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Updated: Mar 23, 2026

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Programmable RNA Tracking in Live Cells with CRISPR/Cas9.

David A Nelles1, Mark Y Fang2, Mitchell R O'Connell3

  • 1Department of Cellular and Molecular Medicine and Institute for Genomic Medicine, University of California, San Diego, La Jolla, CA 92037, USA; Materials Science and Engineering Graduate Program, University of California, San Diego, La Jolla, CA 92093, USA.

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Researchers developed a new CRISPR/Cas9 tool (RCas9) to track RNA in living cells without needing genetic tags. This RNA-targeting Cas9 system allows programmable visualization of endogenous messenger RNA (mRNA) in real-time.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • CRISPR/Cas9 systems from Streptococcus pyogenes are powerful for genome editing.
  • Current RNA targeting methods often require exogenous tags, limiting flexibility.
  • A programmable RNA targeting tool is needed for endogenous RNA analysis and imaging.

Purpose of the Study:

  • To demonstrate a nuclease-inactive CRISPR/Cas9 system for programmable RNA targeting.
  • To enable endogenous RNA tracking and imaging in living cells without genetic tags.
  • To investigate mRNA localization and trafficking dynamics.

Main Methods:

  • Utilized nuclease-inactive Streptococcus pyogenes CRISPR/Cas9 (RCas9) engineered for RNA binding.
  • Employed guide RNAs (sgRNAs) to program RCas9 targeting of specific messenger RNAs (mRNAs).
  • Observed RCas9 localization and mRNA accumulation in living cells using microscopy and fluorescence in situ hybridization.

Main Results:

  • Nuclear-localized RCas9 was exported to the cytoplasm upon binding sgRNAs targeting mRNA.
  • Accumulation of ACTB, CCNA2, and TFRC mRNAs was observed in RNA granules.
  • Demonstrated time-resolved tracking of ACTB mRNA to stress granules, revealing trafficking dynamics.

Conclusions:

  • RCas9 provides a programmable, tag-free method for tracking endogenous RNA in living cells.
  • This RNA-targeting Cas9 system facilitates the study of mRNA localization and dynamics.
  • The findings establish a new tool for RNA biology research analogous to genomic CRISPR tools.