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Genetically Encoded Double-Stranded DNA-Based Nanostructure Folded by a Covalently Bivalent CRISPR/dCas System.

Tiantian Wu1,2, Yuanwei Cao3,4, Qing Liu1

  • 1CAS Key Laboratory of Nanosystem and Hierarchical Fabrication, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing 100190, China.

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Researchers developed a new DNA nanotechnology strategy using clustered regularly interspaced short palindromic repeats (CRISPR) and double-stranded DNA. This method creates novel hybrid nanostructures for potential gene regulation applications.

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Area of Science:

  • Biotechnology
  • Nanotechnology
  • Molecular Biology

Background:

  • DNA nanotechnology commonly uses single-stranded DNA hybridization for nanostructure construction.
  • Existing methods face limitations in creating complex, stable DNA nanostructures.

Purpose of the Study:

  • To introduce a novel strategy for constructing double-stranded DNA-ribonucleoprotein (RNP) hybrid nanostructures.
  • To utilize a covalently bivalent clustered regularly interspaced short palindromic repeats (CRISPR)/nuclease-dead CRISPR-associated protein (dCas) system for DNA folding.

Main Methods:

  • Fusion of dCas9 and dCas12a proteins via a stimuli-responsive peptide linker to create bivalent RNPs.
  • Activation of RNP staples by guide RNAs to target and bind specific sequences on a double-stranded DNA scaffold.
  • Induction of DNA folding through RNP recognition and binding, forming hybrid nanostructures.

Main Results:

  • Successful construction of double-stranded DNA-RNP hybrid nanostructures.
  • Demonstration of the nanostructures' ability to protect genetic information in a folded state.
  • Exhibition of stimuli-responsive gene transcription upon unfolding of the nanostructures.

Conclusions:

  • The developed strategy offers a new approach for DNA nanotechnology by employing double-stranded DNA folding with CRISPR-based RNP systems.
  • This method provides a genetically encoded platform for stable nanostructure formation and controlled gene regulation.
  • The findings open new avenues for designing advanced functional DNA nanodevices.