INVESTIGADORES
MASSA Gabriela Alejandra
artículos
Título:
Comparative potato genome editing: Agrobacterium tumefaciens‑mediated transformation and protoplasts transfection delivery of CRISPR/Cas9 components directed to StPPO2 gene
Autor/es:
GONZÁLEZ, MATÍAS NICOLÁS; MASSA GABRIELA ALEJANDRA; ANDERSSON, MARIETTE; DECIMA ONETO CECILIA; TURESSON, HELLE; LEONARDO STORANI; OLSSON, NIKLAS; FÄLT, ANN-SOFIE; HOFVANDER, PER; FEINGOLD, SERGIO ENRIQUE
Revista:
PLANT CELL TISSUE AND ORGAN CULTURE
Editorial:
SPRINGER
Referencias:
Año: 2021
ISSN:
0167-6857
Resumen:
Delivery of the CRISPR/Cas9 components to the plant cells is a key step in its application as a genome editing tool. Here,we compared Agrobacterium-mediated transformation and protoplast transfection with CRISPR/Cas9 components for potatogenome editing. Two sgRNAs were designed to simultaneously direct Cas9 to the StPPO2 gene encoding for a tuber polyphenoloxidase (PPO). A binary vector (CR-PPO) was utilized for either Agrobacterium-mediated transformation or for transientexpression in protoplasts, while ribonucleoprotein complexes (RNP-PPO) were additionally assayed in protoplasts. Editingefficiency varied, yielding 9.6%, 18.4% and 31.9% of edited lines from Agrobacterium-mediated transformation, RNP-PPOand CR-PPO transient expression in protoplasts, respectively. Furthermore, only the CR-PPO transient expression resulted inlines edited in all four StPPO2 alleles, observed in 46% of the edited lines and confirmed by tuber PPO activity and enzymaticbrowning analysis. Lines with on-target DNA insertions were found from all three approaches and characterized by sequencing.The dual-sgRNA strategy resulted in a low incidence of the targeted deletion, likely due to contrasting efficiencies betweensgRNAs, that was partially evident in the in silico analysis. Our results demonstrate that gene editing efficiency in potatodepends on the CRISPR/Cas9 delivery strategy and provide insights to consider when selecting the appropriate methodology