Abstract
Disorders of the central nervous system (CNS) result in one of the largest economic burdens on society. While research has made enormous contributions in the identification of CNS disease-related genes, the underlying molecular mechanisms associated with disease pathology in human neuronal cells remain unclear. Developing a method to recapitulate disease- and age-associated phenotypes in human neurons grown ex vivo is critical for furthering understanding on CNS disorders and advancing therapeutic development. Our research uses CRISPR guide RNAs (gRNA) with a nuclease-deficient Cas9 (dCas9) fused with a transcriptional activator to upregulate endogenous “neuronal-fate” determining genes in human fibroblasts to facilitate transdifferentiation to induced neurons (iN). We have successfully upregulated endogenous BRN2, ASCL1, MYT1L and NEUROD1 (BAMN) in wild-type fibroblasts following delivery of a CRISPR-dCas9 activation domain and gRNAs targeted to BAMN. Our time course studies show downregulation of fibroblast markers and upregulation of immature, developing and mature neuronal markers via qPCR. Immunocytochemistry revealed subpopulations of cells that are positive for neuronal markers, suggesting transdifferentation to iN. The present study shows the potential of targeted activation of endogenous neuronal-fate determining genes with CRISPR/dCas9, and supports an alternative method for uncovering disease variations and advancing personalized medicine for individuals with CNS disorders.