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. Author manuscript; available in PMC: 2018 Nov 29.
Published in final edited form as: Nat Biotechnol. 2018 May 29;36(9):843–846. doi: 10.1038/nbt.4172

Figure 1. Identifying and addressing factors that limit base editing efficiency in mammalian cells.

Figure 1

(a) Plasmids used to elucidate the relationship between base editor expression and editing efficiency in mammalian cells: mCherry (transfection control), and either BE4 (“in trans”), BE4 and GFP on separate promoters (“in cis”), or BE4–P2A–GFP (“P2A”). (b) Percent mCherry-positive or GFP-positive HEK293T cells 3 days after transfection of the constructs in (a). (c) Target C:G-to-T:A editing in unsorted and sorted HEK293T cells. Sorted in trans cells were mCherry-positive, while sorted in cis and P2A cells were mCherry and GFP double-positive. (d) Effects of six NLS configurations on BE4 editing efficiency at five genomic loci in HEK293T cells. (e) Effects of five codon usages on editing efficiency of bis-bpNLS-BE4 in HEK293T cells. IDT=Integrated DNA Technologies; JC=Jeff Coller; GA=GeneArt; GS=GenScript; IDT-GS=IDT APOBEC+GenScript Cas9 nickase. (f) Phylogenetic tree for ancestral APOBEC reconstruction. (g) Base editing of bis-bpNLS-BE4 variants with GenScript codons using the ancestral APOBEC domains in (f) in HEK293T cells. Values and error bars represent the mean and standard deviation of n=3 biologically independent experiments (dots) 3 days after transfection.