Background The potato tuber starch trait is affected by the composition of amylose and amylopectin. The synthesis of amylose in amyloplasts is catalyzed by granule-bound starch synthase (GBSS). GBSS gene expression was inhibited via CRISPR-Cas9-mediated genome editing in leaves of the Desiree Potato cultivar. Constructs containing the Cas9 gene and sgRNAs targeting the GBSS gene were introduced by Agrobacterium-mediated transformation delivery into protoplasts. Methods and results Outcomes included lines with mutations in all or only some of the homoeoalleles of GBSS genes and lines in which homoeoalleles carried different mutations and multiple alleles that were up to 21 % of regenerated shoots. Mutations were found in one bp from the used guide sequence, verifying the high homology between a guide sequence and a target region near the protospacer adjacent motif (PAM) site. DNA delivery into protoplast resulted in mutants with no detectable Cas9 gene. Microscopic evaluation of iodine-stained starch granules was shown to be a sensitive system for qualitative and quantitative examination of amylose formation in starch granules of transgenic potato tubers. Conclusion This study presented the successful application of CRISPRCas9 to fully knockout a GBSS gene function in a potato tetraploid plant in one round of transfection, without a stable introduction of DNA into the genome. In this study, the region of the GBSS gene was targeted, for driving the guide sequences. all the different experiments yielded high mutation frequencies, in the same order of magnitude, demonstrating the robustness of the CRISPR-Cas9 technique for potato research and breeding. Here, also the use of this new technology was demonstrated to develop a trait of commercial interest, an amylopectin potato starch, with uses in both food and technical applications.