Journal
PLANT CELL TISSUE AND ORGAN CULTURE
Volume 145, Issue 2, Pages 291-305Publisher
SPRINGER
DOI: 10.1007/s11240-020-02008-9
Keywords
CRISPR; Cas9; Genome editing; Potato; Protoplasts; Agrobacterium; Polyphenol oxidases
Funding
- INTA [PNBIO1131024]
- INTA-Fondo de Valorizacion Tecnologica [FVT-63]
- Edicion genica, transgenesis y mutagenesis como generadores de nueva variabilidad en especies de interes agropecuario [INTA PE-I115]
- BECAR program from the Argentinian Ministry of Education and Sports
- Trees and Crops for the Future (TC4F), a Strategic Research Area at SLU - Swedish Government
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Comparisons were made between different delivery methods of CRISPR/Cas9 components for potato genome editing, with transient CR-PPO expression in protoplasts showing the highest efficiency and successfully editing all four StPPO2 alleles. The dual-sgRNA strategy resulted in a low incidence of targeted deletion, potentially due to contrasting efficiencies between sgRNAs. These findings suggest that the CRISPR/Cas9 delivery strategy plays a crucial role in gene editing efficiency in potato.
Key message We compared the StPPO2 gene editing outcomes and efficiencies through Agrobacterium-mediated transformation and protoplasts transfection with DNA or RNPs, and demonstrated that genome editing efficiency depends on the CRISPR/Cas9 delivery approach in potato. 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 potato genome editing. Two sgRNAs were designed to simultaneously direct Cas9 to the StPPO2 gene encoding for a tuber polyphenol oxidase (PPO). A binary vector (CR-PPO) was utilized for either Agrobacterium-mediated transformation or for transient expression in protoplasts, while ribonucleoprotein complexes (RNP-PPO) were additionally assayed in protoplasts. Editing efficiency varied, yielding 9.6%, 18.4% and 31.9% of edited lines from Agrobacterium-mediated transformation, RNP-PPO and CR-PPO transient expression in protoplasts, respectively. Furthermore, only the CR-PPO transient expression resulted in lines edited in all four StPPO2 alleles, observed in 46% of the edited lines and confirmed by tuber PPO activity and enzymatic browning 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 between sgRNAs, that was partially evident in the in silico analysis. Our results demonstrate that gene editing efficiency in potato depends on the CRISPR/Cas9 delivery strategy and provide insights to consider when selecting the appropriate methodology.
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