Fig. 4

Evaluation of base editor variants with BE-FLARE. a Comparison of APOBEC-BE3-T2A-TurboRFP variants with BE-FLARE. PC9 BE-FLARE stable cells were transiently transfected with BE3-T2A-RFP versions where the cytosine deaminase domain of BE3 was rA1, A3A or A3B co-expressing non-targeting gRNA (NT) or BFP gRNA (BFP gRNA). The frequency of GFP-positive cells was quantified by flow cytometry after 72 h. Data are representative of three independent experiments. b Quantification of the experiments described in (a). Mean ± SD from three independent experiments. Unpaired Student’s t test; *P < 0.05. c Representative histograms from flow cytometry analysis of GFP fluorescent signal from the experiments described in (b). Data are representative of three independent experiments. d Base editing of BE-FLARE by rA1-BE3 (left), A3A-BE3 (middle) or A3B-BE3 (right) transfected cells assessed by amplicon sequencing of the BFP locus. GFP-positive cells were FACS sorted 72 h after transfection, and 5 days later, genomic DNA was taken for amplicon sequencing. BE with a non-targeting guide served as a negative control (see Additional file 1: Table S3). Boxed in red is a bystander mutation leading to the introduction of a premature stop codon. Data represent the mean ± SD from two independent experiments and include SNPs found at ≥ 1% of total reads per sample. Read counts can be found in Additional file 1: Table S3. Data from these experiments are also part of Fig. 1c. e Immunoblot analysis of PC9 BE-FLARE cells 24, 48 and 72 h post-transfection with the indicated base editor variants (not T2A-RFP). Base editor expression over time was tracked by Cas9 immunodetection. Data are representative of two independent experiments. Raw data can be found in Additional file 2