Gene Editing Targeting the DUX4 Polyadenylation Signal: A Therapy for FSHD?

Figures

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  Figure 1. TALEN and gRNA design. TALENs and guide RNAs were designed to target the D4Z4 region (A). 5 TALEN pairs were designed, and their positions are indicated (B). Six single guide RNAs (sgRNAs), among them, 4 are complexed to Streptococcus pyogenes Cas9 endonuclease (C) and 2 to Neisseria meningitides Cas9 (D). The PAS is indicated in red.

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  Figure 2. Nuclease selection. U2OS cells were transfected with expression plasmids for TALENs (A) or for guide RNAs and cognate Streptococcus pyogenes Cas9 (SPCas9 in B) or Neisseria meningitides Cas9 (NmCas9 in C) proteins. Two days after transfection, DNA was extracted and the frequency of indels in the targeted DUX4 PAS was analysed by the T7E1 assay. The target region was amplified by PCR, then PCR products were denatured, followed by re-annealing, leading to a population of double strand fragments, some of which containing mismatches that are detected and cleaved by the T7 endonuclease. The cleavage products are visible on an agarose gel. PCR product is 500 bp long and after T7E1 digestion, expected bands are roughly 200 and 300 bp long depending on the position of mutations induced by the nuclease tested. The proportion of indels was estimated from quantification of the different bands corresponding to non-cleaved and cleaved PCR products.

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  Figure 3. Flow chart and primers validation. The flow chart is described (A). Genomic modifications are assessed by sequencing, Primers were designed to specifically recognize chromosomes 4 or 10. The PCR products are similar in length (B) but digestion by MseI (present only on chromosome 4) or XmaI (present only on chromosome 10) confirmed the PCR specificity (C).

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  Figure 4. Characteristics of clone#1. This clone was selected after transfection of TALEN pairs 406-407 with a miR-1 ssDNA oligonucleotide. (A): The miR-1 sequence (in blue) is inserted within the PAS (in yellow). (B): Myoblasts from clone#1 were treated with 0.4 mM chaetocin and harvested 24 h later. DUX4 expression was assessed after RT-PCR Parental FSHD cells and one unmodified clone obtained in parallel to clone#1 were used as controls.

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  Figure 5. Characteristics of clone#2. (A): This clone was selected after transfection of TALEN pair 406-407 with both the miR-5′ and miR-random. The chromosomal modifications are shown. (B): DUX4 mRNA expression was assessed by RT-PCR in different wells with cells of the clone carrying the modification and in the parental unmodified FSHD cells (Par). (C): 3′RACE nested PCR using forward primers located in Exon 3 shows the redirection of the cleavage site in the modified clone (B) compared to the parental cells (Par), as indicated by the red arrows. (D): The sequence shows the redirection of the cleavage site. The PAS is highlighted in yellow. (E): Expression levels of 3 genes downstream of DUX4 (TRIM43, MBD3l2 and LEUTX) were measured in parental cells and wells A, B and C. Data are presented as means ± SD; ****: p < 0.0001; *: p < 0.05 by one-way ANOVA with Tukey’s post hoc test. When the parental cells are compared to the clone, statistical analysis are presented in blue. Other analyses are indicated on the graph. Par: parental.