A System to Investigate Combinatorial Dosage Sensitivity of X/Y Homologous Genes in Human iPSCs

Turner Syndrome is the result of a mitotic or meiotic error in early embryonic development causing most cells in the individual’s body to contain only one sex chromosome. As a result, many genes on X or Y are present in one copy instead of two, which is problematic for haploinsufficient genes. We hypothesize that genes in the PAR region and X/Y ortholog genes are such haploinsufficient genes. To test this, we employ CRISPRi, a modality that can turn down expression of target genes. To this end, we created guide sequences for our genes of interest and cloned them into plasmids. Next, we combined three of those individual inserts into a single construct using a technique entitled Golden Gate Assembly. Finally, we transposed them into a piggyBac vector which will allow us to introduce our sequences to iPSCs carrying inducible CRISPRi in the future.