Development of potato (Solanum tuberosum L.) plants with inactivated StPain-1 gene using CRISPR/Cas9

Storage of potato tubers intended for further processing is complicated by their cold-induced sweetening (CIS). Enzymatic hydrolysis of sucrose into glucose and fructose occurs at low temperatures. The resulting high hexose content adversely affects the quality of processed potato products such as chips and fries and promotes the formation of acrylamide, which is a neurotoxin and carcinogen. During CIS, sucrose hydrolysis is catalyzed by vacuolar invertase encoded by the StPain-1 gene. Previous studies have shown that suppression of the enzyme activity confers potato resistance to CIS without reducing the nutritional value of tubers. In this study, CRISPR/Cas9 technology was used to generate Solanum tuberosum L. cv. Fritella plants with a knockout of StPain-1. Two binary vectors based on pKSE401 were constructed (Vector A and Vector B), each carrying two gRNAs targeting exon 1 (sgRNA-P1.A or sgRNA-P1.B) and exon 3 (sgRNA-P3.A or sgRNA- P3.B). Editing efficiency with each gRNA was evaluated through next-generation sequencing (NGS). Transformation with Vector A produced 48 transformants, 22 of which carried knockouts in all StPain-1 alleles. Transformation with Vector B yielded 26 transformants, including 10 plants with complete StPain-1 knockout. Chips made from tubers of nine edited Fritella plants demonstrated reduced vacuolar invertase activity: chips from StPain-1 knockout lines were lighter compared to the non-edited control sample. Quantitative assessment of glucose, fructose, and sucrose levels, as well as StPain-1 mRNA expression in tubers of four selected transformants (two per vector), confirmed enzyme inactivation. The resulting plants exhibit increased resistance to cold-induced sweetening and can be used as a promising source of nonfunctional StPain-1 alleles for breeding new potato varieties.