A Traceable DNA-Replicon Derived Vector to Speed Up Gene Editing in Potato: Interrupting Genes Related to Undesirable Postharvest Tuber Traits as an Example

• Published in: Plants (Basel) Vol. 10; no. 9; p. 1882
• Main Authors: Acha, Giovana, Vergara, Ricardo, Muñoz, Marisol, Mora, Roxana, Aguirre, Carlos, Muñoz, Manuel, Kalazich, Julio, Prieto, Humberto
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 10.09.2021; MDPI
• Subjects: agrobacterium-mediated transformation; CRISPR; CRISPR/Cas9; Deoxyribonucleic acid; DNA; Enzymes; Enzymic browning; Explants; Fluorescence; Geminiviridae; geminivirus replicons; Gene deletion; Gene transfer; Genes; Genetic modification; genome editing; Genomes; Genomics; Genotypes; Green fluorescent protein; Infiltration; Mutation; potato; Potatoes; Proteins; Reagents; Regeneration; Ribonucleic acid; RNA; Solanum tuberosum; Vegetables; Viruses; genome editing; geminivirus replicons; agrobacterium-mediated transformation; CRISPR/Cas9; green fluorescent protein; potato
• ISSN: 2223-7747; 2223-7747
• DOI: 10.3390/plants10091882

In potato ( L.), protoplast techniques are limited to a few genotypes; thus, the use of regular regeneration procedures of multicellular explants causes us to face complexities associated to CRISPR/Cas9 gene editing efficiency and final identification of individuals. Geminivirus-based replicons contained in T-DNAs could provide an improvement to these procedures considering their cargo capability. We built a -derived replicon vector, pGEF-U, that expresses all the editing reagents under a multi-guide RNA condition, and the ( ) marker gene. -mediated gene transfer experiments were carried out on 'Yagana-INIA', a relevant local variety with no previous regeneration protocol. Assays showed that pGEF-U had transient expression for up to 10 days post-infiltration when leaf explants were used. A dedicated potato genome analysis tool allowed for the design of guide RNA pairs to induce double cuts of genes associated to enzymatic browning ( and ) and to cold-induced sweetening ( and ). Monitoring GFP at 7 days post-infiltration, explants led to vector validation as well as to selection for regeneration (34.3% of starting explants). Plant sets were evaluated for the targeted deletion, showing individuals edited for and genes (1 and 4 lines, respectively), although with a transgenic condition. While no targeted deletion was seen in and plant sets, stable GFP-expressing calli were chosen for analysis; we observed different repair alternatives, ranging from the expected loss of large gene fragments to those showing punctual insertions/deletions at both cut sites or incomplete repairs along the target region. Results validate pGEF-U for gene editing coupled to regular regeneration protocols, and both targeted deletion and single site editings encourage further characterization of the set of plants already generated.