Combined enhancement of ascorbic acid, β-carotene and zeaxanthin in gene-edited lettuce

Lettuce is widely grown and consumed but provides lower nutritional value compared to other leafy greens, particularly in the essential vitamins A and C. To address this, major control points in carotenoid and ascorbic acid (AsA) production were targeted using a viral-based CRISPR/Cas9 system in the commercial lettuce cultivar 'Noga'. Knockout of lycopene ε-cyclase (LCY-ε), the enzymatic gatekeeper opposing production of β-branch carotenoids, increased β-carotene (provitamin A) levels up to 2.7-fold and facilitated zeaxanthin accumulation up to 4.3 microg/g fresh weight. Chlorophyll fluorescence measurements revealed that photosystem II efficiency was unaffected in LCY-ε mutants, though their non-photochemical quenching (NPQ) capacity decreased at light intensities above 400 micromol m² s^-1. However, the gene-edited plants exhibited normal growth and comparable plant mass, despite the absence of two major lettuce xanthophylls, lutein and lactucaxanthin. Modifications in a regulatory region in the upstream ORF of GDP-L-galactose phosphorylase 1 and 2 (uGGP1 and uGGP2), the rate-limiting enzyme in AsA production, resulted in an average 6.9-fold increase in AsA levels. The mutation in uGGP2 was found to dominantly influence AsA over-accumulation. Knockout lines that combined the mutations in LCY-ε, uGGP1, uGGP2 and in carotenoid cleavage dioxygenase 4a (CCD4a), an isozyme involved in β-carotene degradation in lettuce, exhibited significantly enhanced content of AsA, β-carotene and zeaxanthin. Our results demonstrate the potential of multi-pathway gene editing to 'supercharge' economically important crops such as lettuce as a means to address micronutrient deficiencies in modern diets.