CRISPRi/dCas9-KRAB mediated suppression of Solanidine galactosyltransferase (sgt1) in Solanum tuberosum leads to the reduction in α-solanine level in potato tubers without any compensatory effect in α-chaconine.

Potatoes produce two major steroidal glycoalkoids α-solanine and α-chaconine that comprise about 95% of all the glycoalkaloids in the potatoes. Under the stressful conditions production of glycoalkaloids get amplified that can lead to toxic symptoms in humans, particularly when taken in excess of 200–400 mg in adults and 20–40 mg in children. Toxicity may lead number of neuro-metabolic disorders due to anticholinesterase activity and also cause cell membrane disturbance ultimately affecting the digestion and metabolism. Minimizing these glycoalkaloids in potato tubers is crucial to reduce toxicity. Based on the previous reports suggesting α-solanine as a significant inhibitor of acetylcholinesterase, in this study we have analyzed the interaction of α-solanine, with acetylcholinesterase using computer-based docking and molecular-dynamic simulations. Our findings from docking studies with acetylcholinesterase showed a high docking score (−16.630 kcal/mol) for α-solanine, while molecular dynamics simulation suggested that α-solanine forms stable protein-ligand interactions with acetylcholinesterase. After having confirmed interaction through the docking and simulation results, we attempted to suppress the α-solanine level through suppression of the Solanidine galactosyltransferase (sgt1) gene that catalyzes solanidine to α-solanine conversion during the biosynthesis of α-solanine using the CRISPRi/dCas9-KRAB tool. Through CRISPRi/dCas9-KRAB, we intended to specifically target reducing the α-solanine level without disturbing the cellular concentration of α-chaconine. Our developed transgenic potatoes with suppressed sgt1 gene showed low α-solanine and unchanged α-chaconine levels, while nutritional analysis showed no significant changes compared to untransformed plants. • Computer based simulation and molecular dynamics studies suggest that α-solanine interacts with acetylcoline esterase resulting minimal hydrolysis acetylcholine thus interference with neural relay system i.e. acetylcholine mediated transmission of impulse. • Application of CRISPRi/Cas9 tool to suppress sgt1 is quite precise in suppression of α-solanine without any compensatory alteration in α-chaconine level. • Insignificant alteration in the α-chaconine level in the tubers enable plants to sustain moderate level of abiotic stresses. [ABSTRACT FROM AUTHOR]