Tomato protoplasts as cell target for ribonucleoprotein (RNP)-mediated multiplexed genome editing
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RESEARCH NOTE
Tomato protoplasts as cell target for ribonucleoprotein (RNP)‑mediated multiplexed genome editing Alessandro Nicolia1 · Mariette Andersson2 · Per Hofvander2 · Giovanna Festa1 · Teodoro Cardi1 Received: 21 July 2020 / Accepted: 18 October 2020 © Springer Nature B.V. 2020
Abstract The possibility to produce plants edited in multiple genes by means of DNA-free approaches opens new perspectives for breeding purposes and acceptance of resultant genotypes. In this work, we have explored the polyethylene glycol (PEG)mediated delivery of ribonucleoproteins (RNPs) in tomato protoplasts using a multiplexing approach (i.e. two genes targeted simultaneously using two sgRNAs per gene) for the first time. We have analysed the editing outcome in fully developed green calli and demonstrated that tomato protoplasts are a valid cell target for RNP-mediated multiplexed genome editing with high efficiency. Key message RNP could be applied with high efficiency in a multiplexing genome editing approach in tomato protoplasts. Keywords Tomato · Ribonucleoprotein · Genome editing · Protoplast Abbreviations RNP Ribonucleoprotein HFRA High resolution fragment analysis PEG Polyethylene glycol CRISPR Clustered regularly interspaced short palindromic repeats Cas9 CRISPR associated protein 9 Tomato (Solanum lycopersicum L.) is the most cultivated vegetable worldwide after potato and is grown for both fresh and processing markets. It is considered a model plant for Communicated by Joyce Van Eck. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11240-020-01954-8) contains supplementary material, which is available to authorized users. * Alessandro Nicolia [email protected] 1
Council for Agricultural Research and Economics, Research Centre for Vegetable and Ornamental Crops, Via Cavalleggeri 25, 84098 Pontecagnano, Italy
Department of Plant Breeding, Swedish University of Agricultural Sciences, P.O. Box 101, 23053 Alnarp, Sweden
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research because of its simple genetic nature (2n = 2x = 24), medium-size genome, short generation time, routine transformation technology in place and a high availability of plant genetic resources. With the advent of genome editing techniques and CRISPR/Cas9 in particular, many papers have been published on tomato and other vegetables (Cardi et al. 2017a; Wang et al. 2019), the vast majority relying on Agrobacterium tumefaciens (currently renamed to Rhizobium radiobacter) mediated transformation of multicellular explants for the delivery of editing reagents. In tomato, transformation and shoot regeneration is routinely achieved using cotyledonary explants, while alternative direct approaches, such as protoplast or biolistics-based delivery methods, are either not available or have limited applicability (Cardi et al. 2017b; Van Eck 2017). The delivery in plant cells of in vitro pre-assembled ribonucloprotein (RNP) complexes, constituted of protein (i.e. Cas9) bound to ribonucleic acid (i.e. the single guide RNA, sgRNA)
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