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MicroRNA (miRNA) are short, regulatory RNA transcribed from introns—non-coding regions of a gene—or intergenic regions—stretches of DNA present between genes. Several processing steps are required to form biologically active, mature miRNA. The initial transcript, called primary miRNA (pri-mRNA), base-pairs with itself forming a stem-loop structure. Within the nucleus, an endonuclease enzyme, called Drosha, shortens the stem-loop structure into hairpin-shaped pre-miRNA. After...
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MicroRNA (miRNA) are short, regulatory RNA transcribed from introns (non-coding regions of a gene) or intergenic regions (stretches of DNA present between genes). Several processing steps are required to form biologically active, mature miRNA. The initial transcript, called primary miRNA (pri-mRNA), base-pairs with itself, forming a stem-loop structure. Within the nucleus, an endonuclease enzyme, called Drosha, shortens the stem-loop structure into hairpin-shaped pre-miRNA. After the pre-miRNA...
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Multicellular organisms contain a variety of structurally and functionally distinct cell types, but the DNA in all the cells originated from the same parent cells. The differences in the cells can be attributed to the differential gene expression. Liver cells, whose functions include detoxification of blood, production of bile to metabolize fats, and synthesis of proteins essential for metabolism, must express a specific set of genes to perform their functions. Gene expression also varies with...
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Cell-Type-Specific CRISPR Activation with MicroRNA-Responsive AcrllA4 Switch.

Moe Hirosawa1,2, Yoshihiko Fujita1, Hirohide Saito1

  • 1Department of Life Science Frontiers, Center for iPS Cell Research and Application (CiRA) , Kyoto University , Kyoto 606-8501 , Japan.

ACS Synthetic Biology
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Summary

Researchers developed a conditional CRISPR-Cas9 system regulated by microRNAs. This system enables cell-specific genome editing by controlling the expression of an anti-CRISPR protein, offering potential therapeutic applications.

Keywords:
CRISPR-Cas9RNA switchanti-CRISPRmicroRNAsynthetic biology

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Area of Science:

  • Molecular Biology
  • Gene Editing
  • Biotechnology

Background:

  • CRISPR-Cas systems offer precise genome editing capabilities.
  • Controlling CRISPR-Cas activity in specific cell types remains a challenge for therapeutic applications.
  • Anti-CRISPR proteins can inhibit CRISPR-Cas activity, but their application requires precise control.

Purpose of the Study:

  • To develop a cell-type-specific CRISPR-Cas9 activation system.
  • To engineer a synthetic mRNA switch responsive to endogenous microRNA (miRNA) activity.
  • To enable conditional gene knockout or activation based on intracellular miRNA levels.

Main Methods:

  • Designed a synthetic mRNA encoding the anti-CRISPR protein AcrllA4 with a miRNA-binding site in the 5'-UTR.
  • Integrated this miRNA-responsive switch with CRISPR-Cas9 or dCas9-VPR and guide RNA.
  • Demonstrated cell-specific activation of gene editing or activation in the presence of specific miRNAs.

Main Results:

  • The miRNA-responsive AcrllA4 switch successfully inhibited Cas9 activity until a target miRNA was present.
  • The system enabled cell-specific gene knockout using Cas9 or gene activation using dCas9-VPR.
  • Conditional CRISPR-Cas9 activation was achieved by sensing endogenous miRNA activity.

Conclusions:

  • The developed conditional CRISPR-Cas9 ON system provides precise spatiotemporal control over genome engineering.
  • This miRNA-regulated system holds significant promise for future therapeutic applications and targeted genome editing.
  • Sensing intracellular miRNAs offers a novel strategy for cell-specific control of gene editing tools.