Site-specific R-loop formation induces CGG repeat contraction and Fragile X gene reactivation

Fragile X syndrome (FXS) is a neurodevelopmental disorder and a leading cause of intellectual disability. In FXS, the neuronal regulator, FMR1, is epigenetically silenced by a CGG repeat expansion. Here, we investigate conditions under which repeat expansion and gene silencing could be reversed. Surprisingly, inducing formation of R-loops (3-stranded RNA-DNA structures) within FMR1 is sufficient to initiate CGG contraction, promoter demethylation, and FMR1 reactivation. Recruiting RNaseH degrades the R-loop and abolishes the response. Targeting the nascent mRNA for degradation also eliminates the response. Thus, we have identified an exogenous nuclease-free method of contracting CGG repeats and reversing FMR1 silencing. We propose that DNA demethylation, new transcription, and R-loop formation engage in a feed-forward cycle to contract CGG repeats and reactivate FMR1. Four RNA-seq samples consisting of two replicates from the FXS full-mutation iPS cells expressing dCas9 and gNHG3(guide RNA targeting to FMR1 gene) and two replicates with scrambled guide RNA (gScr) as control.