Study 2- RNA-seq of male KOLF2.2J hiPSC-derived trophoblast cell lines homozygous null for seven different transcription factors

This study aims to phenotype iPSC-derived trophoblast lines that contain homozygous null alleles for transcription factors expressed within the extra-embryonic lineage, with a focus on differentiation toward either primitive syncytium or extra-embryonic mesenchymal cells, depending on the TF being analyzed. Null alleles were generated using three distinct genetic engineering approaches: full protein coding region deletion (KO), critical exon deletion (CE), and insertion of a premature termination codon with frameshift (PTC+1). RNA-seq data was generated after 6 days of initiating differentiation from iPSCs. The study included the homozygous null alleles for the following transcription factors: MEIS1, MXD1, MEIS2, RUNX1, MEF2C, NCOA3, and BHLHE40. 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 trophoblast lineage differentiation. The study involved the generation of RNA-seq on 87 samples. Four of the transcription factors were analyzed in the primitive syncytium differentiation scheme and three in the extra-embryonic mesenchymal cell 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 cell fate decisions within the extra-embryonic lineage.