Figure 1.

Wild-type and mutant GFP sequences and ssODN design. (A) The pGFPmut vector contains a single point mutation at base pair +213 of the GFP coding sequence that creates a stop codon in this position. ssODNs were designed to target bases in the stop codon to restore GFP expression. (B) Sequences of targeting (COR^GFP, m⁵CpG^GFP, m⁵CpC^GFP) and control (CTL^GFP) ssODNs. (C) Annealing of the targeting ssODNs to the genomic sequence creates a mismatch (box), whereas the control ssODN pair perfectly. The COR^GFP ssODN is designed to induce an A-to-C conversion that eliminates the stop codon and restores wild-type GFP expression. The m⁵CpG^GFP ssODN creates a G:T mismatch in the context of a m⁵CpG, thus mimicking the delamination of m⁵C. This will induce a T-to-C substitution which will convert the stop codon into an arginine. The m⁵CpC^GFP ssODN targets the same base as COR^GFP but contains an m⁵C modification which will lead to an A-to-G conversion and result in a single amino acid substitution (a tryptophan in place of a cysteine). The CTL^GFP ssODN perfectly complements the mutant sequence and therefore should not induce any genomic alterations.