Fig. 4.
Targeted methylation editing of the Avy locus reverses coat color-related phenotype. (A) Schematic representation of targeting the Avy promoter region by dCas9-Dnmt3a or dCas9-Tet1 with specific sgRNAs to manipulate the coat color-related phenotype. Unmethylated IAP that lies upstream of the agouti gene allows ectopic expression of the gene, resulting in a yellow coat color. Methylated IAP induces expression of the gene under normal developmental control, leading to a pseudoagouti coat color (termed pseudoagouti as the mice are isogenic with fully yellow mice and not genetically agouti). (B) Bisulfite sequencing on three offspring generated from yellow mother oocytes that have been successfully de novo-methylated. (C) Bisulfite sequencing on three offspring generated from pseudoagouti mother oocytes that have been successfully demethylated. For offspring, tail DNA was used for methylation analysis. For offspring phenotype classification, 1–5 represents coat color scoring as described in Methods. “1” represents completely yellow, whereas “5” represents completely pseudoagouti in this figure, respectively. White circles represent unmethylated CpGs, and black circles represent methylated CpGs. Values on each bisulfite grouping indicate the percentage of CpG methylation, with number of clones analyzed in parentheses.