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Researchers engineered novel RNA-binding proteins (RBPs) using CRISPR-Cas systems. These Cas protein-responsive switches enable precise control over gene translation in mammalian cells, expanding synthetic biology tools.

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Area of Science:

  • Synthetic biology
  • Molecular biology
  • Gene regulation

Background:

  • Expanding the repertoire of RNA-binding proteins (RBPs) is crucial for developing sophisticated posttranscriptional regulatory circuits in mammalian cells.
  • CRISPR-Cas systems offer a versatile platform for engineering novel functionalities beyond genome editing.
  • Existing synthetic biology tools for posttranscriptional control have limitations in scope and applicability.

Purpose of the Study:

  • To engineer and validate novel RNA-binding proteins (RBPs) using CRISPR-Cas systems for posttranscriptional gene regulation.
  • To demonstrate the principle of Cas protein-responsive switches for controlling mRNA translation.
  • To expand the toolkit available for synthetic biology applications in mammalian cells.

Main Methods:

  • Designed Cas protein-responsive genetic switches by incorporating CRISPR RNA (crRNA)/single-guide RNA (sgRNA) sequences into the 5' untranslated region (5'UTR) of target mRNAs.
  • Exploited the RNA-binding capacity of Cas proteins, exemplified by Streptococcus pyogenes Cas9 (SpCas9), to regulate translation.
  • Developed both plasmid-based and direct RNA-based versions of these switches for versatile application.
  • Validated the functionality of these switches in HEK293FT mammalian cell lines.

Main Results:

  • Successfully demonstrated that Cas proteins can function as engineered RBPs to control gene expression.
  • Developed functional Cas protein-responsive switches that enable tunable regulation of mRNA translation.
  • Showcased the adaptability of the switch design by using Streptococcus pyogenes Cas9 (SpCas9) as a proof-of-concept.
  • Confirmed the efficacy of these synthetic switches in a mammalian cell context (HEK293FT cells).

Conclusions:

  • CRISPR-Cas systems can be repurposed as versatile RNA-binding proteins (RBPs) to create novel posttranscriptional regulatory tools.
  • Cas protein-responsive switches represent a powerful and adaptable platform for synthetic biology, enabling precise control over gene translation.
  • This approach significantly expands the available genetic parts for constructing complex synthetic circuits in mammalian systems.