Fig. 2

Stepwise optimization of Cas12a-ABE using ITER. a, b Schematic illustration of 12 LbCas12a-ABEs (a) and nine crRNA (b) architectures tested in this study. c, Representative pictures showing mCherry- and GFP-positive cells measured during the optimization campaign of LbCas12a-ABE in wheat protoplasts. A-to-G base editing of the GFP reporter recovers GFP fluorescence. Colored dots represent different iterations and match measurements in d. Scale bars: 200 μm. d Editing efficiencies of LbCas12a ABE as measured by the rate of GFP recovery in wheat cells. BE and crRNA architectures are referred to as numbers and letters, respectively, as shown in a and b. Red and blue lines represent mean efficiency of cells transfected with or without crRNA, respectively. (‘) Five ITER cycles were conducted by sequentially testing adenine deaminases, NLSs, crRNA systems, Cas12a variants, and protein linkers. Best candidates obtained at individual iterations were used as baseline in the following iteration and are depicted with a blue arrowhead. (“) Key Cas12a-BE architectures obtained along the optimization path were compared side by side. Components leading to increased activity are shown at the right of the panel. Editing rates in d were calculated from two to four independent biological replicates that are depicted as dots on plots. Statistical significance is calculated with a one-way ANOVA test followed by a Tukey HSD post hoc test at threshold p<0.05