Abstract
Neurodevelopmental disorders are characterized by motor and cognitive dysfunction, frequently resulting in intellectual disabilities. There are no disease-modifying treatments that tackle the underlying genetics of these disorders. However, the X chromosome has a disproportionate number of genes implicated in neurodevelopmental processes. In female somatic cells, the X chromosomes are under strict control of gene dosage through epigenetic modifications, and one of the two X chromosomes is randomly inactivated through these epigenetic changes. However, many silenced genes escape inactivation, implying there is a path to modify previously inactivated genes. We can target endogenous X-linked intellectual disability (XLID) genes to rescue inactivated wild-type copies using CRISPR-dCas9 technology. Here, an inducible epigenetic editor was developed for reactivating epigenetically silenced X-linked genes. Halmai et al. demonstrated that the XLID gene, cyclin dependent kinase-like 5 (CDKL5), can be reactivated using a demethylase, the catalytic domain of Ten-Eleven Translocation 1 (TET1CD) and the transcriptional activator, VP64 (Halmai et al., 2020). Furthermore, Gao et al. illustrated that targeted gene regulation can be modulated with inducers for temporal control (Gao et al., 2016). Here, we generated a chemically induced proximity ligation-based split CRISPR-dCas9 construct fused to effector domains, miniVPR and TET1CD, that can rewrite epigenetic marks on the inactive X chromosome to resemble the gene signatures of actively transcribed genes following induction. To create this epi-genome editor, we cloned four constructs, ABI-dCas9(N), dCas9(C)-GAI, PYL1-miniVPR, and GID1-TET1CD. To assess the longevity of the epigenetic modifications, we performed time course assays with an established epi-genome editor, miniVPR-dCas9(N) and dCas9N(C)-TET1CD (miniVPR-TET1CD) to observe the effects of targeted reactivation in K562 cells. Gene expression of CDKL5 was analyzed by using qPCR for both evaluating the functionality of the epi-genome editors and durability. We aimed to expand this tool to multiple XLID genes and created a reporter assay to screen guide RNAs. Our results showed that the ABA-inducible epi-genome editor sufficiently reactivates CDKL5. Additionally, miniVPR-TET1CD demonstrated a lack of durability, suggesting that there are challenges in maintaining epigenetic modifications. Overall, creating an inducible epigenetic editing dCas9 tool offers the potential for controlling the timing and extent of epigenetic modifications. Temporal control of epi-genome editors can deepen our understanding of epigenetic markers in gene function and in disease, paving the way for novel potential targeted therapeutic interventions.