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Translational regulation in prokaryotes ensures efficient protein synthesis by controlling ribosome access to mRNA. This regulation is mediated by secondary RNA structures, including translational riboswitches, RNA thermometers, and small RNAs (sRNAs), which respond to intracellular and environmental signals to modulate gene expression.Translational RiboswitchesRiboswitches in the leader region of mRNAs can regulate translation by altering the accessibility of the Shine-Dalgarno (SD) sequence,...
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The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids, forming the foundation for its adaptation as a powerful genome-editing tool. Originally discovered in prokaryotes, this system has been repurposed to revolutionize genetic engineering across a wide range of organisms, including plants, animals, and humans. The core component, Cas9, is an endonuclease derived from Streptococcus pyogenes, capable of introducing...
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The gene expression in cells is regulated at different stages: (i) transcription, (ii) RNA processing, (iii) RNA localization, and (iv) translation. Transcriptional regulation is mediated by regulatory proteins such as transcription factors, activators, or repressors—these control gene expression by initiating or inhibiting the transcription of genes. Once a precursor or pre-mRNA is produced, it undergoes post-transcriptional modification, including 5' capping, splicing, and the...
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Programmable mammalian translational modulators by CRISPR-associated proteins.

Shunsuke Kawasaki1, Hiroki Ono2,3, Moe Hirosawa2

  • 1Department of Life Science Frontiers, Center for iPS Cell Research and Application, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan. kawasaki.shunsuke.5e@kyoto-u.ac.jp.

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|April 19, 2023
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Summary

Researchers repurposed Cas proteins to control gene translation in mammalian cells, creating CARTRIDGE (Cas-Responsive Translational Regulation Integratable into Diverse Gene control). This innovation enables complex synthetic biology circuits with enhanced precision and modularity.

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Area of Science:

  • Synthetic Biology
  • Molecular Biology
  • Gene Regulation

Background:

  • Translational control via RNA-binding proteins is crucial for artificial gene circuits.
  • Efficient and orthogonal RNA-binding proteins for translational regulation are limited.
  • Existing CRISPR technologies offer potential for novel gene control mechanisms.

Purpose of the Study:

  • To develop a novel system for translational modulation in mammalian cells using repurposed Cas proteins.
  • To engineer CARTRIDGE (Cas-Responsive Translational Regulation Integratable into Diverse Gene control) for precise gene expression control.
  • To demonstrate the utility of CARTRIDGE in constructing complex synthetic gene circuits.

Main Methods:

  • Repurposed Cas proteins to act as translational modulators in mammalian cells.
  • Designed mRNAs with a Cas-binding RNA motif in the 5'-untranslated region (UTR).
  • Integrated multiple Cas-mediated translational modulators to build synthetic circuits (e.g., logic gates, cascades).

Main Results:

  • Demonstrated efficient and orthogonal repression or activation of target mRNA translation by Cas proteins.
  • Successfully constructed artificial gene circuits, including logic gates, cascades, and half-subtractor circuits.
  • Showcased the potential of repurposing other CRISPR-related technologies (anti-CRISPR, split-Cas9) for translational control.

Conclusions:

  • CARTRIDGE provides a versatile molecular toolkit for mammalian synthetic biology.
  • Coupling Cas-mediated translational and transcriptional regulation significantly enhances synthetic circuit complexity.
  • This approach offers a powerful new strategy for engineering sophisticated biological systems.