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

Giovana Acha,Ricardo Vergara,Julio Kalazich,Humberto Prieto,Marisol Muñoz,Manuel Muñoz,Carlos Aguirre,Roxana Mora

doi:10.3390/plants10091882

Abstract

In potato (Solanum tuberosum 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 Bean yellow dwarf virus-derived replicon vector, pGEF-U, that expresses all the editing reagents under a multi-guide RNA condition, and the Green Fluorescent Protein (GFP) marker gene. Agrobacterium-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 GFP 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 (StPPO1 and 2) and to cold-induced sweetening (StvacINV1 and StBAM1). 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 StPPO1 and StBAM1 genes (1 and 4 lines, respectively), although with a transgenic condition. While no targeted deletion was seen in StvacINV1 and StPPO2 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.

Highlights

Genome editing strategies in plants based on the Clustered Regularly InterspacedShort Palindromic Repeats and (CRISPR)-Associated Protein 9 System (CRISPR/Cas9)rely on three fundamental aspects: (a) adequate delivery systems for the editing reagents (i.e., guide RNA and Cas9 nuclease), (b) tissue culture procedures that allow for 4.0/).regeneration of the edited cells, and (c) genome knowledge of the cultivar to be edited, avoiding off-target loci effect by the reagents.In potato (Solanum tuberosum L.), different approaches have been used to achieve successful CRISPR/Cas9-based genome editing
These vectors led to the generation of transgenic plants expressing the editing reagents; the transgenic condition constitutes an additional bottleneck in the process considering that the species is highly heterozygous and most of their cultivars show gametophytic self-incompatibility, making transgene segregation difficult by selfing or out-crossing techniques
While these results have shown the feasibility of carrying out gene editing of potato by geminivirus T-DNA vectors, this experimentation has revealed weak points of the strategy which are related mainly to the efficiency for the gene targeting and in the traceability of the mutation from the starting explant to the final edited individual [15,16]

Summary

Introduction

Genome editing strategies in plants based on the Clustered Regularly InterspacedShort Palindromic Repeats and (CRISPR)-Associated Protein 9 System (CRISPR/Cas9)rely on three fundamental aspects: (a) adequate delivery systems for the editing reagents (i.e., guide RNA (gRNA) and Cas nuclease), (b) tissue culture procedures that allow for 4.0/).regeneration of the edited cells, and (c) genome knowledge of the cultivar to be edited, avoiding off-target loci effect by the reagents.In potato (Solanum tuberosum L.), different approaches have been used to achieve successful CRISPR/Cas9-based genome editing. Potato clone of the group Phureja DM1-3 516 R44, hereafter referred to as ‘DM’) genotype, to express the editing reagents allowing for mutated versions of the AUXIN/INDOLE-3ACETIC ACID PROTEIN gene (StIAA2) [1] This type of vectors allowed for CRISPR/Cas targeted mutations in the promoter region of StFLORE, a long noncoding RNA, and showed the regulator role of this molecule in flowering and drought tolerance [2]. Success in protoplast protocols is far from routine in the current plant tissue culture state of the art, and regular gene transfer procedures, based mostly on regeneration protocols from multicellular explants such as leaves, internodes, and somatic embryos, represent a conditioning factor for gene editing which is under continuous improvement [6] In this regard, new approaches for expressing the editing reagents coupled with regular gene transfer procedures using multicellular explants could contribute to massive use of the technique

Results

Discussion

Conclusion