CRISPR mediated targeting of DUX4 distal regulatory element represses DUX4 target genes dysregulated in Facioscapulohumeral muscular dystrophy

Abstract

Facioscapulohumeral muscular dystrophy (FSHD) is a debilitating muscle disease that currently does not have an effective cure or therapy. The abnormal reactivation of DUX4, an embryonic gene that is epigenetically silenced in somatic tissues, is causal to FSHD. Disease-specific reactivation of DUX4 has two common characteristics, the presence of a non-canonical polyadenylation sequence within exon 3 of DUX4 that stabilizes pathogenic transcripts, and the loss of repressive chromatin modifications at D4Z4, the macrosatellite repeat which encodes DUX4. We used CRISPR/Cas9 to silence DUX4 using two independent approaches. We deleted the DUX4 pathogenic polyadenylation signal, which resulted in downregulation of pathogenic DUX4-fl transcripts. In another approach, we transcriptionally repressed DUX4 by seeding heterochromatin using the dCas9-KRAB platform within exon 3. These feasibility of targeting DUX4 experiments were initially tested in a non-myogenic carcinoma cell line that we have previously characterized. Subsequently, in an immortalized patient myoblast cell line, we demonstrated that targeting DUX4 by either approach led to substantial downregulation of not only pathogenic DUX4 transcripts, but also a subset of its target genes that are known biomarkers of FSHD. These findings offer proof-of-concept of the effect of silencing the polyadenylation sequence on pathogenic DUX4 expression.

The KRAB Domain of ZNF10 Guides the Identification of Specific Amino Acids That Transform the Ancestral KRAB-A-Related Domain Present in Human PRDM9 into a Canonical Modern KRAB-A Domain

“…Here and throughout the manuscript amino acids with nonpolar side-chains are colored black, those with polar side-chains are in green, ones with basic side-chains are blue and those with acidic side-chains are red. [30][31][32] along with the HMM scores (E-values; the lower, the better) against the different KRAB-A models. Selection based on best hits of a group member vs the different KRAB-A models, inclusion of phylogenetically oldest species and added outliers (names in red).…”

“…Of major interest is the role of KZNF proteins in the restriction of retroviral genomic sequences, in particular during development, and for exaptation, i.e., their repurposing for novel regulatory principles [16,25,26]. In conjunction with CRISPR/Cas the ZNF10-KRAB is used as a tool for modulating and redirecting transcriptional gene regulation in basic research (CRISPRi; [27,28]), and applied in oncology (e.g., [29,30]), in human genetics (e.g., [31]) and virology (e.g., [32]).…”

DUX4 Role in Normal Physiology and in FSHD Muscular Dystrophy

“…One obvious advantage over murine transgenic models is that human-to-mouse muscle xenografts are comprised almost exclusively of human tissue and thus provide the best source of mature human muscle, outside of the clinic, to study the specificity and efficacy of drugs designed to treat FSHD. In particular, xenograft models of FSHD are the only setting in which therapeutic approaches targeting the FSHD locus and DUX4 epigenetic or transcriptional regulation [ 157 , 159 , 160 , 161 , 162 , 163 , 164 , 165 ] can be tested in vivo.…”

“…Another strategy to increase epigenetic silencing at the FSHD locus is to use an enzymatically inactive “dead” Cas9 (dCas9) fused to an effector domain from an epigenetic repressor (CRISPRi) [ 181 ]. Using this approach, repression of DUX4 and its targets has been achieved in cell lines and in FSHD muscle cells [ 160 , 163 , 165 ].…”

Downstream events initiated by expression of FSHD-associated DUX4: Studies of nucleocytoplasmic transport, γH2AX accumulation, and Bax/Bak-dependence

“…As potential therapies for FSHD, multiple groups are developing technologies to inhibit the function or expression of DUX4-FL (e.g. Bosnakovski et al, 2019 ; Bouwman et al, 2021 ; Das and Chadwick, 2021 ; Himeda et al, 2020 ; Lim et al, 2020 , 2021 ; Lu-Nguyen et al, 2021 ; Mariot et al, 2020 ; Mellion et al, 2021 ; Oliva et al, 2019 ; Rashnonejad et al, 2020 ; Šikrová et al, 2021 ). Direct targeting of DUX4 will likely be the primary therapeutic strategy for FSHD, because once begun, any technique that inhibits DUX4 expression or function should block multiple pathogenic changes that are dependent on DUX4 transcriptional activity ( Mitsuhashi et al, 2018 ).…”