Generation of CRISPRi Cell Lines to Elucidate the Impact of Cholesterol Metabolism on DVL2 Signaling — 42a — William DeLaughter^1,2, Sonali Sengupta², Jazmine D. W. Yaeger², Kevin R. Francis^2,3

1 Sanford Program for Undergraduate Research (SPUR) from Central Methodist University, Fayette, MO, USA

2 Cellular Therapies and Stem Cell Biology Group, Sanford Research, Sioux Falls, SD, USA

3 Department of Pediatrics, Sanford School of Medicine, University of South Dakota, Sioux Falls, SD, USA

Loss of Wnt signaling homeostasis broadly contributes to developmental deficits and promotes tumorigenesis. The Dishevelled (DVL) family of proteins are important regulators of canonical and non-canonical Wnt signaling, localizing to the cholesterol-rich plasma membrane and functioning as a protein scaffold for Wnt ligand receptors. We previously determined that plasma membrane accumulation of the sterol 7-dehydrocholesterol (7DHC) perturbs Dishevelled 2 (DVL2) protein function due to reduced binding affinity of DVL2’s PDZ domain for 7DHC compared to cholesterol. We recently found that accumulation of 7DHC shifts the subcellular localization of DVL2 from the plasma membrane to the nucleus. While nuclear DVL2 is likely to promote non-canonical WNT signaling, how nuclear DVL2 localization occurs is unclear. We hypothesize that the chaperone protein FoxK2 is responsible for nuclear DVL2 shuttling. Thus, delineating the role of FoxK2 in nuclear transport of DVL2 within cholesterol limiting conditions will help define the cellular mechanisms at play. To analyze FoxK2 requirements for nuclear DVL2 localization, we utilized a CRISPRi/dCas9 model to repress cellular transcription of FoxK2. We designed optimized single guide RNA (sgRNA) oligos targeting FoxK2 that were annealed and cloned into a previously digested lentiviral expression vector. Clones were screened by PCR and validated by Sanger sequencing. We are currently performing a lentiviral transduction of sgRNA constructs into a ready-to-transduce human induced pluripotent stem cell (hiPSC) line constitutively expressing dCas9 and validated for pluripotent protein expression. FoxK2Ai cell lines will be validated for efficient transcriptional repression using qPCR, western blotting, and microscopy, as well as analyzed for impacts on cholesterol-regulated DVL2 localization. Generation of FoxK2 repressed cellular models will help us elucidate the mechanism of DVL2 nuclear transport during disrupted cholesterol metabolism with implications to developmental disorders of interest and cancer.  

Sanford Research
Kevin R. Franics