Extended Data Figure 1 : Effects of deaminase, linker length, and linker composition on base editing.
From: Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage
a, Gel-based deaminase assay showing activity of rat APOBEC1 (rAPOBEC1), lamprey CDA1 (pmCDA1), human AID (hAID), human APOBEC3G (hAPOBEC3G), rAPOBEC1-GGS-dCas9, rAPOBEC1-(GGS)3-dCas9, and dCas9-(GGS)3-rAPOBEC1 on ssDNA. Enzymes were expressed in a mammalian cell lysate-derived in vitro transcription-translation system and incubated with 1.8 μM dye-conjugated ssDNA and USER enzyme (uracil DNA glycosylase and endonuclease VIII) at 37 °C for 2 h. The resulting DNA was resolved on a denaturing polyacrylamide gel and imaged. The positive control is a sequence with a U synthetically incorporated at the same position as the target C. b, Coomassie-stained denaturing PAGE of the expressed and purified proteins used in c–f. c–f, Gel-based deaminase assay showing the deamination window of base editors with deaminase–Cas9 linkers of GGS (c), (GGS)3 (d), XTEN (e), or (GGS)7 (f). Following incubation of 1.85 μM deaminase–dCas9 fusions complexed with sgRNA with 125 nM dsDNA substrates at 37 °C for 2 h, the dye-conjugated DNA was isolated and incubated with USER enzyme at 37 °C for 1 h to cleave the DNA backbone at the site of any Us. The resulting DNA was resolved on a denaturing polyacrylamide gel, and the dye-conjugated strand was imaged. Each lane is numbered according to the position of the target C within the protospacer, or labelled with ‘–’ if no target C is present. 8U is a positive control sequence with a U synthetically incorporated at position 8. For uncropped gel data, see Supplementary Fig. 1.
