Efficient CRISPR/Cas9-based genome editing in carrot cells

Klimek-Chodacka, Magdalena; Oleszkiewicz, Tomasz; Lowder, Levi G.; Qi, Yiping; Barański, Rafał

doi:10.1007/s00299-018-2252-2

Cited by 129 publications

(60 citation statements)

References 52 publications

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“…It can also serve as a source of released cells and protoplasts, and is amenable to genetic transformation (Baranski 2008). These features make carrot callus a convenient laboratory-based model system that has been recently demonstrated for precise gene editing purposes (Klimek-Chodacka et al 2018). Genetically modified callus was also valuable for studying physiological processes and their molecular control in other species, including carotenoid biosynthesis, regulation and accumulation (Kim et al 2013; Schaub et al 2018).…”

Section: Introductionmentioning

confidence: 99%

Unique chromoplast organisation and carotenoid gene expression in carotenoid-rich carrot callus

Oleszkiewicz

Klimek-Chodacka

Milewska‐Hendel

et al. 2018

Planta

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Main conclusionThe new model orange callus line, similar to carrot root, was rich in carotenoids due to altered expression of some carotenogenesis-associated genes and possessed unique diversity of chromoplast ultrastructure.Callus induced from carrot root segments cultured in vitro is usually pale yellow (p-y) and poor in carotenoids. A unique, non-engineered callus line of dark orange (d-o) colour was developed in this work. The content of carotenoid pigments in d-o callus was at the same level as in an orange carrot storage root and nine-fold higher than in p-y callus. Carotenoids accumulated mainly in abundant crystalline chromoplasts that are also common in carrot root but not in p-y callus. Using transmission electron microscopy, other types of chromoplasts were also found in d-o callus, including membranous chromoplasts rarely identified in plants and not observed in carrot root until now. At the transcriptional level, most carotenogenesis-associated genes were upregulated in d-o callus in comparison to p-y callus, but their expression was downregulated or unchanged when compared to root tissue. Two pathway steps were critical and could explain the massive carotenoid accumulation in this tissue. The geranylgeranyl diphosphate synthase gene involved in the biosynthesis of carotenoid precursors was highly expressed, while the β-carotene hydroxylase gene involved in β-carotene conversion to downstream xanthophylls was highly repressed. Additionally, paralogues of these genes and phytoene synthase were differentially expressed, indicating their tissue-specific roles in carotenoid biosynthesis and metabolism. The established system may serve as a novel model for elucidating plastid biogenesis that coincides with carotenogenesis.Electronic supplementary materialThe online version of this article (10.1007/s00425-018-2988-5) contains supplementary material, which is available to authorized users.

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Section: Introductionmentioning

confidence: 99%

Unique chromoplast organisation and carotenoid gene expression in carotenoid-rich carrot callus

Oleszkiewicz

Klimek-Chodacka

Milewska‐Hendel

et al. 2018

Planta

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“…Clustered regularly interspaced short palindromic repeats (CRISPR) along with CRISPR-associated proteins (Cas) acts as a type of adaptive immunity system in prokaryotes and provides sequence-specific protection against foreign DNA or RNA [28]. In general, the CRISPR/Cas9 technology is based on the location and identification of foreign DNA sequences by small guide RNAs (gRNA) and its cleavage by an associated DNA endonuclease (Cas9).…”

Section: Crispr/cas9 System: Toward Genome Editing In Plant In Vitromentioning

confidence: 99%

“…In order to demonstrate the application of the CRISPR/Cas9 system, a carrot cell culture was used by Klimek-Chodacka et al [28]. The gene F3H, encoding flavonone-3-hydroxylase (F3H; EC 1.14.11.9) in the anthocyanin pathway (expressing a purple-colored carrot callus), was blocked by multiplexing CRISPR/Cas9 vectors.…”

Section: Crispr/cas9 System: Toward Genome Editing In Plant In Vitromentioning

confidence: 99%

Green (cell) factories for advanced production of plant secondary metabolites

Marchev

Yordanova

Georgiev

2020

Critical Reviews in Biotechnology

113

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For centuries plants have been intensively utilized as reliable sources of food, flavoring, agrochemical and pharmaceutical ingredients. However, plant natural habitats are being rapidly lost due to climate change and agriculture. Plant biotechnology offers a sustainable method for the bioproduction of plant secondary metabolites using plant in vitro systems. The unique structural features of plant-derived secondary metabolites, such as their safety profile, multi-target spectrum and "metabolite likeness," have led to the establishment of many plant-derived drugs, comprising approximately a quarter of all drugs approved by the Food and Drug Administration and/or European Medicinal Agency. However, there are still many challenges to overcome to enhance the production of these metabolites from plant in vitro systems and establish a sustainable large-scale biotechnological process. These challenges are due to the peculiarities of plant cell metabolism, the complexity of plant secondary metabolite pathways, and the correct selection of bioreactor systems and bioprocess optimization. In this review, we present an integrated overview of the possible avenues for enhancing the biosynthesis of high-value marketable molecules produced by plant in vitro systems. These include metabolic engineering and CRISPR/Cas9 technology for the regulation of plant metabolism through overexpression/repression of single or multiple structural genes or transcriptional factors. The use of NMR-based metabolomics for monitoring metabolite concentrations and additionally as a tool to study the dynamics of plant cell metabolism and nutritional management is discussed here. Different types of bioreactor systems, their modification and optimal process parameters for the labor industrial-scale production of plant secondary metabolites are specified.

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“…Disease resistance through genome editing is also an emerging area of research in vegetables and is beginning to attract attention now that the CRISPR/Cas system has become regular for the genome editing of several plant species. A tomato plant resistant to powdery mildew disease was generated by utilizing two sgRNAs that introduced specific mutations (48-bp deletion in a homozygous configuration) in the MLO1 locus, the major contributor to susceptibility to the fungal pathogen Oidium neolycopersici [123]. Broad-spectrum bacterial disease resistant tomato plants were also obtained by editing the tomato SlDMR6-1 (downy mildew resistance 6 gene) ortholog [138].…”

Section: Genome Editing In Vegetable Cropsmentioning

confidence: 99%

Development of Improved Fruit, Vegetable, and Ornamental Crops Using the CRISPR/Cas9 Genome Editing Technique

Corte

Mahmoud

Moraes

et al. 2019

Plants

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Horticultural crops, including fruit, vegetable, and ornamental plants are an important component of the agriculture production systems and play an important role in sustaining human life. With a steady growth in the world's population and the consequent need for more food, sustainable and increased fruit and vegetable crop production is a major challenge to guarantee future food security. Although conventional breeding techniques have significantly contributed to the development of important varieties, new approaches are required to further improve horticultural crop production. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has emerged as a valuable genome-editing tool able to change DNA sequences at precisely chosen loci. The CRISPR/Cas9 system was developed based on the bacterial adaptive immune system and comprises of an endonuclease guided by one or more single-guide RNAs to generate double-strand breaks. These breaks can then be repaired by the natural cellular repair mechanisms, during which genetic mutations are introduced. In a short time, the CRISPR/Cas9 system has become a popular genome-editing technique, with numerous examples of gene mutation and transcriptional regulation control in both model and crop plants. In this review, various aspects of the CRISPR/Cas9 system are explored, including a general presentation of the function of the CRISPR/Cas9 system in bacteria and its practical application as a biotechnological tool for editing plant genomes, particularly in horticultural crops.

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Efficient CRISPR/Cas9-based genome editing in carrot cells

Cited by 129 publications

References 52 publications

Unique chromoplast organisation and carotenoid gene expression in carotenoid-rich carrot callus

Unique chromoplast organisation and carotenoid gene expression in carotenoid-rich carrot callus

Green (cell) factories for advanced production of plant secondary metabolites

Development of Improved Fruit, Vegetable, and Ornamental Crops Using the CRISPR/Cas9 Genome Editing Technique

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