Epsilon Archive for Student Projects

Abstract

tarch is a fundamental biopolymer with diverse applications across food, industrial, and biomedical sectors. The composition and structure of its two key polysaccharides, amylose, and amylopectin, govern its functional properties, and efforts to optimize starch quality have increasingly turned to genetic modification. This thesis investigates CRISPR/Cas9-mediated genome editing in potato (Solanum tuberosum) to enhance the expression of Starch Synthase I (SSI) in tuber tissue by introducing the GBSS promoter upstream of the stSSI gene, with a genetic background of gbss, ssII, and ssIII, allowing SSI to act as the dominant starch synthase.
The project employs phenotypic and genotypic characterization to assess the effects of these edits, combining enzymatic assays and genome analysis to validate intended outcomes. The off-target activity was evaluated through PCR-based Sanger sequencing and TIDE analysis, confirming the precision of CRISPR edits. Zymography protocols were optimized for in vitro-grown microtubers, enabling early detection of enzymatic activity. The edited lines demonstrated increased SSI activity in most cases, supporting the success of the promoter-driven gene expression strategy.
This work contributes to the broader aim of producing genetically tailored, high-performance potato starch and highlights the value of promoter editing for precise trait enhancement in polyploid crops. It also establishes a more time and resource-efficient pipeline for early-stage evaluation of gene-edited lines.