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Updated: May 8, 2025

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development
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Optoribogenetic Modulation of Transcription.

Christian Renzl1, Günter Mayer2,3

  • 1LIMES, University of Bonn, Bonn, Germany. crenzl@uni-bonn.de.

Methods in Molecular Biology (Clifton, N.J.)
|December 26, 2024
PubMed
Summary
This summary is machine-generated.

This study presents a novel optogenetic CRISPR/dCas9 system for light-controlled gene expression. The method enables precise, reversible activation of gene overexpression, applicable to endogenous genes.

Keywords:
AptamersCRISPRaGene regulationOptoribogeneticsPhotoreceptor protein

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Area of Science:

  • Molecular Biology
  • Optogenetics
  • Gene Regulation

Background:

  • Optogenetic tools offer precise spatiotemporal control over biological processes.
  • CRISPR/dCas9 systems are powerful tools for gene editing and regulation.
  • Existing methods for gene expression control lack precise light-dependent activation.

Purpose of the Study:

  • To develop a light-inducible CRISPR/dCas9 system for controlled gene expression.
  • To demonstrate the feasibility of light-dependent gene overexpression using a proof-of-concept experiment.
  • To establish a protocol for rapid, in vitro application of this optogenetic system.

Main Methods:

  • Integration of the PAL photoreceptor and aptamer 53 into a CRISPR/dCas9 framework.
  • Development of a protocol involving cell seeding, transfection, and flow cytometry analysis.
  • Utilizing eBFP as a reporter for proof-of-concept validation.

Main Results:

  • Successful implementation of light-dependent gene overexpression in vitro.
  • Demonstration of precise and reversible control over gene expression using light.
  • Validation of the system's applicability for upregulating endogenous genes.

Conclusions:

  • The developed optogenetic CRISPR/dCas9 system provides a robust platform for light-controlled gene activation.
  • This method offers subcellular resolution and broad applicability for gene expression studies.
  • The 3-day protocol facilitates efficient and rapid experimentation.