Fig. 2: Gene correction of two distinct mutant alleles within a single human cell.

a ArrayEdit-based isolation of iPSC clones corrected at either or both loci. CRISPR S1mplex design for the gene correction of compound heterozygous mutations. S1mplexes targeting 1441delT mutant were labeled with an AlexaFluor488 compound while S1mplexes targeting the 2237G>A mutation were labeled with an AlexaFluor647. These ribonucleoprotein genome editors were mixed prior to transfecting into cells and subsequently plated on the ArrayEdit platform to conduct high-content analysis (see Supplementary Fig. 1). b Left: LysoSensor quantification per µFeature of two mock transfections after 7 days of growth. Normal control hPSCs were significantly more intense than unedited, Pompe diseased iPSCs on ArrayEdit. Bottom right: the growth rate of unedited and control hPSCs following a mock transfection to establish a baseline for growth. Growth rates were calculated by measuring the per-day change in the number of cells of the µFeature. Top right: LysoSensor intensity was plotted against growth rate per µFeature to identify edited colonies. Dashed lines indicate regions of interest. (n = 145 independent cell lines). c Magnification of quadrant II from (b). µFeatures in this region were selected for genomic analysis to isolate edited clones. (n = 17 independent cell lines). d Sanger sequencing traces of corrected cell lines. The unedited line contains mutations at both alleles: 1441delT mutation causes a breakdown of sequence trace, whereas a single point mutation demonstrates a heterozygosity 2237G>A locus. SpyCas9 cut site is denoted by a dotted line. e Karyotypes of all isolated gene-corrected lines as well as unedited cells. No abnormalities were detected at a band resolution of 500. f Immunocytochemistry of pluripotency markers in gene-corrected lines. All lines were positive for pluripotency markers NANOG and TRA-1-60 (scale bar: 100 µm). g Schematic of long PCR covering both SpyCas9 cut sites. Arrows denote primers. The expected PCR amplicon is 7959 bp in length. h Gel analysis of long-range PCR described in (g) in each isolated cell line. No significant deviances from the expected length were detected, and no other notable bands were observed. WA09 control cells are hPSCs.