Fig. 2: RfxCas13d can target the G₄C₂ repeat-containing RNA and reduce RNA foci in C9-BACexp mice.

a Cartoon of the injection scheme. Created in BioRender. Gaj, T. (2024) https://BioRender.com/a93i238. b The experimental plan to analyze C9ORF72 mRNA in EGFP-KASH⁺ nuclei isolated by fluorescence-activated cell sorting (FACS). Created in BioRender. Gaj, T. (2024) https://BioRender.com/w54s355. c Representative immunofluorescent staining of the hippocampus (HPC) and motor cortex (MC) in C9-BACexp mice two-months after injection with 2 × 10¹⁰ GCs each of PHP.eB-RfxCas13d-crRNA and PHP.eB-EGFP-KASH. Scale bar, 20 µm. Relative all-V and V3 mRNA in d the HPC and e MC of EGFP-KASH⁺ nuclei from C9-BACexp mice injected with 2 × 10¹⁰ GCs each of PHP.eB-RfxCas13d-crRNA and PHP.eB-EGFP-KASH. All-V and V3 for each crRNA normalized to the non-targeted (NTG) control (n ≥ 7). f Representative RNA FISH for the G₄C₂ repeat RNA (magenta) from the HPC of C9-BACexp mice injected with 2 × 10¹⁰ GCs each of PHP.eB-RfxCas13d-crRNA and PHP.eB-EGFP-KASH. Scale bar, 5 µm. Quantification of g the number of RNA foci per EGFP-KASH⁺ cell in the HPC and h the percentage of EGFP-KASH⁺ cells with 0, 1–2, 3–4, or >5 foci (n ≥ 4). g, h The total number of cells counted per biological replicate is described in Supplementary Table 1. RNA foci were counted by a blinded investigator. Values indicate means and error bars indicate SD. d, e All-V and V3 for each crRNA compared to NTG using a one-tailed unpaired t-test, with exact P values shown. g crRNAs 13 and 7 compared to NTG using a one-tailed unpaired t-test, with exact P values shown. All data points are biologically independent samples. Source data are provided in the Source Data file.