Study 3- RNA-seq of male KOLF2.2J hiPSC-derived cortical brain organoids homozygous null for six different transcription factors

This study aims to phenotype iPSC-derived cortical brain organoids that contain homozygous null alleles for transcription factors expressed in the early neuroectoderm. Null alleles were generated using three distinct genetic engineering approaches: full coding region deletion (KO), critical exon deletion (CE), and insertion of a premature termination codon with frameshift (PTC+1). RNA-seq data was generated at the indicated day post initiation of differentiation from hiPSCs. The study included the homozygous null alleles for the following transcription factors: LHX9, RFX4, PAX6, OTX1, LHX5, and FEZF2. This analysis provided insight into the differences between the various CRISPR-Cas9-based approaches and the impact of loss of function for these transcription factors in cortical brain organoid differentiation. The study involved the generation of RNA-seq on 179 samples at different time points of differentiation to cortical brain organoids. At least three different time points were selected for each line and the chosen time points were selected based on the known initiation of transcription of each respective transcription factor in wildtype cells within this differentiation scheme. Three biological replicates were run for each genetic engineering approach for each gene. 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. This comprehensive design allowed for a robust comparison of genetic engineering methods and the effects of transcription factor manipulation on brain organoid formation.