Transgene-Free Genome Editing in Tomato and Potato Plants Using Agrobacterium-Mediated Delivery of a CRISPR/Cas9 Cytidine Base Editor

Abstract

Genome editing tools have rapidly been adopted by plant scientists for gene function discovery and crop improvement. The current technical challenge is to efficiently induce precise and predictable targeted point mutations valuable for crop breeding purposes. Cytidine base editors (CBEs) are CRISPR/Cas9 derived tools recently developed to direct a C-to-T base conversion. Stable genomic integration of CRISPR/Cas9 components through Agrobacterium-mediated transformation is the most widely used approach in dicotyledonous plants. However, elimination of foreign DNA may be difficult to achieve, especially in vegetatively propagated plants. In this study, we targeted the acetolactate synthase (ALS) gene in tomato and potato by a CBE using Agrobacterium-mediated transformation. We successfully and efficiently edited the targeted cytidine bases, leading to chlorsulfuron-resistant plants with precise base edition efficiency up to 71% in tomato. More importantly, we produced 12.9% and 10% edited but transgene-free plants in the first generation in tomato and potato, respectively. Such an approach is expected to decrease deleterious effects due to the random integration of transgene(s) into the host genome. Our successful approach opens up new perspectives for genome engineering by the co-edition of the ALS with other gene(s), leading to transgene-free plants harboring new traits of interest.

Keywords: ALS; Agrobacterium; CRISPR/Cas9; cytidine base editor; potato; tomato; transgene-free.

Figure 1

Targeted modifications in tomato StALS1 gene. (a) SlALS1 and SlALS2 target region. The SlALS1 Pro186 codon is highlighted in green and the SlALS2 Pro184 codon in blue. The single mismatch in SlALS2 locus is written in red. The three targeted cytidines (C₋₂₀, C₋₁₄ and C₋₁₃) are written in green and in blue in the SlALS1 and StALS2 sequences, respectively. The protospacer adjacent motif (PAM) site is highlighted in yellow. Green and grey arrows indicate the relative positions of the PCR primers; (b) T-DNA physical map of the cytidine base editor (CBE) binary vector. Colored arrows indicate the relative positions of the PCR primers; (c) multiplex PCR analyses of 14 independent chlorsulfuron resistant plants, the wild-type (WT) and the positives controls (T+). L: molecular marker; (d) high resolution melting (HRM) assay using primers (grey arrows) flanking the targeted SlALS1 region. The color-label represents groups of plants (40 plantlets) that harbor similar melting curve shapes. The wild-type curves are colored in red. (e) chromatograms of the targeted region of the WT and of six independent chlorsulfuron resistant plants. The arrows indicate modifications; (f) histograms indicating the number of unedited plants (black), edited plants (grey) and plants with indels (in-del, striped), independently for C₋₂₀, C₋₁₄ or C₋₁₃ (not for the entire sequence), found on a total of 105 mutated plants in the targeted SlALS1 region; (g) percentage of each type of nucleotide changes found on cytidines C₋₂₀, C₋₁₄ or C₋₁₃. The total number of edited plants used in this analysis was 58 for C₋₂₀, 78 for C₋₁₄, 13 for C₋₁₃; (h) percentage of single, double and triple editing events in 75 base edited plants; (i) histogram indicating the number of unedited plants (in black), edited plants (in grey), and plants with indels (in-del, striped) in the SlALS2 locus, independently for C₋₂₀, C₋₁₄ or C₋₁₃ (not for the entire sequence), found on a total of 51 mutated plants on the SlALS1 locus.

Figure 2

Targeted modifications in potato StALS gene. (a) target region in the StALS1 and StALS2 genes of the cultivar Desiree. The StALS1 Pro186 codon is highlighted in green and the StALS2 Pro184 codon in blue. The single mismatch in the StALS2 locus is highlighted in red. Targeted C₋₂₀, C₋₁₄ and C₋₁₃ are represented in green and blue for StALS1 and StALS2, respectively. PAM site is highlighted in yellow. Green and grey arrows indicate the relative positions of the PCR primers; (b) T-DNA physical map of the CBE binary vector. Colored arrows indicate the relative positions of the PCR primers; (c) HRM analysis using primers targeting both StALS1 and StALS2 loci. Plants (20) are color-labeled according to their melting curve shape. The wild-type curve is colored in red; (d) summary of mutation efficiency and outcomes for 20 regenerated plants. The number of edited and transgenic or T-DNA-free plants is indicated; (e) Sanger chromatograms of the targeted region (StALS1 and StALS2) of five plants that do not harbor indels. The wild-type sequence is also provided. The black arrows indicate the localization of targeted cytidines; (f) PCR analyses of five chlorsulfuron resistant plants and the wild-type using primers matching the T-DNA (localization is indicated by the colored arrows); (g) Sanger chromatograms and rooting test of the three plants that did not amplify the NptII fragment. The wild-type and a positive control are also included. The black arrows indicate the localization of the targeted cytidines.