Potato plants (Solanum tuberosum L.) with reduced tuber enzymatic browning developed by genome editing with the CRISPR/Cas9 system

GONZÁLEZ, MATÍAS NICOLÁS; MASSA GABRIELA ALEJANDRA; ANDERSSON, MARIETTE; LEONARDO STORANI; DECIMA ONETO CECILIA; HOFVANDER, PER; FEINGOLD SERGIO ENRIQUE

Simposio; X ENCUENTRO LATINOAMERICANOY DEL CARIBE DE BIOTECNOLOGÍA AGROPECUARIA Y XI SIMPOSIO REDBIO ARGENTINA; 2019

Enzymaticbrowning of potato tubers (Solanumtuberosum L.) is the cause of undesirable changes in organolepticproperties and the loss of nutritional value of fresh and processed products,which represents as a serious quality deficiency for industry and consumers. Thisprocess is a consequence of Polyphenol oxidases (PPOs) activity, which catalyzethe conversion of phenolic substrates to quinones, leading to the formation ofdark-colored precipitates. In potato, PPOs are encoded by a multi-gene family,with at least four genes expressed in the tuber. In this work, we have producedthe specific knock out of a PPO genein the tetraploid cultivar Desiree, by inducing mutations with a CRISPR/Cas9system. Ribonucleoprotein complexes (RNPs), formed by two single-guide RNAs (sgRNAs)and Cas9 nuclease, were transfected to potato protoplasts, and complete plantswere regenerated. Mutations were screened by High Resolution Fragment Analysis(HRFA) on the target gene of 92 regenerated lines, and the genome editing efficiencyobtained was 40%, with 24% of the edited lines carrying mutations in the fouralleles of the target gene. Even though the majority of mutations were smalldeletions - ranging from 1 to 18 bp -, large fragment deletions were obtained coincidentlywith the loss of the sequence between the target sites of both sgRNAs.  No off target mutations were detected inother members of the PPO gene familyof selected edited lines. Lines carrying mutations in all the four alleles ofthe target gene displayed a significant reduction in PPO activity and enzymaticbrowning in the tubers, when compared to the control. Our results demonstratethat CRISPR/Cas9 can be applied to develop transgene-free potato varieties thatmaintain the organoleptic, antioxidant and nutritional properties duringharvest and post-harvest procedures.