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bioRxiv · 10.1101/2025.04.05.646707

Perturbing RNA localization for functional study in neurons

Qi, S.·Han, M.·Fu, M. L.·Zhu, Y.·Choi, A. A.·Li, E.·Bezney, J.·Cai, S.·Miles, L.·Ma, Y.
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Abstract

Spatial RNA organization plays a pivotal role in diverse cellular processes and diseases, but the functional implications of spatial RNA localization remain underexplored. We present CRISPR-mediated transcriptome organization (CRISPR-TO) that harnesses RNA-guided, nuclease-dead dCas13 for programmable control of RNA localization in live cells. CRISPR-TO enables targeted localization of RNAs to diverse subcellular compartments, including p-bodies, stress granules, telomeres, and nuclear stress bodies, across cell types. In primary cortical neurons, we demonstrate that repositioned mRNAs undergo local translation along neurites and at neurite tips and co-transport with ribosomes, with {beta}-actin mRNA localization enhancing the formation of dynamic filopodial protrusions and inhibiting axonal regeneration. Furthermore, CRISPR-TO-enabled parallel screening in primary neurons identifies Stmn2 mRNA localization as a driver of neurite outgrowth. By enabling large-scale perturbation of the spatial transcriptome, CRISPR-TO bridges a critical gap left by current sequencing and imaging technologies, offering a versatile platform for high-throughput functional interrogation of RNA localization in living cells and organisms.

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Qi, S., Han, M., Fu, M. L., Zhu, Y., Choi, A. A., Li, E., Bezney, J., Cai, S., Miles, L., Ma, Y.. 2025-04-08. Perturbing RNA localization for functional study in neurons. https://doi.org/10.1101/2025.04.05.646707

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