PMC

(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.

(a) Comparison at three genomic loci in HEK293T cells across eight plasmid doses. (b) Comparison of ABE7.10, bis-bpNLS-ABE with IDT codons, and ABEmax at three genomic loci in HEK293T cells across eight plasmid doses. (c) C:G-to-T:A editing for the correction of MPDU1 Leu119Pro by BE4, BE4max, or AncBE4max in unsorted or sorted fibroblasts derived from congenital disorder of glycosylation (CDG) type 1f patients. BE4 samples were sorted for mCherry and BE4max–P2A–GFP and AncBE4max–P2A–GFP samples for GFP-positive cells. (d) C:G-to-T:A editing of the 3′ splice acceptor in intron 6 of SCN9a in mouse N2a neuroblastoma cells, unsorted or sorted as described in (c). (e) A:T-to-G:C editing for the installation of -116 A>G and -113 A>G in the HBG fetal hemoglobin promoters by ABE7.10 or ABEmax in HEK293T cells. ABE samples were sorted for mCherry-positive cells, and ABEmax–P2A–GFP samples were sorted for mCherry and GFP double-positive cells. (f) A:T-to-G:C editing to install HBG promoter mutation -175 T>C by ABE7.10 or ABEmax in HEK293T cells, unsorted or sorted as described in (e). Values and error bars represent the mean and standard deviation of n=3 biologically independent experiments (dots) 3 days after transfection.