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Single-molecule live-cell RNA imaging with CRISPR-Csm.

Chenglong Xia1,2, David Colognori2,3, Xueyang Jiang2,3

  • 1California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, CA USA.

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|July 29, 2024
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Summary

Researchers developed single-molecule live-cell fluorescence in situ hybridization (smLiveFISH) to visualize unmodified RNA in living cells. This method tracked endogenous NOTCH2 and MAP1B mRNA, revealing distinct cellular localization mechanisms.

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Area of Science:

  • Molecular Biology
  • Cell Biology
  • Genetics

Background:

  • Understanding RNA dynamics in single cells requires high-resolution, real-time imaging.
  • Previous methods could not achieve live-cell imaging of unmodified endogenous RNA molecules.

Purpose of the Study:

  • To develop a novel method for direct visualization of single, unmodified endogenous RNA molecules in living cells.
  • To investigate the localization mechanisms of specific mRNA transcripts in real-time within single cells.

Main Methods:

  • Developed single-molecule live-cell fluorescence in situ hybridization (smLiveFISH).
  • Utilized the CRISPR-Csm complex with multiplexed guide RNAs for RNA targeting and visualization.
  • Applied smLiveFISH to track endogenous NOTCH2 and MAP1B mRNA in various cell types, including primary cells.

Main Results:

  • Successfully visualized and tracked individual endogenous NOTCH2 and MAP1B mRNA transcripts in living cells.
  • Identified co-translational translocation of NOTCH2 mRNA at the endoplasmic reticulum.
  • Observed directional transport of MAP1B mRNA toward the cell periphery.

Conclusions:

  • smLiveFISH enables robust, high-resolution live-cell imaging of unmodified endogenous RNA.
  • The study revealed distinct spatiotemporal organization mechanisms for native transcripts.
  • This method offers potential for uncovering principles of RNA localization in cellular function and disease.