Deletion of the CTG Expansion in Myotonic Dystrophy Type 1 Reverses <i>DMPK</i> Aberrant Methylation in Human Embryonic Stem Cells but not Affected Myoblasts

Yanovsky‐Dagan, Shira; Bnaya, Ester; Diab, Manar Abu; Handal, Tayma; Zahdeh, Fouad; Broek, W.J.A.A. van den; Epsztejn‐Litman, Silvina; Wansink, Derick G.; Eiges, Rachel

doi:10.1101/631457

Cited by 8 publications

(18 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The success of the repeat excision strategy is confirmed in a number of studies by other laboratories [21,22,23,24]. Together, these reports show the reliability and robustness of the CRISPR/Cas technique, since different choices were made regarding the sgRNA sequences, located closer to or further away from the (CTG•CAG)n sequence (Table 1).…”

Section: Crispr/cas-mediated Genome Editing In the Dm1 Locussupporting

confidence: 53%

“…Notably, we and others also discovered that a DSB close to the expanded repeat (<50 bp) induces uncontrolled deletion of large repeat segments, thereby resulting in unpredictable repeat contraction [18,21,24]. This phenomenon, not observed when sgRNAs are directed further away from the repeat (>200 bp) [22], relates probably to the occurrence of unstable slipped-strand structures at (CTG•CAG)n tracts in or close to a DSB [27,28] (see for an excellent review on this topic [29]).…”

Section: Crispr/cas-mediated Genome Editing In the Dm1 Locusmentioning

confidence: 99%

“…In most studies, one or more of these molecular measures were tested and these indeed improved after removal of the expansion (Table 1). One particular DNA biomarker for CDM, that deserves special attention, is hypermethylation of the CpG island surrounding the repeat in the DM1 locus [24]. This abnormal chromatin structure is commonly seen in patient cells with repeat lengths of over a few hundred triplets.…”

Section: Crispr/cas-mediated Genome Editing In the Dm1 Locusmentioning

confidence: 99%

“…This abnormal chromatin structure is commonly seen in patient cells with repeat lengths of over a few hundred triplets. In a collaborative project between the Eiges laboratory and our group, we compared CpG methylation before and after repeat removal in ESCs and immortalized myoblasts, both carrying CDM-size repeats with corresponding hypermethylation [24]. To our surprise, excision of the repeat in undifferentiated stem cells resets the methylation status in the locus, but methylation levels remain unchanged in affected myoblasts after deletion of the large expansion.…”

Section: Crispr/cas-mediated Genome Editing In the Dm1 Locusmentioning

confidence: 99%

See 3 more Smart Citations

CRISPR/Cas Applications in Myotonic Dystrophy: Expanding Opportunities

Raaijmakers

Ripken

Ausems

et al. 2019

IJMS

Self Cite

View full text Add to dashboard Cite

CRISPR/Cas technology holds promise for the development of therapies to treat inherited diseases. Myotonic dystrophy type 1 (DM1) is a severe neuromuscular disorder with a variable multisystemic character for which no cure is yet available. Here, we review CRISPR/Cas-mediated approaches that target the unstable (CTG•CAG)n repeat in the DMPK/DM1-AS gene pair, the autosomal dominant mutation that causes DM1. Expansion of the repeat results in a complex constellation of toxicity at the DNA level, an altered transcriptome and a disturbed proteome. To restore cellular homeostasis and ameliorate DM1 disease symptoms, CRISPR/Cas approaches were directed at the causative mutation in the DNA and the RNA. Specifically, the triplet repeat has been excised from the genome by several laboratories via dual CRISPR/Cas9 cleavage, while one group prevented transcription of the (CTG)n repeat through homology-directed insertion of a polyadenylation signal in DMPK. Independently, catalytically deficient Cas9 (dCas9) was recruited to the (CTG)n repeat to block progression of RNA polymerase II and a dCas9-RNase fusion was shown to degrade expanded (CUG)n RNA. We compare these promising developments in DM1 with those in other microsatellite instability diseases. Finally, we look at hurdles that must be taken to make CRISPR/Cas-mediated editing a therapeutic reality in patients.

show abstract

Section: Crispr/cas-mediated Genome Editing In the Dm1 Locussupporting

confidence: 53%

Section: Crispr/cas-mediated Genome Editing In the Dm1 Locusmentioning

confidence: 99%

Section: Crispr/cas-mediated Genome Editing In the Dm1 Locusmentioning

confidence: 99%

Section: Crispr/cas-mediated Genome Editing In the Dm1 Locusmentioning

confidence: 99%

See 2 more Smart Citations

CRISPR/Cas Applications in Myotonic Dystrophy: Expanding Opportunities

Raaijmakers

Ripken

Ausems

et al. 2019

IJMS

Self Cite

View full text Add to dashboard Cite

show abstract

“…This is consistent with the report of Yanovsky-Dagan et al (2019) demonstrating demethylation upon CTG repeat excision in DM1 hESCs. However, they suggest that in undifferentiated hESCs, CpG methylation is re-established de novo after demethylation during DNA replication 37 .…”

Section: Discussionmentioning

confidence: 99%

MSH2knock-down shows CTG repeat stability and concomitant upstream demethylation at theDMPKlocus in myotonic dystrophy type 1 human embryonic stem cells

Franck

Barbé

Ardui

et al. 2020

Preprint

View full text Add to dashboard Cite

Myotonic dystrophy type 1 (DM1) is caused by expansion of a CTG repeat in the DMPK gene, where expansion size and somatic mosaicism correlates with disease severity and age of onset. While it is known that the mismatch repair protein MSH2 contributes to the unstable nature of the repeat, its role on other disease-related features, such as CpG methylation upstream of the repeat, is unknown. In this study, we investigated the effect of an MSH2 knock-down (MSH2KD) on both CTG repeat dynamics and CpG methylation pattern in human embryonic stem cells (hESC) carrying the DM1 mutation. Repeat size in MSH2 wild type (MSH2WT) and MSH2KD DM1 hESC was determined by PacBio sequencing and CpG methylation by bisulfite massive parallel sequencing. We found stabilization of the CTG repeat concurrent with a gradual loss of methylation upstream of the repeat in MSH2KD cells, while the repeat continued to expand and upstream methylation remained unchanged in MSH2WT control lines. Repeat instability was re-established and biased towards expansions upon MSH2 transgenic re-expression in MSH2KD lines while upstream methylation was not consistently re-established. We hypothesize that the hypermethylation at the mutant DM1 locus is promoted by the MMR machinery and sustained by a constant DNA repair response, establishing a potential mechanistic link between CTG repeat instability and upstream CpG methylation. Our work represents a first step towards understanding how epigenetic alterations and repair pathways connect and contribute to the DM1 pathology.

show abstract

Time-controlled and muscle-specific CRISPR/Cas9-mediated deletion of CTG-repeat expansion in the DMPK gene

Cardinali

Provenzano

Izzo

et al. 2022

Molecular Therapy - Nucleic Acids

View full text Add to dashboard Cite

CRISPR/Cas9-mediated therapeutic gene editing is a promising technology for durable treatment of incurable monogenic diseases such as myotonic dystrophies. Gene-editing approaches have been recently applied to in vitro and in vivo models of myotonic dystrophy type 1 (DM1) to delete the pathogenic CTG-repeat expansion located in the 3′ untranslated region of the DMPK gene. In DM1-patient-derived cells removal of the expanded repeats induced beneficial effects on major hallmarks of the disease with reduction in DMPK transcript-containing ribonuclear foci and reversal of aberrant splicing patterns. Here, we set out to excise the triplet expansion in a time-restricted and cell-specific fashion to minimize the potential occurrence of unintended events in off-target genomic loci and select for the target cell type. To this aim, we employed either a ubiquitous promoter-driven or a muscle-specific promoter-driven Cas9 nuclease and tetracycline repressor-based guide RNAs. A dual-vector approach was used to deliver the CRISPR/Cas9 components into DM1 patient-derived cells and in skeletal muscle of a DM1 mouse model. In this way, we obtained efficient and inducible gene editing both in proliferating cells and differentiated post-mitotic myocytes in vitro as well as in skeletal muscle tissue in vivo .

show abstract

Deletion of the CTG Expansion in Myotonic Dystrophy Type 1 Reverses DMPK Aberrant Methylation in Human Embryonic Stem Cells but not Affected Myoblasts

Cited by 8 publications

References 38 publications

CRISPR/Cas Applications in Myotonic Dystrophy: Expanding Opportunities

CRISPR/Cas Applications in Myotonic Dystrophy: Expanding Opportunities

MSH2knock-down shows CTG repeat stability and concomitant upstream demethylation at theDMPKlocus in myotonic dystrophy type 1 human embryonic stem cells

Time-controlled and muscle-specific CRISPR/Cas9-mediated deletion of CTG-repeat expansion in the DMPK gene

Contact Info

Product

Resources

About