a, Gel-based deaminase assay showing activity of rAPOBEC1, pmCDA1, hAID, 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), (d), (e), and (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 with – if no target C is present. 8U is a positive control sequence with a U synthetically incorporated at position 8. For gel source data, see .