A modular CRISPRa system for molecular therapy of FOXG1 syndrome

To date, there is an unmet need for novel approaches that address haploinsufficiency disorders in a targeted and robust manner. Here, we developed a novel, modular tool for transcriptional regulation of genes involved in neurodevelopmental disease in the absence of creating double-stranded breaks. We utilized this novel platform technology to identify sgRNAs that efficiently regulate expression of the intellectual disability gene FOXG1 that utilizes an optimized protein intein system. Using a modular assembly of CRISPR trans-activators, including single and dual VP64 dCas9 fusion proteins, sgRNA-mediated MS2 p65 HSF (MPH) recruitment or the addition of the VPR tripartite effector (VP64, p65, Rta) can induce dynamic gene regulation. We further increased mRNA levels of FOXG1 using duplexed sgRNAs paired with different combinations of transcriptional activators. We demonstrate that FOXG1 gene upregulation results in a significant increase in FoxG1 protein translation. Following treatment of mutant FOXG1 neural stem cells with the CRISPR activators, we observed a significant increase in FOXG1 expression with no-off targets as determined by in silico sgRNA binding prediction paired with RNA-sequencing. This approach holds great promise as a modular platform technology that is readily adapted for gene therapy delivery for those affected by rare genetic conditions. Overall design: RNAseq from healthy neural stem cells, mutant neural stem cells and CRISPR corrected, isogenic neural stem cells following CRISPRa or no effector transfection