Generation of a CRISPR/dCas9 transcriptional repression system to examine the oncogenic function of SWI/SNF loss in neural progenitor cells
The SWI/SNF chromatin remodeling complex is implicated in a wide range of human cancers, particularly in malignant rhabdoid tumors (MRTs) and a rare form of ovarian cancer known as small cell carcinoma of the ovary-hypercalcemic type (SCCOHT), which are driven by the universal loss of SMARCB1 and SMARCA4/A2, respectively. At least nine SWI/SNF complex members have been found to be recurrently mutated in cancers, and ~20% of all cancers bear at least one mutation in the SWI/SNF complex. The SWI/SNF complex modifies nucleosome positioning, an essential function in regulating gene expression. SCCOHT primarily affects young women with the mean age at diagnosis being 24 (ranging from 14 months to 48 years), typically presenting at a late stage with poor response to chemotherapy. While SCCOHT is known to be driven by the loss of the two mutually exclusive ATPase subunits SMARCA4 and SMARCA2, the putative histogenesis from a neural progenitor cell has not been experimentally modeled in a cell or animal model. Towards this end, this work sought to employ a modified version of the traditional CRISPR system, CRISPR-dCas9-KRAB, to individually target the promoters of the nine frequently mutated SWI/SNF genes for repression. This is accomplished through the design of unique small guide RNAs (sgRNA) which we cloned into a lentiviral expression vector through a series of PCR and restriction enzyme-based cloning methods, requiring a number of protocol modifications to optimize target sequence insertion. Functional assays will be employed to experimentally determine the effect of SWI/SNF subunit inhibition on the development of these cells. Further experiments to study the inhibition of these nine target SWI/SNF genes may provide valuable insights not only into the SCCOHT cell-of-origin, but also to the histogenesis of other SWI/SNF-mutant cancers.