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

Abstract

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.

CRISPR-based editing strategies to rectifyEYA1complex genomic rearrangement linked to haploinsufficiency

“…These include small indels, large deletion, substantial deletions, or inversions 44 . More specifically, it is plausible to posit that the formation of an inversion concurrently negates PAM sites and excises a fraction of the sgRNAs, liberating the cleavage sites 43 .…”

“…Alternatively, when Cas9 nucleases simultaneously cleave at both target loci the subsequent ligation could induce a large deletion, inversion, or translocation of the genomic fragment intervening between the target sites. Documented instances of such inversions suggest a scenario where both sgRNAs interact with the Cas protein, forming a single complex capable of excising gene segments 43 . This process facilitates the complex in generating a nick in both sgRNAs, thereby presenting three potential pathways for DNA repair.…”

“…The copyright holder for this this version posted November 15, 2023. ; https://doi.org/10.1101/2023.11.14.23298277 doi: medRxiv preprint suggest a scenario where both sgRNAs interact with the Cas protein, forming a single complex capable of excising gene segments 43 . This process facilitates the complex in generating a nick in both sgRNAs, thereby presenting three potential pathways for DNA repair.…”

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

“…The genome editing of β-cyclase was also used to develop the β-carotene-enriched banana variety [ 91 ]. In our case, in Citrus , the knockout of β-LCY2 , through a dual-single guide approach, was aimed at producing loss-of-function mutants to induce lycopene accumulation in anthocyanin-rich sweet oranges [ 92 ]. For the first time, genome editing has been adopted to improve citrus fruit qualitative traits.…”