CRISPR Cas9-ALC Binary Vector Construct

Background

The long-shelf-life is a critical trait for the quality of fleshy fruit, and it is one of the major objectives in breeding programs as it influences fruit marketability for both farmer’s and consumer’s perception. The term ALC was coined from ‘Alco Baca’ Portugal cultivar of tomato from which slow ripening gene alc was discovered in 1960. ALC gene in its heterozygous mutation form was found to be significantly slow down the ripening of tomato with prolonged shelf with acceptable color development and flavor attributes. The present innovation named "CRISPR/Cas9-ALC binary vector construct" was created to generate tomato fruits with long shelf life.

Technology Details

The long-shelf-life is a critical trait for the quality of fleshy fruit, and it is one of the major objectives in breeding programs as it influences fruit marketability for both farmer’s and consumer’s perception. Tomato (Solanum Lycopersicum) has been mostly studied as classic climacteric model species with fleshy fruits to unravel the molecular basis of fruit softening. The term ALC was coined from ‘Alcobaca’ Portugal cultivar of tomato from which slow ripening gene alc was discovered in 1960. ALC gene in its homozygous mutation form was found to be significantly slow down the ripening of tomato with prolonged shelf life but with inferior flavor and poor color development. However, in plants heterozygous for these alleles shown to have a prolonged shelf with acceptable color development and flavor attributes. To generate tomato fruits with long shelf life, CRISPR/Cas9-ALC binary vector construct was created. CRISPR/Cas9 technology was exploited to knockout the ALC gene (Solyc10g006880.2.1). ALC consists of 3 exons and its encoded protein has two domains viz., NAM domain (17-143 amino acid) and NAC domain (11-179 amino acid). Synthetic gRNA was designed to target the second exon of ALC (Figure 1A). It was incorporated into a Cas9 expressing binary vector and confirmed by restriction analysis and sequencing