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Nils Schneider1,2, Christoph Gäbelein1,2, Julius Wiener1,2

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This study introduces a novel method combining genetic code expansion and CRISPR/Cas9 to label proteins within cells. This technique allows for precise tracking of protein synthesis and localization without disrupting normal cell function.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Cell Biology

Background:

  • Accurate spatiotemporal labeling of endogenous proteins is crucial for understanding cellular dynamics.
  • Existing methods may lack precision or interfere with physiological processes.

Purpose of the Study:

  • To develop and validate a method for high-resolution, spatiotemporal labeling of endogenous proteins.
  • To enable quantification of protein synthesis rates and localization at the single-cell level.

Main Methods:

  • Utilized genetic code expansion with an orthogonal tRNA/tRNA synthetase pair.
  • Employed CRISPR/Cas9 genome engineering for precise protein labeling.
  • Incorporated noncanonical amino acids for stop codon read-through events.
  • Applied proximity ligation assay and real-time imaging for analysis.

Main Results:

  • Successfully pulse-labeled endogenous β-actin and tumor protein p53 with an HA tag.
  • Demonstrated seamless quantification of protein synthesis rates and spatial localization.
  • Confirmed no interference with physiological cellular expression control.
  • Observed no perturbation of endogenous protein functions.

Conclusions:

  • The combined genetic code expansion and CRISPR/Cas9 method provides a powerful tool for studying endogenous protein dynamics.
  • This approach offers high spatiotemporal resolution and minimal disruption to cellular processes.
  • Enables precise quantification of protein synthesis and localization in real-time.