Development of a porcine model of phenylketonuria with a humanized R408W mutation for gene editing
Fig 5
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.