Figure 1: Design and targeted deaminase activity of chimeric deaminases in E.coli.
From: Engineering and optimising deaminase fusions for genome editing
(a) Schematic representation of the design of targeted deaminases. The DNA binding domain (DBD), either ZF or TALE, was fused to the N-terminus of the deaminase with a certain linker. (b) Experimental overview: we integrated a GFP cassette (top) consisting of a broken start codon ACG, DNA binding sequence and the GFP coding sequence into the bacterial genome. We subsequently transformed targeted deaminases (middle) in pTrc-kan plasmid into the strain and induced protein expression. Targeted deamination of the C in the broken start codon leads to a ACG→ATG transition (bottom), rescuing GFP translation which is quantifiable via flow cytometry. (c) ZF-deaminases were tested for targeted deaminase activity by measuring GFP rescue. ZF, ZF-APOBECs (ZF-APOBEC1, ZF-APOBEC3F, ZF-APOBEC3G) or ZF-AID indicate cells transformed with plasmids that express ZF, ZF-APOBECs or ZF-AID respectively. All error bars indicate s.d. (All t-tests compare ZF-deaminases against the ZF control. *Pvalue<0.05, **Pvalue<0.01, ***Pvalue<0.001, n=4). (d) GFP rescue by ZF-AID and TALE-AID in the ZF-reporter and TALE-reporter strains.(All t-tests compare the fusion deaminases against the AID control. *Pvalue<0.05, **Pvalue<0.01, ***Pvalue<0.001, n=4). (e) GFP rescue by ZF-AIDs and TALE-AID in (wild type), (Δung), and (ΔmutS Δung) strains. All error bars indicate s.d. (All t-tests compare the fusion deaminases against the AID control. *Pvalue<0.05, **Pvalue<0.01, ***Pvalue<0.001, n=4). (f) E.coli (ΔmutS Δung) cells imaged under fluorescence (upper) and phase contrast (lower) after expression of ZF-AID or AID for 10 h. Top, Scale bar, 20 μm. More detailed structures and sequences of the fusion proteins and reporters are shown in Fig. 2a,c, Supplementary Fig. 1 and Supplementary Notes 1–7.
