Figure 5. AAV-delivered autoregulated Cas13d reduces CUG_n RNA accumulation in human DM1 myoblasts.

(A) Fluorescent western blot of Cas13d in AAV-transduced DM1 patient-derived myoblasts. Cas13d is visualized with α-HA antibody (green), and HSP70 is included as a control (pink). Protein ladder (L) is shown.

(B) Representative 20× confocal images of AAV-treated DM1 myoblasts stained for Cas13d mRNA (HCR FISH, magenta) and protein (α-HA IF, yellow). Nuclei stained with DAPI (cyan). Scale bars, 10 μm.

(C) Mean nuclear intensity of Cas13d HCR FISH in unregulated and GENO NT conditions. Dots represent nuclei, black line indicates median. n > 33 nuclei, >3 images. ***p < 0.001, two-sided Mann-Whitney U test. Gray line indicates mean baseline nuclear FISH signal in PBS-treated myoblasts, and gray shaded region indicates SD. n = 26 nuclei, 3 images.

(D) Mean nuclear intensity of α-HA IF in unregulated and GENO NT conditions. Dots represent nuclei, black line indicates median. n > 33 nuclei, >3 images. ***p < 0.001, two-sided Mann-Whitney U test. Gray line and shaded region calculated as in (C).

(E) Representative 40× confocal images of AAV-treated DM1 myoblasts stained for Cas13d mRNA (HCR FISH, magenta) and CUG_n RNA (CAG₁₀ FISH probe, grayscale). Nuclei stained with DAPI (cyan). Scale bars, 10 μm.

(F) Mean nuclear intensity of CUG_n FISH in GENO targeting and NT conditions. Dots represent nuclei, black line indicates median. n > 43 nuclei, 21 images. *p < 0.05, one-sided Mann-Whitney U test.

(G) Cumulative distribution function (c.d.f.) of FISH spot intensity of individual CUG_n RNA foci in GENO targeting (magenta, solid) and NT (gray, solid) conditions. PBS-treated condition is also shown (gray, dashed). n > 745 spots. ***p < 0.001, two-sided Mann-Whitney U test.