Targeted CRISPR/dCas9-Mediated Reactivation of Epigenetically Silenced Genes Suggests Limited Escape from the Inactive X Chromosome

In the wake of genome engineering, rewriting of the epigenome has risen as a promising alternative for precision medicine. This approach is even more relevant in tissues that traditionally have been more difficult to edit at the genomic level, such as neurons. Neurological diseases are a heterogeneous group of disorders caused by alterations in nervous system function and many of these disorders can be attributed to genetic factors such as chromosomal aberrations or gene mutations. The neurodevelopmental disorder CDKL5 deficiency is caused by de novo mutations in the CDKL5 gene on the X-chromosome. Due to random X-chromosome inactivation, females affected by the disorder form a mosaic of cells expressing mutant and wild type alleles. Despite the availability of small molecule drugs that can globally reactivate silenced genes from the inactive X chromosome, locus specific approaches remain elusive. Our research is focused on methods to specifically reactivate the healthy CDKL5 allele on the silenced X-chromosome in human neuronal-like cell lines using CRISPR/dCas9 fused to epigenetic effector domains . Our group has been the first to identify proximal cis regulatory elements in the CDKL5 core promoter for VP64-mediated programmable transcription in the female neuronal-like cell line SH-SY5Y. Allele-specific deep sequencing shows that the observed increase in gene expression in stable SHSY5Y cells is predominantly due to superactivation of the active allele in combination with limited reactivation of the silenced allele. Epigenetic editing of the CDKL5 core promoter using Tet1-mediated targeted DNA demethylation suggests a significant increase in reactivation of the inactive allele accompanied by a reduction in methylated CpG diresidues as demonstrated by bisulfite amplicon sequencing. Further elucidating and understanding the epigenetics underlying X chromosome inactivation holds great potential for children suffering from CDKL5 deficiency and other X-linked disorders.