Impeding Transcription of Expanded Microsatellite Repeats by Deactivated Cas9

Abstract

Summary Transcription of expanded microsatellite repeats is associated with multiple human diseases, including myotonic dystrophy, Fuchs' endothelial corneal dystrophy, and C9orf72-ALS/FTD. Reducing production of RNA and proteins arising from these expanded loci holds therapeutic benefit. Here, we tested the hypothesis that deactivated Cas9 enzyme impedes transcription across expanded microsatellites. We observed a repeat length-, PAM-, and strand-dependent reduction of repeat-containing RNAs upon targeting dCas9 directly to repeat sequences; targeting the non-template strand was more effective. Aberrant splicing patterns were rescued in DM1 cells, and production of RAN peptides characteristic of DM1, DM2, and C9orf72-ALS/FTD cells was drastically decreased. Systemic delivery of dCas9/gRNA by adeno-associated virus led to reductions in pathological RNA foci, rescue of chloride channel 1 protein expression, and decreased myotonia. These observations suggest that transcription of microsatellite repeat-containing RNAs is more sensitive to perturbation than transcription of other RNAs, indicating potentially viable strategies for therapeutic intervention.

A CTG repeat-selective chemical screen identifies microtubule inhibitors as selective modulators of toxic CUG RNA levels

“…Generation of Stable DM1 HeLa (CTG)480 Cell Lines. Stable barcoded HeLa cell lines expressing r(CUG)0 and r(CUG)480 were generated as described (26). Briefly, (CTG)0 and (CTG)480 DMPK (exons 11-15) expression cassettes (37) containing a 22-bp unique sequence plus an additional 8-bp unique barcode for pooled RNA-seq were inserted into pAC156 (gift from Albert Cheng, The Jackson Laboratory, Farmington, CT), a plasmid with PiggyBac transposon terminal repeats as well as a puromycin selection cassette.…”

“…Many preclinical therapeutic studies focus on targeting nuclear r(CUG) EXP :MBNL aggregates using small molecules, antisense oligonucleotides, RNA interference, and proteins such as RNAtargeting Cas9 to free MBNL proteins and rescue DM1-associated missplicing (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23). Several studies have targeted transcription of the expanded CTG repeat tract using small molecules and deactivated Cas9, providing proof of principle for blocking r(CUG) EXP production as a viable therapeutic strategy in DM1 (24)(25)(26). Using the CRISPR/Cas9 system, several studies have also demonstrated the potential for contraction or complete removal of the expanded CTG repeat tract (27)(28)(29)(30).…”

Targeted Reactivation of FMR1 Transcription in Fragile X Syndrome Embryonic Stem Cells

“…Alternatively, there may be differences between hESCs and HEK293T cells in their basal transcriptomes that make them differentially sensitive to CGG repeat targeted gRNAs. This approach of directly targeting the repeats leverages the very nature of the repeat to achieve greater efficacy and specificity and has recently been used in other repeat expansion disorders to great effect (Batra, Nelles et al 2017, Pinto, Saxena et al 2017.…”

Experimental DNA- or RNA-Directed Therapies for Trinucleotide Repeat Disease

“…Pinto et al [37] tested the hypothesis that deactivation of the Cas9 enzyme impedes transcription across expanded microsatellites in DM1 cells. Systemic delivery of dCas9/gRNA by adeno-associated virus led to a reduction in mutant RNA and decreased myotonia [37].…”

“…Pinto et al [37] tested the hypothesis that deactivation of the Cas9 enzyme impedes transcription across expanded microsatellites in DM1 cells. Systemic delivery of dCas9/gRNA by adeno-associated virus led to a reduction in mutant RNA and decreased myotonia [37]. These results indicate that transcription of microsatellite repeat-containing RNAs is more sensitive to the action of Cas9 than transcription of other RNAs, making it a potential strategy for therapeutic intervention.…”