Study 7- RNA-seq of male KOLF2.2J hiPSC-derived differentiated cells for lines in which the original mutation to create null alleles was reverted back to wildtype

This study aimed to phenotype iPSC-derived cortical brain organoids that contained corrections (reversions to wt) for homozygous null alleles for transcription factor PAX6 and to phenotype iPSC-derived extra-embryonic cell lineages that had contained homozygous null alleles for transcription factors PPARG, EPAS1, GCM1, and GRHL1. The original homozygous mutant lines were created by introducing a premature termination codon and frameshift (PTC+1) approach. Here in these clones, these mutations were reverted back to wt and restored gene function. The homozygous null alleles for the five genes studied here showed pronounced transcriptomic changes compared to the parental line, the data provided here indicate, upon reversion of the PTC+1 mutations back to wt, the normal wt transcriptomic response was largely restored indicating specificity of the engineered alleles in manipulating the respective gene’s function. The study involved the generation of RNA-seq on 30 samples, comprising cells differentiated into cortical brain organoids (with dorsal forebrain patterning) and differentiated into the trophoblast lineage, promoting the formation of a primitive syncytium. Three biological replicates were run for each gene KO line. The hypoxia-inducible factor (EPAS1) was differentiated under both 20% oxygen and 3% (~ level present at peri-implantation) oxygen concentrations. Additionally, three biological replicates were run of wild-type KOLF2.2J cells (the parental male cell line in which the KOs were generated) under the same differentiation conditions.