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

Tiantian WuYuanwei CaoQing LiuXiaohui WuYingxu ShangJiafang PiaoYujie LiYuanchen DongDongsheng LiuHaoyi WangJianbing LiuBaoquan Ding
Published in: Journal of the American Chemical Society (2022)
DNA nanotechnology has been widely employed in the construction of various functional nanostructures. However, most DNA nanostructures rely on hybridization between multiple single-stranded DNAs. Herein, we report a general strategy for the construction of a double-stranded DNA-ribonucleoprotein (RNP) hybrid nanostructure by folding double-stranded DNA with a covalently bivalent clustered regularly interspaced short palindromic repeats (CRISPR)/nuclease-dead CRISPR-associated protein (dCas) system. In our design, dCas9 and dCas12a can be efficiently fused together through a flexible and stimuli-responsive peptide linker. After activation by guide RNAs, the covalently bivalent dCas9-12a RNPs (staples) can precisely recognize their target sequences in the double-stranded DNA scaffold and pull them together to construct a series of double-stranded DNA-RNP hybrid nanostructures. The genetically encoded hybrid nanostructure can protect genetic information in the folded state, similar to the natural DNA-protein hybrids present in chromosomes, and elicit efficient stimuli-responsive gene transcription in the unfolded form. This rationally developed double-stranded DNA folding and unfolding strategy presents a new avenue for the development of DNA nanotechnology.
Keyphrases
  • circulating tumor
  • single molecule
  • nucleic acid
  • cell free
  • binding protein
  • genome wide
  • gene expression
  • circulating tumor cells
  • dna methylation
  • drug delivery
  • protein protein