Fig 1: In vitro targeting of humanized PAHR408W and correction by HDR.A) Sequence alignment of humanized pig PAHhR408W (top) showing the guide RNA for the RNPs (grey arrow) as well as a homology template modified to revert the mutation and disrupt the PAM to prevent re-cutting (bottom). The location of the R408W mutation (red letters) and engineered BsaI site are also indicated. B) T7 endonuclease assay of PAH PCR products derived from hR408W fibroblasts, fibroblasts treated with RNPs containing the PAH gRNA, and a positive control for T7 cutting. The presence of the lower/lighter bands indicate mismatched double-stranded DNA indicative of NHEJ resulting from the RNPs. C) Quantitation of amplicon sequences derived from unedited (blue) and RNP treated (red) PCR of the PAHhR408W locus as well as the top 12 predicted off-target cutting sites for the gRNA employed. Only the PAH locus showed indels above background detection levels when compared to untreated samples. D) RFLP with BsaI was negative for untreated hR408W fibroblasts, while the diagnostic bands were present when cells were co-transfected with the RNP and the single stranded oligo template for HDR. Positive control was based on a synthesized dsDNA encoding the intended HDR product. Relative position of the BsaI cut site is diagramed below the gel for reference. E) HDR-specific PCR showed the presence of the anticipated product in co-transfected cells, but not untreated hR408W fibroblasts. The positive control from D, synthetic DNA created to produce the target HDR sequence for analysis, was also evaluated. F) The predominant sequence in untreated cells was R408W, which is reduced in both RNP and RNP/HT co-transfected cells. G) Pie chart indicating the relative presence of unedited (R408W), NHEJ, HDR (W408R and BsaI site), and incomplete HDR (not all corrections present) species in PAH locus amplicon sequencing from panel F. Gel images presented in B, D, and E are from a single gel each that was abbreviated for presentation as indicated by black vertical lines.
Fig 2: 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 PAHhR408W/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 (PAHhR408W/hR408W, middle right) and PKU founders gestated on NTBC (PAHhR408W/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 PAHhR408W/hR408W in the same background.
Fig 3: Colocalization of mutant PAH in Pah-R261Q mice with autophagic markers.Immunofluorescence micrographs showing the codistribution of PAH (green) with autophagy markers p62 phosphorylated at Ser403 (P-p62, red) in (a) or LC3 (red) in (b) in hepatic tissue from WT, Pah-R261Q and Enu1 mice. Both markers were increased in Pah-R261Q when compared to both WT and Enu1. The fluorescence intensity (mean ± SD) calculated in 14 stacks of confocal images, relative to WT (=1), was 1.326 ± 0.121 (Pah-R261Q) and 0.778 ± 0.158 (Enu1) for P-p62 (a), and 2.277 ± 0.174 (Pah-R261Q) and 1.535 ± 0.175 (Enu1) for LC3 (b). Pah-R261Q but not WT or Enu1 showed clear colocalization (yellow) of PAH with both P-p62 (a) and LC3 (b), as highlighted in the insets. Hoechst was used for nuclear staining (blue). All micrographs are representative for n = 3 biological replicates in each mice group. Source data are provided as a Source Data file.
Fig 4: Distribution of PAH in hepatic tissue of WT and mouse models Pah-R261Q and Enu1.Immunofluorescence of PAH and ubiquitin (Ub) detection in hepatic tissue of WT, Pah-R261Q, and Enu1 mice, revealing the distribution pattern of PAH (green) and Ub (red). PAH was strongly reduced in both Enu1 and Pah-R261Q when compared to WT, whereas Ub was highly expressed in both mutant mice. The micrographs are representative for n = 3 biological replicates in each mice group. The fluorescence intensity (mean ± SD) calculated in 14 stacks of confocal images, relative to WT (=1), was 0.264 ± 0.105 (Pah-R261Q) and 0.154 ± 0.029 (Enu1) for PAH, and 1.315 ± 0.035 (Pah-R261Q) and 1.405 ± 0.103 (Enu1) for Ub. Colocalization of PAH and ubiquitin (yellow) was observed in both mutant mice, as highlighted in the inset. DAPI was used for nuclear staining (blue). Source data are provided as a Source Data file.
Fig 5: Histology of WT and Pah KO livers in mouse and pig.Standard H&E and Masson’s trichrome sections show no appreciable difference between WT and Pahenu2/enu2 mice or WT and PAHR408W/R408W pigs. Similarly, immunohistochemistry showed similar levels and ubiquitous distribution of expression of the wild type and mutant protein in both models/species.
Supplier Page from Abcam for Anti-PAH antibody [EPR12380]