Programmable control of spatial transcriptome in live cells and neurons

Mengting Han, Maylin L. Fu, Yanyu Zhu, Alexander A. Choi, Emmy Li, Jon Bezney, Sa Cai, Leann Miles, Yitong Ma and Lei S. Qi ()
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Mengting Han: Stanford University
Maylin L. Fu: Stanford University
Yanyu Zhu: Stanford University
Alexander A. Choi: Stanford University
Emmy Li: Stanford University
Jon Bezney: Stanford University
Sa Cai: Stanford University
Leann Miles: Stanford University
Yitong Ma: Stanford University
Lei S. Qi: Stanford University

Nature, 2025, vol. 643, issue 8070, 241-251

Abstract: Abstract Spatial RNA organization has a pivotal role in diverse cellular processes and diseases1–4. However, functional implications of the spatial transcriptome remain largely unexplored due to limited technologies for perturbing endogenous RNA within specific subcellular regions1,5. Here we present CRISPR-mediated transcriptome organization (CRISPR-TO), a system that harnesses RNA-guided, nuclease-dead dCas13 for programmable control of endogenous RNA localization in live cells. CRISPR-TO enables targeted localization of endogenous RNAs to diverse subcellular compartments, including the outer mitochondrial membrane, p-bodies, stress granules, telomeres and nuclear stress bodies, across various cell types. It allows for inducible and reversible bidirectional RNA transport along microtubules via motor proteins, facilitating real-time manipulation and monitoring of RNA localization dynamics in living cells. In primary cortical neurons, we demonstrate that repositioned mRNAs undergo local translation along neurites and at neurite tips, and co-transport with ribosomes, with β-actin mRNA localization enhancing the formation of dynamic filopodial protrusions and inhibiting axonal regeneration. CRISPR-TO-enabled 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 sequencing and imaging technologies, offering a versatile platform for high-throughput functional interrogation of RNA localization in living cells and organisms.

Date: 2025
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DOI: 10.1038/s41586-025-09020-z

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