We were particularly interested in the synthetic activation of gene expression from the NRNX1 locus, given the size and diverse alternative splice repertoire of its gene products; we were surprised at our inability to increase expression. We can only speculate that known (Runkel et al., 2013) or unknown epigenetic effects near the NRXN1 promoter prevented us from increasing expression of this key neural gene in NPCs. Numerous factors have been hypothesized to affect gRNA efficacy for genome-editing purposes, including nucleosome positioning and 3D genome architecture of different cell types (Smith et al., 2016), which can limit access of the gRNA to the target site. As our understanding of the epigenome grows, and with it our ability to better predict gRNA targets, we hope that dCas9-mediated transcriptional modulation will become a more robust and scalable technology.
