Figure 3.

Confirming LOF in Mutant iPSC Lines

(A) Fibroblasts that underwent simultaneous HPRT1 CRISPR and iPSC reprogramming were treated with 6-TG (genotype was determined after survival analysis), and percent viability was calculated by determining the CellTiter Blue signal ratio between 6-TG-treated and -untreated cells. The −Cas9 cells were 6-TG-treated iPSC lines generated in parallel without the HPRT1 CRISPR plasmid. Statistical analysis between wild-type (WT) and indel lines was performed by t test. Error bars denote SD. The number of clones for each group in the graph are n = 11, 35, 19, 5, and 2, respectively.

(B) A ROC curve was generated from the WT and indel lines (frameshift and in-frame) to choose the ideal discrimination value for the assay (76%, depicted as the green point on the ROC curve and as the dotted line in panel A). The two heterogeneous lines were found to have both WT and mutant sequence despite HPRT1 being on the X chromosome in male cell lines.