Fig. 3: Transcription and protein levels within gene-corrected cells.

a Schematic indicating the genotypes of the iPSC lines generated and mRNA transcripts collected. Deep sequencing from qRT-PCR around both diseased loci in isolated cell lines. Reads were classified as WT, mutant, or corrected (PAM wobble) and mapped to either allele 1 (top bar) or allele 2 (bottom bar). When neither allele was corrected, both alleles were expressed at approximately the same rate. However, when either mutation was corrected, the corresponding allele was expressed at a higher rate than the one that still possessed a mutation. When both alleles were corrected, the fraction of reads making up the population was evenly distributed. Observations at individual alleles were consistent across both assayed loci. b Western blot for GAA protein. Each of the corrected lines expressed high levels of active protein as well as detectable levels of the precursor protein. Unedited cells expressed significantly lower levels of GAA protein, but the level was still above the limit of detection. c GAA activity in cell lysate as measured by 4-MUG cleavage in acidic conditions. Unedited cells have significantly lower activity showing there was little to no active protein. Corrected cell lines include Double Correct.c1 (isolated via ArrayEdit), Double Correct.c21, Double Correct.c28, Double Correct.c29, Double Correct.c30 (isolated via sequential correction of 2237G>A allele followed by S1mplex electroporation correction of 1440delT) and Double Correct.c73 (isolated via sequential correction of 2237G>A allele followed by using transient puromycin based correction of 1440delT). All corrected lines had significantly higher activity than unedited cells but were indistinguishable from each other (n = 5 technical replicates, ****p = 3.59 × 10⁻¹², two-tailed t-test, α = 0.05, heteroscedastic; mean ± s.d).