Fig. 3

XMAS-TREE enables highly efficient multiplex adenosine base editing in HEK293 cells. a Cells were transfected with pEF-XMAS, pCMV-ABEmax, and a pMT-sgRNA that simultaneously targeted Site-1/Site-3/Site-4 or Site-5/HBG1/HBG2. Base editing was quantified in unsorted as well as reporter-positive and reporter-negative cell populations. One-way ANOVA with Tukey post hoc test was performed. Statistical significance is only shown for the comparisons made between cells positive for the transfection reporter but not the editing reporter (i.e., mCherry-positive/GFP-negative) and cells positive for both (i.e., mCherry/GFP double positive); *p < 0.05, **p < 0.01, ***p < 0.001. n = 3. b Schematic for employing XMAS-TREE for generation of clonal lines that have been simultaneously edited at multiple loci. HEK293 cells are co-transfected with pEF-XMAS, pCMV-ABEmax, and pMT-sgRNA. At 48 h post-transfection, single mCherry/GFP double-positive cells are sorted into 96-well plates. After expansion, target clones are identified by Sanger sequencing at the target sites. c Analysis of clonal editing efficiency at multiple independent genomic sites using XMAS-TREE. A total of 30 clones derived from the mCherry/GFP double-positive and mCherry-positive/GFP-negative cell populations were examined at each locus. White box indicates no editing observed a specified locus, half-filled box indicates mono-allelic targeting at the genomic site, and full box indicates bi-allelic editing at the target locus