Fig 2: Creation of a Pah-KO mouse model. (A) The mouse genomic locus on chromosome 10 and the murine targeted allele after CRISPR gene editing are shown. Exon 1 contains the translation initiation codon. NCBI GeneID: 18478; RefSeq transcript: NM_008777. Exon 1 sequence is shadowed gray, the start codon (ATG) is framed, gRNA is shown as white letters on a black background, the asterisks and arrow indicate the position of PAM and G>T transition. The donor oligo for CRISPR targeting is in bold and underlined. (B) Sequencing genomic DNA samples of the founder 5349. The point mutation of G to T is indicated in the black boxes. (C) Western blot analyses to detect PAH protein in liver or kidney of wildtype (wt) or Hom (ko) mice. Solubilized proteins (60 µg) from mouse liver or kidney were fractionated and immune-blotted with an anti-PAH antibody. The PAH protein (red; expected size 50 kDa) is evident in wildtype mice (lanes 1, 3, 5 and 7) but is absent in Hom mice (2, 4, 6 and 8); lanes 1, 2, 5, 6) are from kidney; lanes 3, 4, 7, 8 are from liver. Equal loading is indicated by probing with antibody to beta-actin (green). M, molecular weight marker lane (bands not visible in this image).

Fig 3: Characterization of Hom and Het Pah-KO mice for 6 months. (A) The Hom mice showed lower bodyweights than the Het mice throughout the study (B) Summary of blood Phe at various timepoints is shown (n = 7/group) (C) Summary of blood Tyr at various timepoints is shown (n = 7/group). The graphical data are represented as mean ± SD (Phe and Tyr levels in Hom vs Het mice p < 0.0001) (n = 7 mice/group). (D) Western blot analysis of liver homogenate using anti-PAH antibody is shown. Note the lack of PAH protein in the Hom mice whereas Het mice showed a corresponding band (~ 50 kDa) for mouse PAH protein. Each lane contained 135 µg protein. Beta actin was used as an internal control. Full-length blots/gels are presented in Supplementary Fig. 2. (E) Summary of % PAH positive hepatocytes in the Hom and Het mice are shown. The graphical data are represented as mean ± SD (**p = 0.0012). (F) Representative liver and kidney PAH-IHC images show lack of IHC signal in a Hom mouse whereas liver from a Het mouse exhibit strong IHC signal in hepatocytes, mostly in the centrilobular region. Renal cortical epithelial cells from a Hom mouse show no IHC signal as compared to Het mouse, where most proximal convoluted tubular epithelium exhibit strong signal.

Fig 4: Analysis of Phe, Tyr and neurotransmitters in brains of Hom and Het Pah-KO mice at 6 months. Scatter plot charts show brain Phe, Tyr, serotonin, dopamine, L-DOPA, DOPAC, NE, 5-HIAA, and HVA. n = 6 Hom, 7 Het. Bars indicate group mean and the error bars indicate standard deviation (*p = 0.01, **p = 0.003—0.007, ***p = 0.0001, ****p < 0.0001).