Comparative analysis of CRISPR/Cas9 delivery approaches for polyphenol oxidase 2 gene editing in potato Matías Nicolás González(1,2), Gabriela Alejandra Massa(1,2,3), Mariette Andersson(4), Cecilia Andrea Décima Oneto(1,3), Helle Turesson(4), Leonardo Storani(1,2), Niklas Olsson(4), Ann‑Sofie Fält(4), Per Hofvander(4) and Sergio Enrique Feingold(1) (1) Laboratorio de Agrobiotecnología, Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible (INTA-CONICET), Ruta 226, Km 73.5, Balcarce, B7620, Argentina (2) Consejo Nacional de Investigaciones Científicas y Técnicas, C1425FQB, Godoy Cruz 2290, CABA, Argentina (3) Facultad de Ciencias Agrarias, Universidad Nacional de Mar del Plata, Ruta 226, Km 73.5, Balcarce, B7620, Argentina (4) Department of Plant Breeding, Swedish University of Agricultural Sciences, P.O. Box 101, SE–23053 Alnarp, Sweden The components of the CRISPR/Cas9 bacterial immune system have been widely adopted as a genome editing tool, accelerating basic plant research and crop improvement. Delivery of the components to the cell and further regeneration of edited plants are key steps in the use of this technology. Here, we compared Agrobacterium-mediated transformation and protoplast transfection with CRISPR/Cas9 to mediate potato polyphenol oxidase 2 (StPPO2) gene editing in the tetraploid cultivar Desiree. StPPO2 is a member of the StPPO gene family, previously identified as the principal contributor to the polyphenol oxidase activity in the tuber. A DNA vector was either used for Agrobacterium-mediated transformation or transient expression in protoplasts, while ribonucleoprotein complexes (RNPs) were additionally assayed in protoplasts. Editing efficiency varied among the three approaches, with 9.6%, 18.4%, and 31.9% of edited lines obtained from Agrobacterium-mediated transformation, RNP, and DNA vector transfection in protoplasts, respectively. Only the latter resulted in StPPO2 tetra-allelic edited lines, which was observed in 46% of the total edited lines. On-target DNA insertions were found in edited lines from all three approaches. Further phenotypic analysis of a StPPO2 tetra-allelic edited line, confirmed the loss of function of the StPPO2 protein, by revealing a significant reduction in both the total polyphenol oxidase activity and the enzymatic browning in the tubers. Our results demonstrate that StPPO2 gene editing efficiency in potato depends on the CRISPR/Cas9 delivery strategy and provide insights to consider when selecting the appropriate methodology.
Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible (INTA-CONICET)
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