Dual-targeting CRISPR-CasRx reducesC9orf72ALS/FTD sense and antisense repeat RNAs in vitro and in vivo

Abstract

AbstractThe most common genetic cause of both frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) is a G4C2repeat expansion in intron 1 of theC9orf72gene. This repeat expansion undergoes bidirectional transcription to produce sense and antisense repeat RNA species. Both sense and antisense-derived repeat RNAs undergo repeat-associated non-AUG translation in all reading frames to generate five distinct dipeptide repeat proteins (DPRs). Importantly, toxicity has been associated with both sense and antisense repeat-derived RNA and DPRs. This suggests targeting both sense and antisense repeat RNA may provide the most effective therapeutic strategy. The RNA-targeting CRISPR-Cas13 systems offer a promising avenue for simultaneous targeting of multiple RNA transcripts, as they mature their own guide arrays, thus allowing targeting of more than one RNA species from a single construct. We show that CRISPR-Cas13d originating fromRuminococcus flavefaciens(CasRx) can successfully reduceC9orf72sense and antisense repeat transcripts and DPRs to background levels in HEK cells overexpressingC9orf72repeats. CRISPR-CasRx also markedly reduced the endogenous sense and antisense repeat RNAs and DPRs in three independentC9orf72patient-derived iPSC-neuron lines, without detectable off-target effects. To determine whether CRISPR-CasRx is effectivein vivo, we treated two distinctC9orf72repeat mouse models using AAV delivery and observed a significant reduction in both sense and antisense repeat-containing transcripts. Taken together this work highlights the potential for RNA-targeting CRISPR systems as therapeutics forC9orf72ALS/FTD.