Fig. 4: High-fidelity RfxCas13d can target the G4C2 repeat-containing RNA and improve deficits in C9-BACexp mice.
From: A high-fidelity CRISPR-Cas13 system improves abnormalities associated with C9ORF72-linked ALS/FTD
a Cartoon of the injection scheme. Created in BioRender. Gaj, T. (2024) https://BioRender.com/a93i238. b 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-N2V8-crRNA and PHP.eB-EGFP-KASH. Scale bar, 30 µm. Relative all-V and V3 mRNA in EGFP-KASH+ nuclei from c the HPC and d MC of C9-BACexp mice injected with 2 × 1010 GCs each of PHP.eB-RfxCas13d-N2V8-crRNA with PHP.eB-EGFP-KASH. All-V and V3 for crRNA-13 normalized to the non-targeted (NTG) control (n ≥ 6). e Representative FISH for the G4C2 repeat RNA (magenta) in EGFP-KASH+ cells from the HPC of C9-BACexp mice injected with 2 × 1010 GCs each of PHP.eB-RfxCas13d-N2V8-crRNA and PHP.eB-EGFP-KASH. Scale bar, 5 µm. f Quantification of the number of RNA foci per EGFP-KASH+ cell in the (left) HPC and (right) MC of injected C9-BACexp mice (n ≥ 6). The total number of cells counted per biological replicate is described in Supplementary Table 3. g Soluble poly(GP) in the HPC of injected C9-BACexp mice (n ≥ 6). Volcano plot of the RNA-seq analysis from C9-BACexp mice injected with h PHP.eB-RfxCas13d-N2V8-NTG or i PHP.eB-RfxCas13d-N2V8-crRNA-13 and compared to wild-type littermates (n = 3–7). Lines denote a > 1.2-fold change (FC) and an FDR-adjusted P < 0.05. FC of the (j) down-regulated or (k) up-regulated DEGs from the analysis in (h, i). Values indicate means and error bars indicate SD. c, d All-V and V3 for crRNA-13 compared to NTG using a one-tailed unpaired t-test, with exact P values shown. f, g crRNA−13 compared to NTG using a one-tailed unpaired t-test, with exact P values shown. h, i FDR-adjusted P values were determined by a global FDR correction across pairwise comparisons. j, k crRNA-13 compared to NTG using a two-tailed unpaired t-test. All data points are biologically independent samples. Source data are provided in the Source Data file.
