CRISPR-Cas9 Mediated Anthocyanin Biofortification in Tomato (Solanum lycopersicum): A Precision Genome Editing Approach

Dietary anthocyanins are vital antioxidants with well-documented health benefits, offering protection against chronic diseases such as cardiovascular disorders, metabolic syndrome, and neurodegenerative conditions. However, the suboptimal levels of anthocyanins in consumed foods limit their potential health benefits. Leveraging commonly consumed crops for anthocyanin biofortification offers a practical and sustainable strategy for incorporating anthocyanins into daily diets to improve public health. Tomato (Solanum lycopersicum) is an ideal candidate for anthocyanin biofortification due to its worldwide cultivation, widespread consumption, and dietary prominence. Cultivated tomatoes have lost their ability to synthesize anthocyanins due to domestication-driven genetic modifications. However, some wild tomato species exhibit light-dependent anthocyanin accumulation in fruit. Anthocyanin fruit (Aft) locus was characterized in wild tomato, which is responsible for anthocyanin accumulation. Aft encodes a functional anthocyanin activator AN2like (R2R3-MYB transcription factor). Cultivated tomatoes contain a non-functional allele of AN2like and therefore fail to produce anthocyanins. To reintroduce anthocyanin biosynthesis in cultivated tomatoes, the AN2likeAft gene from anthocyanin-rich (Aft) tomatoes can be precisely integrated into the non-functional AN2likeaft locus via CRISPR-Cas9 mediated homology directed repair (HDR). This study presents a novel approach for restoring anthocyanin biosynthesis in cultivated tomatoes using CRISPR-Cas9 HDR mediated gene replacement.