Gene Editing to Tackle Facioscapulohumeral Muscular Dystrophy

Figures

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  FIGURE 1. Schematic structure and activity of TALEN and CRISPR/cas9 for genome editing. (A): TALENs and CRISPR/cas9 are engineered to bind a target sequence of interest by assembly from TALE repeat units specific to each base pair or simple cloning into sgRNA expression vector, respectively. (B): Nuclease-induced genome editing. Double strand break can be repaired by Non-homologous end joining (NHEJ) or homology directed repair (HDR). NHEJ can lead to small insertions and deletions (indels) at the DBS site, whereas HDR can lead to more precise mutations from a DNA donor template. (C): Schematic representation of the CRISPR-Cas9 system where HNH and RuvC represents 2 nuclease domains of Cas9. The single guide RNA (sgRNA) molecule directs Cas9 protein to a specific DNA target. The Cas 9 cleaves genomic DNA upstream to the PAM (Protospacer Adjacent Motif) sequence. The Cas9 can be fused to different modulators or editors to mediate gene expression regulation or base editing.

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  FIGURE 2. The locus D4Z4 and molecular mechanisms leading of FSHD. The D4Z4 locus is composed of 11–100 repeats in healthy individuals and is highly methylated. FSHD1 patients present a contraction of the D4Z4 array (1–10 units left) leading to chromatin relaxation. FSDH2 patients present also a chromatin relaxation that is mediated by mutations in epigenetic modifier genes. FSHD occurs when the D4Z4 array is relaxed and DUX4 expressed. DUX4 expression is uniquely associated with the 4qA haplotype, which carries the DUX4 p(A).

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  FIGURE 3. DUX4 is a toxic protein. DUX4 is composed of 2 DNA-binding homeodomains (HD1 and HD2) localized in its N-terminal region. The C-terminal part of the protein includes the transcription transactivation domain (TAD). DUX4 expression perturbs many cellular pathways involved leading to in myofiber death, increased sensitivity to oxidative stress, defects in myogenesis, muscle atrophy, inhibition of the non-sense mediated decay and the aberrant expression of hundreds of genes.

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  FIGURE 4. Position of the different sgRNAs used that target the 3′UTR of DUX4. The DUX4 poly(A) sequence is materialized by a red box. SgRNAs sequences are indicated upper or lower the main sequence (orientation dependant) by arrow. Sequences in Das 2021, Joubert 2020 or Sikrová 2021 are indicated in blue, green, purple respectively. Sequences are indicated in the lower part of the figure.