Figure 1. Cas13d reduces accumulation of CUG_n repeat RNA and rescues MBNL-dependent splicing in a HeLa cell-culture model of DM1.

(A) Diagram of cotransfection experiments to test Cas13d on-target activity and MBNL-dependent splicing. Plasmids encoding Cas13d and EGFP, gRNA, CUG₄₈₀ target RNA, and an MBNL1 exon 5 splicing minigene are expressed in HeLa cells prior to CUG_n RNA FISH and RT-PCR.

(B) gRNAs tested, including three CUG-targeting gRNAs and a non-targeting (NT) control.

(C) Representative FISH images for CUG_n RNA (grayscale) in targeting and NT conditions. Cells stained with DAPI (nuclei, cyan) and CellMask (cells, magenta) for segmentation. Scale bars, 10 μm.

(D) Magnified images of CUG_n RNA FISH (grayscale) from (C) in targeting and NT conditions. Outlines of nuclei (blue) computed by automatic thresholding. Scale bars, 10 μm.

(E) Median CUG_n FISH signal per nucleus, relative to NT. Error bars show 95% confidence interval (CI), estimated by bootstrapping. n > 7 nuclei. *p < 0.05, non-overlapping CI. n.s., not significant (p > 0.05).

(F) Diagram of splicing minigene assay. Sequestration of MBNL by CUG_n RNA increases MBNL1 exon 5 inclusion ratio (ψ). CUG-targeting Cas13d aims to rescue ψ to unperturbed levels.

(G) MBNL1 exon 5 ψ for targeting and NT conditions, measured by RT-PCR. Transcriptional inhibition by deactivated Cas9 (dCas9) with matching spacers is also shown. Error bars show standard deviation (SD). n = 3. *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed Student’s t test.