Abstract
Focal cortical dysplasia (FCD) is a common cause of focal epilepsy and is often caused by somatic, mosaic mutations. The sparse nature of mutated cells (1-6% of cells in resective tissue at best) presents many experimental barriers to FCD gene discovery. Consequently, identifying novel genes is extremely challenging and may not be possible in many cases using patient tissues. The small number of known FCD genes were identified by hypothesis-driven targeted sequencing rather than an unbiased approach. Therefore, we have developed an unbiased screening platform for identifying novel FCD genes in vitro using a genome-wide inhibitory CRISPR library. Using iPSCs stably-expressing doxycycline inducible KRAB-dCAS9 and NGN1/2, we can simultaneously inhibit gene expression and differentiate into neurons. Using an antibody for an FCD biomarker (phospho-S6) and FACS sorting, we were able to obtain genomic DNA from pS6-high and -low cell populations. To validate our screen, we constructed a test library of positive, negative, or neutral regulators of pS6. The abundance of each gRNA sequence was then assessed using Next-gen sequencing. As expected, the gRNAs for FCD genes were significantly enriched in the pS6-high sample. A genome-wide gRNA library is now being applied to identify novel genes. Validated FCD gene candidates will offer additional targets for sequencing from patient tissues and the potential for new therapeutic strategies for FCD-associated epilepsies.
