CRISPR interference interrogation of COPD GWAS genes
This project developed to address limitations in our understanding of the genetic factors that predispose a subset of individuals exposed to environmental injury to develop COPD, a major contributor to morbidity and mortality worldwide. While genome-wide association studies (GWAS) have now identified 82 variants across the genome as significantly associated with risk of developing COPD, little is known about the functions of COPD GWAS genes in lung cell types. To determine their functional role either at homeostasis or in response to injury, we developed an inducible CRISPR interference (CRISPRi) system that allows inducible knockdown of genes of interest in patient induced pluripotent stem cells (iPSCs) or their differentiated progeny.
After working with collaborators to identify a set of 9 COPD GWAS genes likely to be expressed and biologically relevant in type 2 alveolar epithelial cells, we iteratively knocked them down with CRISPRi in iPSC-derived type 2 alveolar epithelial cells (iAT2s) and evaluated their effects on iAT2 proliferation, maturation, and differentiation. We found that knocking down one gene in particular, desmoplakin (DSP), dysregulated each of these processes. In further experiments, we found that DSP knockdown leads to a structural reorganization of iAT2s characterized by disruption of tight junctions, adherens junctions, and desmosomes together with increased proliferation through ERK-MAPK signaling. Finally, we found that DSP expression levels in human iAT2s alter their response to disease-relevant injurious stimuli, such as cigarette smoke injury. In summary, we developed an approach to identify the functional significance of genes of interest in differentiated human lung cell types and applied it first to screen the effects of a set of disease-relevant genes and then to characterize one gene in depth in human type 2 alveolar epithelial cells.
The images above show decreased DSP immunostaining (panels A-B) in DSP knockdown when induced with doxycycline. Desmosomes are present by EM in control iAT2s in panel C but absent in DSP knockdown iAT2s in the presence of doxycycline (+Dox).