59. Multiplex Gene Activation by CRISPR/Cas9-Based Transcription Factors for the Direct Conversion of Fibroblasts to a Neuronal Phenotype
Authors: Joshua Black, Andrew Adler, Hunter Hutchinson, Honggang Wang, Geoffrey Pitt, Kam Leong, Charles Gersbach
Journal: Molecular Therapy (2015)

The reprogramming of cell lineage between mature somatic cell types has a significant potential to advance disease modeling, drug discovery, and gene and cell therapies for regenerative medicine. Several groups have established methods to reprogram cell phenotype through the ectopic delivery of cDNAs encoding master regulatory transcription factors. These factors can target epigenetically silenced genes and activate transcriptional networks specific to other cell lineages. The recent advances made in engineering the CRISPR/Cas9 system as a programmable transcriptional regulator provide an opportunity to study alternate methods to achieve cell reprogramming. The dCas9-based transcription factors (dCas9-TFs) can attain multiplex activation and repression of endogenous genes. It's simplicity of use enables a standardized method of regulating endogenous transcriptional networks in any cell type of interest. The ability of dCas9-TFs to target virtually any region of the genome within the native chromatin context provides an opportunity to study how non-coding regulatory elements and epigenetic signatures influence reprogramming outcomes. Here we demonstrate the capacity of dCas9-TFs to drive the direct conversion of murine embryonic fibroblasts (MEFs) to cells of a neuronal phenotype. We utilized a dCas9-TF with both N-terminal and C-terminal VP64 transactivation domains and demonstrated a 10-fold improvement in activation of target genes compared to dCas9-TFs with only a single VP64. Using this dCas9-TF variant, we demonstrated the multiplex activation of BRN2, ASCL1, and MYT1L (BAM factors) in MEFs. These three factors have been shown to generate functional neurons when expressed ectopically in MEFs. When compared to overexpression of the transgenes, dCas9-TFs induced significantly less mRNA and protein expression of the BAM factors, as determined by qRT-PCR and immunofluorescence staining. However, by day 14 in culture following the delivery of dCas9-TFs targeting the endogenous BAM factors, we identified cells positive for the pan-neuronal markers TUJ1 and MAP2 that exhibited complex neuronal morphologies. Furthermore, we found that dCas9-TFs were able to generate 23% more TUJ1/MAP2-co-positive cells than that achieved through the ectopic delivery of the BAM factors. Electrophysiological recordings of these cells identified single, action potential-like responses to step-depolarization by current clamp in the whole cell configuration. The cells also displayed calcium transients in response to KCl-induced depolarization, measured by monitoring fluorescence intensity of a GCaMP reporter. Ongoing work is focused on improving the functional maturity of the cells and addressing the kinetics of gene activation and epigenetic reprogramming at the target loci. We hypothesize that the targeted activation at the endogenous loci may more deterministically reprogram the chromatin to marks of active transcription that stabilize gene expression.