Fig. 2

SsdA_tox-CBE variants enable C-to-T editing of a GUS^G537 reporter in N. benthamiana. a Schematic of the GUS^G537 cytosine base editing reporter. The C-to-T (highlighted in red) editing in GUS^G537 can alter the glycine codon (GGA) to a glutamic acid codon (GAA) and restore GUS activity. The protospacer is shown with gray background, and the PAM is in blue. b Ten different A. tumefaciens transformants were co-inoculated together with the GUS⁵³⁷ reporter into N. benthamiana leaves. Leaf disks were harvested 2 dpi then stained in GUS staining solution and de-stained in ethanol. Blue color indicates restored GUS activity. Dark blue leaf disk from clone #7 are marked by triangles. WT GUS: wild-type GUS, positive control. GUS^G537: negative control. c Sanger sequencing results from A. tumefaciens clone #7 between days 1 and 10. Mismatches to the wild-type sequence are highlighted in red and indicated by triangles. d Top: Architectures of CBEs containing SsdA^G103S-v1, SsdA^G103R-v1, SsdA^G103A-v1, or SsdA^G103C-v1. An intron was introduced into the coding sequences of these SsdA variants to prohibit translation in bacteria. Bottom: C-to-T editing efficiencies of SsdA-CBE variants of the GUS^G537 reporter. GUS activities were measured and normalized to 2 × 35S::GUS (WT GUS, positive control). Values and error bars indicate the mean ± SEM, n = 3. * P < 0.05; ** P < 0.01 using Student’s two-tailed unpaired t-test