Abstract 506: Modulation of ZIC3 Expression in Pluripotent Stem Cells Using CRISPR-interference to Model Heterotaxy

Abstract

Failure of proper left-right patterning during embryogenesis is an underlying mechanism of congenital heart disease (CHD). Heterotaxy (HTX) is a complex laterality syndrome in which multiple organs, including the heart, are abnormally arranged and mal-rotated. Loss-of-function mutations in ZIC3 , a zinc finger transcription factor within the GLI superfamily, have been associated with X-linked HTX. The mechanism behind how ZIC3 causes laterality defects remains unknown. To investigate this relationship, we developed a method to directly explore ZIC3 using induced pluripotent stem cells (iPSCs) at a molecular, cellular, and functional level through utilization of CRISPR interference (CRISPRi). A stable CRISPRi transgenic line was generated through lentiviral transduced vectors encoding inducible endonuclease-dead Cas9 and an sgRNA targeting the ZIC3 promoter. As ZIC3 is necessary for iPSC pluripotency, this inducible system allowed for a controllable knockdown while maintaining parent cell line pluripotency. RT-PCR confirmed that doxycycline induced a robust knockdown of ZIC3 expression over 72 hours with a fold change of 0.12 compared to non-induced. RNA-Seq was used to generate a gene expression profile of ZIC3 loss. This showed that 446 genes were significantly differentially expressed (adjusted p < 0.01), and of those, 163 genes had an absolute fold change of greater than 1.5. Of the top 15 most significantly differentially expressed genes, 47% (7 of 15) are associated with the cytoskeleton, including gamma actin 1 and myosin light chain 9. These results were confirmed by RT-PCR. Confocal imaging revealed significant changes to the actin cytoskeleton in response to ZIC3 knockdown, with a notable reduction in filamentous actin. Current ongoing studies are focusing on the impact of ZIC3 knockdown on the structural and mechanical differences in differentiated cardiomyocytes. In summary, we have developed a method by which to explore CHD using iPSCs in order to better understand the genetic mechanisms of heterotaxy, and in future studies, other forms of CHD.