BioProtoc.2685\">10.21769/BioProtoc.2685</a>.","apa":"Li, L., Krens, G., Fendrych, M., &#38; Friml, J. (2018). Real-time analysis of auxin response, cell wall pH and elongation in Arabidopsis thaliana Hypocotyls. <i>Bio-Protocol</i>. Bio-protocol. <a href=\"https://doi.org/10.21769/BioProtoc.2685\">https://doi.org/10.21769/BioProtoc.2685</a>","ieee":"L. Li, G. Krens, M. Fendrych, and J. Friml, “Real-time analysis of auxin response, cell wall pH and elongation in Arabidopsis thaliana Hypocotyls,” <i>Bio-protocol</i>, vol. 8, no. 1. Bio-protocol, 2018.","ista":"Li L, Krens G, Fendrych M, Friml J. 2018. Real-time analysis of auxin response, cell wall pH and elongation in Arabidopsis thaliana Hypocotyls. Bio-protocol. 8(1).","ama":"Li L, Krens G, Fendrych M, Friml J. Real-time analysis of auxin response, cell wall pH and elongation in Arabidopsis thaliana Hypocotyls. <i>Bio-protocol</i>. 2018;8(1). doi:<a href=\"https://doi.org/10.21769/BioProtoc.2685\">10.21769/BioProtoc.2685</a>"},"title":"Real-time analysis of auxin response, cell wall pH and elongation in Arabidopsis thaliana Hypocotyls","type":"journal_article"},{"publication":"Bio-protocol","external_id":{"pmid":["27331080"]},"article_processing_charge":"No","intvolume":"         5","publication_status":"published","year":"2015","OA_type":"gold","publist_id":"6816","day":"20","pmid":1,"author":[{"first_name":"Peter","last_name":"Marhavy","orcid":"0000-0001-5227-5741","id":"3F45B078-F248-11E8-B48F-1D18A9856A87","full_name":"Marhavy, Peter"},{"orcid":"0000-0002-8510-9739","last_name":"Benková","first_name":"Eva","id":"38F4F166-F248-11E8-B48F-1D18A9856A87","full_name":"Benková, Eva"}],"publication_identifier":{"eissn":["2331-8325"]},"tmp":{"legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode","name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)","image":"/images/cc_by.png","short":"CC BY (4.0)"},"acknowledgement":"European Research Council with a Starting Independent Research grant: ERC-2007-Stg-207362-HCPO, Czech Science Foundation: GA13-39982S\r\nWe thank Matyas Fendrych for critical reading and comments. The protocol was developed based on previously published work of De Rybel et al. (2010) and Laskowski et al. (2008). ","oa_version":"Published Version","issue":"8","DOAJ_listed":"1","month":"04","date_created":"2018-12-11T11:48:44Z","date_updated":"2026-05-19T09:39:29Z","volume":5,"doi":"10.21769/BioProtoc.1446","_id":"832","extern":"1","status":"public","oa":1,"user_id":"ba8df636-2132-11f1-aed0-ed93e2281fdd","language":[{"iso":"eng"}],"article_type":"original","OA_place":"publisher","citation":{"chicago":"Marhavý, Peter, and Eva Benková. “Real Time Analysis of Lateral Root Organogenesis in Arabidopsis.” <i>Bio-Protocol</i>. Bio-protocol LLC, 2015. <a href=\"https://doi.org/10.21769/BioProtoc.1446\">https://doi.org/10.21769/BioProtoc.1446</a>.","apa":"Marhavý, P., &#38; Benková, E. (2015). Real time analysis of lateral root organogenesis in arabidopsis. <i>Bio-Protocol</i>. Bio-protocol LLC. <a href=\"https://doi.org/10.21769/BioProtoc.1446\">https://doi.org/10.21769/BioProtoc.1446</a>","mla":"Marhavý, Peter, and Eva Benková. “Real Time Analysis of Lateral Root Organogenesis in Arabidopsis.” <i>Bio-Protocol</i>, vol. 5, no. 8, Bio-protocol LLC, 2015, doi:<a href=\"https://doi.org/10.21769/BioProtoc.1446\">10.21769/BioProtoc.1446</a>.","short":"P. Marhavý, E. Benková, Bio-Protocol 5 (2015).","ieee":"P. Marhavý and E. Benková, “Real time analysis of lateral root organogenesis in arabidopsis,” <i>Bio-protocol</i>, vol. 5, no. 8. Bio-protocol LLC, 2015.","ista":"Marhavý P, Benková E. 2015. Real time analysis of lateral root organogenesis in arabidopsis. Bio-protocol. 5(8).","ama":"Marhavý P, Benková E. Real time analysis of lateral root organogenesis in arabidopsis. <i>Bio-protocol</i>. 2015;5(8). doi:<a href=\"https://doi.org/10.21769/BioProtoc.1446\">10.21769/BioProtoc.1446</a>"},"type":"journal_article","title":"Real time analysis of lateral root organogenesis in arabidopsis","quality_controlled":"1","main_file_link":[{"open_access":"1","url":"https://doi.org/10.21769/BioProtoc.1446"}],"date_published":"2015-04-20T00:00:00Z","abstract":[{"text":"Plants maintain capacity to form new organs such as leaves, flowers, lateral shoots and roots throughout their postembryonic lifetime. Lateral roots (LRs) originate from a few pericycle cells that acquire attributes of founder cells (FCs), undergo series of anticlinal divisions, and give rise to a few short initial cells. After initiation, coordinated cell division and differentiation occur, giving rise to lateral root primordia (LRP). Primordia continue to grow, emerge through the cortex and epidermal layers of the primary root, and finally a new apical meristem is established taking over the responsibility for growth of mature lateral roots [for detailed description of the individual stages of lateral root organogenesis see Malamy and Benfey (1997)]. To examine this highly dynamic developmental process and to investigate a role of various hormonal, genetic and environmental factors in the regulation of lateral root organogenesis, the real time imaging based analyses represent extremely powerful tools (Laskowski et al., 2008; De Smet et al., 2012; Marhavy et al., 2013 and 2014). Herein, we describe a protocol for real time lateral root primordia (LRP) analysis, which enables the monitoring of an onset of the specific gene expression and subcellular protein localization during primordia organogenesis, as well as the evaluation of the impact of genetic and environmental perturbations on LRP organogenesis.","lang":"eng"}],"publisher":"Bio-protocol LLC"}]