Reduced Enzymatic Browning in Potato Tubers by Specific Editing of a Polyphenol Oxidase Gene via Ribonucleoprotein Complexes Delivery of the CRISPR/Cas9 System

GONZÁLEZ, MATÍAS NICOLÁS; MASSA, GABRIELA ALEJANDRA; ANDERSSON, MARIETTE; TURESSON, HELLE; OLSSON, NIKLAS; FÄLT, ANN-SOFIE; STORANI, LEONARDO; DÉCIMA ONETO, CECILIA ANDREA; HOFVANDER, PER; FEINGOLD, SERGIO ENRIQUE

Frontiers in Plant Science

Frontiers Media

Año: 2020 vol. 10

Polyphenol Oxidases (PPOs) catalyze the conversion of phenolic substrates to quinones,leading to the formation of dark-colored precipitates in fruits and vegetables. This process,known as enzymatic browning, is the cause of undesirable changes in organolepticproperties and the loss of nutritional quality in plant-derived products. In potato (Solanumtubersoum L.), PPOs are encoded by a multi-gene family with different expressionpatterns. Here, we have studied the application of the CRISPR/Cas9 system to inducemutations in the StPPO2 gene in the tetraploid cultivar Desiree. We hypothesized that thespecific editing of this target gene would result in a lower PPO activity in the tuber with theconsequent reduction of the enzymatic browning. Ribonucleoprotein complexes (RNPs),formed by two sgRNAs and Cas9 nuclease, were transfected to potato protoplasts. Up to68% of regenerated plants contained mutations in at least one allele of the target gene,while 24% of edited lines carried mutations in all four alleles. No off-target mutations wereidentified in other analyzed StPPO genes. Mutations induced in the four alleles of StPPO2gene, led to lines with a reduction of up to 69% in tuber PPO activity and a reduction of73% in enzymatic browning, compared to the control. Our results demonstrate that theCRISPR/Cas9 system can be applied to develop potato varieties with reduced enzymaticbrowning in tubers, by the specific editing of a single member of the StPPO gene family