CRISPR/Cas9 directed editing of lycopene epsilon-cyclase modulates metabolic flux for β-carotene biosynthesis in banana fruit

Banana is one of the most economically important fruit crops worldwide. Genetic improvement in banana is a challenging task due to its parthenocarpic nature and triploid genome. Genetic modification of crops via the CRISPR/Cas9 module has emerged as a promising tool to develop important traits. In the present work, a CRISPR/Cas9-based approach was used to develop the beta-carotene-enriched Cavendish banana cultivar (cv.) Grand Naine (AAA genome). The fifth exon of the lycopene epsilon-cyclase (LCY epsilon) gene was targeted. The targeting specificity of the designed guide-RNA was also tested by its ability to create indels in the LCY epsilon gene at the A genome of cv. Rasthali (AAB genome). Sequence analysis revealed multiple types of indels in the genomic region of Grand Naine LCY epsilon (GN-LCY epsilon). Metabolic profiling of the fruit pulp of selected edited lines showed enhanced accumulation of beta-carotene content up to 6-fold (similar to 24 mu g/g) compared with the unedited plants. These lines also showed either an absence or a drastic reduction in the levels of lutein and alpha-carotene, suggesting metabolic reprogramming, without any significant effect on the agro-morphological parameters. In addition, differential expression of carotenoid pathway genes was observed in the edited lines in comparison to unedited plants. Overall, this is the first report in banana to improve nutritional trait by using a precise genome editing approach.