Fig. 4
Erythropoietic protoporphyria disease models created by MMEJ display ALAS2 gain-of-function and FECH loss-of-function. a Phenotypic consequences of ALAS2 gain-of-function and FECH loss-of-function mutations on the accumulation of metabolites in the heme synthesis pathway. 5-Aminolevulinic acid (5-ALA), Protoporphyrin IX (PPIX). b Altered protein sequences of ALAS2 and FECH caused by deletion mutations. c Sequence verification of a precise 4 bp deletion mutation in ALAS2 and a 5 bp deletion in FECH generated by either plasmid or RNP transfection. Deletion (dotted line), DSB location (pink bolt), µH (green), SpCas9 PAM (underline). d Schematic for cell culture conditions during erythroid differentiation. Differentiation day (D). e Ratio of erythroid cells in differentiated hiPSC populations on D26, as indicated by CD235a-FITC and CD71-APC markers, measured by FACS. f Gene expression levels measured by qRT-PCR of ALAS2 and FECH in undifferentiated cells and D26 cell populations normalized to cord blood cells. g PPIX-Qdot® 605 fluorescence intensity in erythroid cell populations of mutant and normal cell lines on day 26, measured by FACS. h Dose-response curve of PPIX accumulation in response to 5-ALA treatment in undifferentiated hiPSC disease model clones and the isogenic parental line. Median fluorescence intensity of PPIX-Qdot® 605 measured by FACS and normalized to the parental cell line at 0.5 mM 5-ALA. Means ± s.d. for n = 3 biological replicates. Source data are in the Source Data file
