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. 2023 Jan 13;24:6. doi: 10.1186/s13059-022-02836-2

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© The Author(s) 2022

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 1 — ITER is an efficient platform for testing base editing in wheat and maize protoplasts. a Schematic illustration of ITER. Wheat or maize protoplasts are co-transfected in 96-well format. Cells are imaged and analyzed by automated high-content imaging. Resulting data are interpreted and inform the subsequent optimization cycle. Validation of base editor activity at endogenous targets is performed by genotyping after fluorescence-activated cell sorting (FACS). b Co-transfection efficiency as measured by the number of GFP over the number of mCherry positive cells in wheat protoplasts. Negative controls (−) do not contain vector DNA. The grid shows the 96-well plate positions and the gradient colors coding represents co-transfection efficiencies. c, d Base editing efficiencies for multiple nCas9-CBE (c) or nCas9-ABE (d) configurations in wheat protoplasts. Efficiencies were measured as the percentage of cells showing BFP to GFP conversion for C-to-T editing (c) or GFP recovery for A-to-G editing (d). Red and blue lines represent mean efficiency of cells transfected with or without gRNA, respectively. Representative pictures show mCherry or GFP fluorescence in protoplasts. n indicates the total number of cells measured after image segmentation in the mCherry or GFP channel. Scale bars: 200 μm. e Multiplex C-to-T base editing of A3A-nCas9-UGI or A3A-nCas9-UGI-P2A-GFP at three wheat targets. One sub-genome was analyzed by Sanger sequencing and EditR analysis. Asterisks represents significant difference in efficiency as measured by Student’s t test (***: p<0.001). f, g Multiplex C-to-T base editing of A3A-nCas9-UGI at 6 (f) or 12 (g) maize targets. h Multiplex A-to-G base editing efficiency of BP-TadA8e-nCas9-BP ABE at 6 maize targets. Transfection of the cells with a GFP expressing vector (no BE) was used as a negative control in e–h. x-axis in e–h indicates targeted Cytosines (e, f, g) or Adenines (h) at different positions along the protospacer, with the PAM-distal base being position 1. Editing rates were calculated from three to 6 independent biological replicates that are depicted as dots on plots (c–h)