Fig. 1
From: Deciphering TAL effectors for 5-methylcytosine and 5-hydroxymethylcytosine recognition
The screening platform to completely evaluate all potential TALE RVDs in recognizing modified cytosines. a Chemical structures of C, 5mC, and 5hmC. b The screening system for novel RVDs against modified cytosine is composed of a TALE activator and a GFP expression reporter. The TALEs contain 14.5 repeats fused with VP64. The 7th to 9th RVDs are substituted by the testing RVD XX′, which makes an archive of 420 TALEs. The X and X′ represent for the 12th and 13th residues, respectively. The 5mC and 5hmC reporters are linear DNA fragments amplified utilizing a forward primer with 5mC or 5hmC embedded on position 7th to 9th and a normal reverse primer. The C and T reporters are circular DNA described as previous13. c When the customized TALE does not bind to the reporter, for instance, between TALE-(E*)3 and the 5mC reporter, the GFP expression is at a basal level. In contrast, when the TALE binds tightly to the reporter, as shown for TALE-(G*)3 to the 5mC reporter, the GFP expression is up-regulated. mCherry intensity implies the quantity of transfected TALE-(XX′)3 plasmids
