Department of Genetics

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Browsing Department of Genetics by Subject "650 0CRISPR-associated protein 9"

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    Development of genome editing of potato to repress cold-induced sweetening
    (Stellenbosch : Stellenbosch University, 2023-12) Schwegler, David Alexander; Lloyd, James; Stellenbosch University. Faculty of AgriSciences. Department of Genetics & Institute of Plant Biotechnology.
    ENGLISH ABSTRACT: In South Africa, the potato (Solanum tuberosum L.) has become one of the country’s most significant food sources. Its tubers are an abundant source of dietary carbohydrates for human consumption and contain starch, a polymeric carbohydrate composed of amylose and amylopectin that also has numerous industrial uses. Potatoes are often stored at cold temperatures after harvesting, leading to starch being degraded to form the reducing sugars glucose and fructose. When heated to high temperatures, these sugars react with amino acids in the Maillard reaction to produce acrylamide that is neurotoxic and potentially carcinogenic. It is therefore desirable to produce plants with tubers that don’t produce reducing sugars when stored at cold temperatures. An enzyme that plays a critical role in starch degradation is α-glucan, water dikinase 1 (GWD1), and plants without GWD1 activity have demonstrated impaired starch degradation including in cold-stored potato tubers. Clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 is a multipurpose technology for genetic engineering and provides new opportunities for developing novel plant characteristics. This system provides a novel method to mutate StGWD1 to prevent starch degradation. To accomplish this, single guide RNAs were designed to target regions of the gene before a key catalytic phosphohistidine site that is essential for enzyme activity. The guides were transcribed in vitro and complexed with Cas9 to assess cleavage of a target-containing sequence. The in vitro efficacy assay demonstrated that one of the guides successfully introduced a double-stranded break to the target sequence. Two strategies were used to try to mutate StGWD1. The first was a transgene-free method for gene editing that required successful protoplast isolation and regeneration. After successfully isolating viable potato protoplasts, micro-calli were generated from tissue culture of these cells. Unfortunately, regeneration did not proceed further and so a transgenic approach using Agrobacterium- mediated transformation was then used to transform leaf explants. This resulted in transgenic calli, although sequencing reactions and Tracking of Indels by Decomposition (TIDE) analysis revealed no editing at the target sequences.