Figure 3: Test of the specificity of AID fusions.
From: Engineering and optimising deaminase fusions for genome editing
(a) Test of ZF-8aa-AID sequence specificity using a GFP reporter with point-mutated ZF binding sequences. t-tests compare each mutated site against the unmodified site (top). *Pvalue<0.05, **Pvalue<0.01, ***Pvalue<0.001, n=4. All error bars indicate s.d. (b) Test of TALE-C1-AID sequence specificity using a GFP reporter with point-mutated TALE binding sites. t-tests compare each mutated site against the unmodified site (top). *Pvalue<0.05, **Pvalue<0.01, ***Pvalue<0.001, n=4. All error bars indicate s.d. Note that we altered the first nucleotide, a TALE-N terminus-specified thymine, to three other nucleotides individually, while we changed other nucleotides in the TALE recognition domain to the nucleotide mostly likely to be recognized3. (c) Mutation location and spectrum in the GFP gene of GFP+ and GFP− cells collected after ZF-8aa-AID induction. A schematic structure of the GFP gene is shown above the mutation frequency along the gene’s length among 200 Sanger sequenced colonies of each cell population. Grey lines indicate positions of C/G nucleotides; red lines indicate occurrences of the AID preferred motif (WRC). (d) Mutation spectrum on the GFP gene of GFP+ and GFP− cells collected after TALE-C1-AID induction. (e) Whole-genome SNV profiles of strains with/without ZF-AID induction. SNVs that may stem from cytosine deamination (C/G→T/A) are in either green (if C was in the AID-preferred WRC motif) or blue (all other Cs) bars. (f) Whole-genome SNVs profiles of strains with/without TALE-AID induction. Colour schematic is the same as e.
