Generation of Transfer-DNA-Free Base-Edited Citrus Plants

Alquézar, Berta; Bennici, Stefania; Carmona, Lourdes; Gentile, Alessandra; Peña, Leandro

doi:10.3389/fpls.2022.835282

Cited by 16 publications

(18 citation statements)

References 47 publications

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“…In citrus, the few studies that have applied CRISPR/Cas9 technology used the Golden Gate strategy. This approach has been adopted to introduce resistance to Xanthomonas citri (the causal agent of citrus canker disease) to susceptible citrus species ( Jia et al., 2016 ; Jia et al., 2017 ), to produce a double thorn phenotype in ‘Carrizo’ citrange ( Zhang et al., 2021b ), and to induce resistance against the herbicide imazapyr ( Alquézar et al., 2022 ).…”

Section: Discussionmentioning

confidence: 99%

A dual sgRNA-directed CRISPR/Cas9 construct for editing the fruit-specific β-cyclase 2 gene in pigmented citrus fruits

Salonia

Ciacciulli²,

Pappalardo³

et al. 2022

Front. Plant Sci.

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CRISPR/Cas9 genome editing is a modern biotechnological approach used to improve plant varieties, modifying only one or a few traits of a specific variety. However, this technology cannot be easily used to improve fruit quality traits in citrus, due to the lack of knowledge of key genes, long juvenile stage, and the difficulty regenerating whole plants of specific varieties. Here, we introduce a genome editing approach with the aim of producing citrus plantlets whose fruits contain both lycopene and anthocyanins. Our method employs a dual single guide RNA (sgRNA)-directed genome editing approach to knockout the fruit-specific β-cyclase 2 gene, responsible for the conversion of lycopene to beta-carotene. The gene is targeted by two sgRNAs simultaneously to create a large deletion, as well as to induce point mutations in both sgRNA targets. The EHA105 strain of Agrobacterium tumefaciens was used to transform five different anthocyanin-pigmented sweet oranges, belonging to the Tarocco and Sanguigno varietal groups, and ‘Carrizo’ citrange, a citrus rootstock as a model for citrus transformation. Among 58 plantlets sequenced in the target region, 86% of them were successfully edited. The most frequent mutations were deletions (from -1 to -74 nucleotides) and insertions (+1 nucleotide). Moreover, a novel event was identified in six plantlets, consisting of the inversion of the region between the two sgRNAs. For 20 plantlets in which a single mutation occurred, we excluded chimeric events. Plantlets did not show an altered phenotype in vegetative tissues. To the best of our knowledge, this work represents the first example of the use of a genome editing approach to potentially improve qualitative traits of citrus fruit.

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Section: Discussionmentioning

confidence: 99%

A dual sgRNA-directed CRISPR/Cas9 construct for editing the fruit-specific β-cyclase 2 gene in pigmented citrus fruits

Salonia

Ciacciulli²,

Pappalardo³

et al. 2022

Front. Plant Sci.

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“…We first investigated if we could obtain transgene-free, gene-edited tomato in the T0 generation by base-editing SlALS1 (Solyc03g044330) alone. Previous studies suggested such a possibility, but the putative transgene-free plants were not confirmed by whole genome sequencing 10,15,28 . Here, we constructed the CBE-Cas12a-GFP-SlALS1 construct to edit the SlALS1 gene using CBE to target the proline residue at position 186 (Pro186) (Fig.…”

Section: Als Gene and Gene Of Interestmentioning

confidence: 94%

Efficient transgene-free genome editing in plants in the T0 generation based on a co-editing strategy

Huang

Jia

et al. 2023

Preprint

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Transgene-free genome editing of plants in the T0 generation is highly desirable but challenging, especially in perennials and vegetatively propagated plants. Here, we investigated the co-editing strategy for generating transgene-free, gene-edited plants viaAgrobacterium-mediated transient expression of cytosine base editor (CBE)/gRNA-Cas12a/crRNA-GFPin planta. Specifically, CBE/gRNA was used to base edit theALSgene to confer resistance to herbicide chlorsulfuron as a selection marker, which has no negative effects on plant phenotypes; Cas12a/crRNA was used for editing genes(s) of interest; GFP was used for selecting transgene-free transformants. Using this approach, transgene-free genome-edited plants were efficiently generated for various genes (either individual or multiplex) in tomato, tobacco, potato, and citrus in the T0 generation. The biallelic/homozygous transgene-free mutation rates for target genes among herbicide-resistant transformants ranged from 8% to 50%. Whole genome sequencing further confirmed transgene-free and absence of off-target mutations in the edited plants. The co-editing strategy is efficient for generating transgene-free, genome-edited plants in the T0 generation, thus being a potent tool for plant genetic improvement.

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“…In Carrizo citrange, a genome-editing approach was developed to produce a double thorn phenotype [ 80 ]. Very recently, Alquézar and colleagues [ 81 ] induced through base editing resistance against the herbicide selection agent imazapyr, bypassing the limitations of the presence of the transgene for the selection and obtaining transgene free plants.…”

Section: Improving Citrus Fruit Quality By Ngt Approaches—the ...mentioning

confidence: 99%

The Role of Italy in the Use of Advanced Plant Genomic Techniques on Fruit Trees: State of the Art and Future Perspectives

Nerva

Costa

Ciacciulli³

et al. 2023

IJMS

Self Cite

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Climate change is deeply impacting the food chain production, lowering quality and yield. In this context, the international scientific community has dedicated many efforts to enhancing resilience and sustainability in agriculture. Italy is among the main European producers of several fruit trees; therefore, national research centers and universities undertook several initiatives to maintain the specificity of the ‘Made in Italy’ label. Despite their importance, fruit crops are suffering from difficulties associated with the conventional breeding approaches, especially in terms of financial commitment, land resources availability, and long generation times. The ‘new genomic techniques’ (NGTs), renamed in Italy as ‘technologies for assisted evolution’ (TEAs), reduce the time required to obtain genetically improved cultivars while precisely targeting specific DNA sequences. This review aims to illustrate the role of the Italian scientific community in the use of NGTs, with a specific focus on Citrus, grapevine, apple, pear, chestnut, strawberry, peach, and kiwifruit. For each crop, the key genes and traits on which the scientific community is working, as well as the technological improvements and advancements on the regeneration of local varieties, are presented. Lastly, a focus is placed on the legal aspects in the European and in Italian contexts.

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Generation of Transfer-DNA-Free Base-Edited Citrus Plants

Cited by 16 publications

References 47 publications

A dual sgRNA-directed CRISPR/Cas9 construct for editing the fruit-specific β-cyclase 2 gene in pigmented citrus fruits

A dual sgRNA-directed CRISPR/Cas9 construct for editing the fruit-specific β-cyclase 2 gene in pigmented citrus fruits

Efficient transgene-free genome editing in plants in the T0 generation based on a co-editing strategy

The Role of Italy in the Use of Advanced Plant Genomic Techniques on Fruit Trees: State of the Art and Future Perspectives

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