The Solanum tuberosum GBSSI gene: a target for assessing gene and base editing in tetraploid potato
The StGBSSI gene was successfully and precisely edited in the tetraploid potato using gene and base-editing strategies, leading to plants with impaired amylose biosynthesis.
Genome editing has recently become a method of choice for basic research and functional genomics, and holds great potential for molecular plant-breeding applications. The powerful CRISPR-Cas9 system that typically produces double-strand DNA breaks is mainly used to generate knockout mutants. Recently, the development of base editors has broadened the scope of genome editing, allowing precise and efficient nucleotide substitutions. In this study, we produced mutants in two cultivated elite cultivars of the tetraploid potato (Solanum tuberosum) using stable or transient expression of the CRISPR-Cas9 components to knock out the amylose-producing StGBSSI gene. We set up a rapid, highly sensitive and cost-effective screening strategy based on high-resolution melting analysis followed by direct Sanger sequencing and trace chromatogram analysis. Most mutations consisted of small indels, but unwanted insertions of plasmid DNA were also observed. We successfully created tetra-allelic mutants with impaired amylose biosynthesis, confirming the loss of function of the StGBSSI protein. The second main objective of this work was to demonstrate the proof of concept of CRISPR-Cas9 base editing in the tetraploid potato by targeting two loci encoding catalytic motifs of the StGBSSI enzyme. Using a cytidine base editor (CBE), we efficiently and precisely induced DNA substitutions in the KTGGL-encoding locus, leading to discrete variation in the amino acid sequence and generating a loss-of-function allele. The successful application of base editing in the tetraploid potato opens up new avenues for genome engineering in this species.
KeywordsGenome editing CRISPR-Cas9 Cytidine base editor Potato GBSS HRM
We thank Dr Puchta and his team (Botanical Institute II, Karlsruhe Institute of Technology, Karlsruhe, Germany) for providing the pDeCas9 plasmid via Marianne Mazier (INRA-UR1052, Montfavet, France) and Keiji Nishida for providing the pDicAID_nCas9-PmCDA_NptII_Della plasmid. We thank Fabien Nogué and Peter Rogowsky for their efficient management of the GENIUS project and their constructive discussions. We are grateful to Gabriel Guihard for his contribution in the sequencing of Desiree alleles. We thank the BrACySol BRC (INRA Ploudaniel, France) that provided us with the plants that were used in this study and Gisèle Joly and Catherine Chatot from Florimond Desprez/Germicopa (France) for helpful discussions and choice of the starch elite cultivar Furia. We are grateful to Jean-Louis Rolot from CRAW (Belgium) for providing us with the procedure to obtain in vitro microtubers as well as to Marie-Ange Dantec and all the INRA Ploudaniel greenhouse staff for acclimation and cultivation of the regenerated plantlets. Finally, we are thankful to Carolyn Engel-Gautier and Marina Perez Benitez for their help in correcting the manuscript.
Author contribution statement
FV, LC and JEC designed the experiments. FV, LC, MPK, ZT, FS, MM and NS performed the experiments. FV and JLG wrote the article. FV, LC, FS, NS, PD, JLG and JEC discussed the data and revised the article. All authors approved the final manuscript.
This work was supported by the INRA UMR IGEPP and the Investissement d’Avenir program of the French National Agency of Research for the project GENIUS (ANR-11-BTBR-0001_GENIUS). ZT is funded by a CIFRE PhD grant from SYNGENTA.
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Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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