Figure 1.

Improvement of gene editing and GT using ttLbCas12a‐i. (a) Cas12a expression cassettes for gene editing. The D156R mutation is highlighted, introns are represented by grey bars. The target sequences and regions are given. (b) Table showing the editing efficiencies determined by TIDE analysis as mean values for the respective target sites and variants. Editing efficiencies of the variants were determined for each of the tested target sites and individual transgenic plants grown at 22 °C. (c) Distribution of editing efficiencies of individual plants for both ttLbCas12a variants. Illustrated are the editing efficiencies of each of the analysed transgenic plants at the tested target sites (T1‐T7) for both variants at 22 °C. Data are presented as boxplots, each box represents the 25th and 75th percentile, the median is indicated by a black line, whiskers encompass the 1.5× interquartile range and data beyond, that is shown as outliers. Fold increases for ittLbCas12a‐i compared with ttLbCas12a at each target site are shown. T1: n = 20, T2: n = 20, T3: n = 17, T4: n = 17, T5: n = 18, T6: n = 20, T7: n = 20. P values were calculated using the one‐way ANOVA test: *P < 0.05, **P < 0.01, ****P < 0.0001. (d) Table showing the GT efficiencies and molecular analysis of GT events. The number of analysed T1 lines, the number and percentage of positive T1 lines and the total GT efficiency are shown on the left side of the table. The total GT efficiency is defined as the average of GT efficiencies of each individual line including the lines without GT events. The number of analysed positive T2 plants and the number and percentage of T2 plants with perfect GT events are shown on the right side of the table. (e) Distribution of GT efficiencies of the individual lines for both variants at 22 °C. The GT efficiencies of the individual positive lines are arranged in descending order for both variants. GT events were detected for 83 out of 100 and 87 out of 100 lines in case of ttLbCas12a and ttLbCas12a‐i, respectively.