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

摘要

DNA nanotechnology has been widely employed in theconstruction of various functional nanostructures. However, most DNAnanostructures rely on hybridization between multiple single-stranded DNAs.Herein, we report a general strategy for the construction of a double-strandedDNA-ribonucleoprotein (RNP) hybrid nanostructure by folding double-stranded DNA with a covalently bivalent clustered regularly interspaced shortpalindromic repeats (CRISPR)/nuclease-dead CRISPR-associated protein (dCas) system. In our design, dCas9 and dCas12a can beefficiently fused together through aflexible and stimuli-responsive peptide linker. After activation by guide RNAs, the covalentlybivalent dCas9-12a RNPs (staples) can precisely recognize their target sequences in the double-stranded DNA scaffold and pullthem together to construct a series of double-stranded DNA-RNP hybrid nanostructures. The genetically encoded hybridnanostructure can protect genetic information in the folded state, similar to the natural DNA-protein hybrids present inchromosomes, 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.

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