Multiplex Epigenome Editing ofMECP2to Rescue Rett Syndrome Neurons

Abstract

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused by loss-of-function heterozygous mutations of Methyl CpG-binding Protein 2 (MECP2) on the X chromosome in girls. Reactivation of the silent wild-type MECP2 allele from the inactive X chromosome (Xi) represents a promising therapeutic opportunity for female patients with RTT. Here, we applied a multiplex epigenome editing approach to reactivate MECP2 from Xi in RTT human embryonic stem cells (hESCs) and derived neurons. Demethylation of the MECP2 promoter by dCas9-Tet1 with target sgRNA reactivated MECP2 from Xi in RTT hESCs without detectable off-target effects at the transcriptome level. Neurons derived from methylation-edited RTT hESCs maintained MECP2 reactivation and reversed the smaller soma size and electrophysiological abnormalities. In RTT neurons, insulation of the methylation edited MECP2 locus by dCpf1-CTCF with target CRISPR RNA enhanced MECP2 reactivation and rescued RTT-related neuronal defects, providing a proof-of-concept study for epigenome editing to treat RTT and potentially other dominant X-linked diseases.