Fig. 3: RfxCas13d reduces ATXN2 mRNA in the brain and spinal cord.
From: Mitigating a TDP-43 proteinopathy by targeting ataxin-2 using RNA-targeting CRISPR effector proteins
a Schematic of the AAV vector encoding RfxCas13d and its crRNA. ITR, inverted terminal repeat; CBh, chicken β-actin promoter; NLS, nuclear localization signal; BGH, bovine growth hormone polyadenylation signal. b Overview of the experimental plan. ICV, intracerebroventricular; IV, intravenous. c–g Representative immunofluorescence staining of the c dorsal sagittal section of the brain (scale bar, 1 mm), d motor cortex (MC; scale bar, 50 µm), e hippocampus [HPC; scale bar, (left) 100 µm, (right) 50 µm], f striatum (STR; scale bar, 50 µm), g spinal cord [SC; scale bar, (left) 500 µm, (right) 100 µm]. c, g Images were acquired by tile-scanning using an AxioScan.Z1 (Carl R. Woese Institute for Genomic Biology, Univ. Illinois) and subsequently stitched together to display a larger view. h Quantification of the percentage of NeuN+ cells positive for the HA epitope tag. Values represent means and error bars indicate SD (n = 3). >104 NeuN+ cells counted were per replicate. i Relative mATXN2 mRNA in B6SJLF1/J mice injected with AAV9-CBh-RfxCas13d-mATXN2. Data are normalized to relative mATXN2 mRNA from B6SJLF1/J mice injected with AAV9-CBh-RfxCas13d-NTG. Values represent means and error bars indicate SD (n = 4 for the MC; n = 5 for NTG and n = 4 for mATXN2 in the HPC, n = 4 for the STR, n = 5 for NTG and n = 4 for mATXN2 in the BS; n = 4 for the SC). Data were compared using a one-tailed unpaired t-test, with the exact P-values shown. All analyses were conducted four weeks post-injection. All data points are biologically independent samples.
