Fig. 5

AI-guided promoter editing for ZmVTE4. A The editing scheme of the ZmVTE4 promoter. A total of 10 peaks were identified as the candidate CREs within the region of 2-Kbp upstream and 200-bp downstream of the TSS of ZmVTE4 (top panel). An interpretability method of in silico tiling deletion (see the “Methods” section) was employed to highlight potential regulatory regions and proposed a candidate functional locus, the deletion of which could increase ZmVTE4 expression level (12 ~ 21-bp downstream of the TSS, grey shade and red arrow). B Editing plasticity of ZmVTE4. The model simulated the predicted expression changes on deletion with a single peak and deletion with each combination of all peaks. The number of deleted peaks (x-axis) and predicted expression levels (y-axis) are demonstrated. C Experimental editing results for AI-guided editing scheme of ZmVTE4 promoter. The left panel shows five promoter-edited alleles of ZmVTE4. The right panel shows relative predicted expression levels, LUC activities, ZmVTE4 expression levels, and α-tocopherol contents of WT and five edited alleles. Repeated experimental results about LUC activities, ZmVTE4 expression levels, and α-tocopherol contents in the right panel are significantly higher (vte4-cr4 allele) or lower (vte4-cr5 allele) compared to WT, determined by two-sided Student’s t test at P < 0.05. D Luciferase activity validation of the vte4-cr4 allele. The schematic diagrams of the ZmVTE4 constructs (left), relative predicted expression, and relative LUC activity are displayed (right). LUC, firefly luciferase; REN, Renilla luciferase