Extended Data Fig. 8: Additional data related to the control of endogenous mRNA transport in primary mouse cortical neurons via CRISPR-TO in Fig. 3.

a, Quantification of cell viability for neurons under different treatments. b, Quantification of the ratio of ATF4 average fluorescent intensity between the nucleus and cytoplasm for neurons under different treatments to examine ER stress⁶⁹. In the right diagram, neurons were transfected with CRISPR-TO components (dCas13, KIF5A*-PYL1, and different gRNAs) and CAAX-mScarlet3. Cell populations with and without dCas13 expression were analyzed. For a-b, data was presented as mean values ± standard deviation. Each dot represents one biological replicate. n = 3 or 4 wells per group. Two-sided, unpaired Student’s t-test. c, Representative microscopic images showing the dramatic decrease of astrocytes after fluorodeoxyuridine (FUDR) treatment in the in vitro mouse cortical neuron culture system. Scale bar, 400 µm. d, Representative microscopic images showing very few microglial cells in the in vitro mouse cortical neuron culture system after FUDR treatment. Scale bar, 400 µm. e-f, Representative confocal microscopic images of a single neuron in the gT+DMSO group (e) or the gNT+ABA group (f). The left image shows the shape of a neuron. Scale bar, 100 µm. Right images show magnification of dotted boxes in the left image. Scale bar, 50 µm. g, Histogram of the fluorescence intensity of Actb mRNA particles stained by RNA FISH. n = 196 mRNA particles. h, Quantification of the fluorescence intensity of endogenous Actb mRNA (red) and Gap43 mRNA (green) at neurite tips after CRISPR-TO perturbation with different gRNAs, related to Fig. 3h. n indicates the number of quantified neurite tips for each group. The black bar indicates the mean value. Two-sided, unpaired Student’s t-test. i-j, Quantification of the integrated fluorescence intensity of Actb mRNA FISH signals in the cell body of primary mouse cortical neurons expressing gT/gNT only (i) or dCas13+gT/gNT (j). k, Quantification of the integrated fluorescence intensity of Actb mRNA FISH signals and dCas13 protein signals in the cell body of primary mouse cortical neurons expressing CRISPR-TO components and treated with ABA or DMSO. Black bar, mean. n, cell body. Two-sided, unpaired Student’s t-test. The number above each group is the fold change between two groups. l, Representative microscopic images showing similar Actb mRNA level inside somas (blue arrows) of neurons in gT+DMSO and gT+ABA group, and dCas13 proteins remaining inside somas after ABA treatment in the gT+ABA group. Scale bar, 50 µm. Calibration bar indicates the fluorescence intensity of Actb mRNA FISH signals and dCas13 proteins. Inset images surrounded by the dotted line represent magnification of the region indicated by the white arrow, showing Actb mRNA enriched at neurite tips in the gT+ABA group after CRISPR-TO perturbation.

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