Fig 1. Production of PAH-targeted piglets.

A) TALENs were designed to target Exon 8 of sus scrofa (ss) PAH. The location of the right monomer was strategically placed to contain a mismatch following a successful R408W HDR event. B) Sequence alignment of human (hs, top) and wild type pig (ss, second) shows the high similarity surrounding the target R408W mutation (red box). A homology template (HT, third) was designed to produce the R408W mutation in addition to introducing the 5 SNPs (black boxes) needed to “humanize” the sequence (hR408W) around the mutation to allow for targeting with human-translatable gene editing reagents. Lastly, genotype confirmation verified all piglets were positive for R408W and the 5 humanizing SNPs. C) Picture of wild type Ossabaw piglet (left) and Ossabaw PAH^{hR408W/hR408W} founder piglet (middle left, No. 1769) showing hypopigmentation with hR408W mutation consistent with that observed in PKU mouse models. Additionally, large white/landrace PKU founders (PAH^{hR408W/hR408W}, middle right) and PKU founders gestated on NTBC (PAH^{hR408W/A403GfsX47}, far right) are also presented. D) Birth weights of these PAH-targeted piglets are lower than historical values for wildtype piglets of their respective genetic background strains in the absence of NTBC during gestation. * p < 0.05 compared to untreated PAH^{hR408W/hR408W} in the same background.