Researchers Construct Genetically Encoded Double-Stranded DNA-Based Nanostructure by a Bivalent CRISPR/dCas System

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As genetic information carriers, nucleic acid molecules can be employed to accurately assemble nanostructures with specific sizes and shapes on the basis of complementary base pairing. Due to excellent structural designability and biocompatibility, nucleic acid nanostructures have been widely applied in the field of biomedicine research. With continuous development of nucleic acid manipulation technology and nucleic acid chemical biology, DNA nanostructures based on chemical modification and controllable self-assembly can efficiently load various drugs to realize targeted delivery and controlled release for intelligent and accurate treatment, which provide new ideas for disease diagnosis and treatment.

Previously, a research team led by Prof. DING Baoquan from the National Center for Nanoscience and Technology (NCNST) has constructed various gene therapy systems based on chemical modification and controllable self-assembly of nucleic acid (J. Am. Chem. Soc. 2021, 143, 19893; Angew. Chem. Int. Ed. 2021, 60, 1853; J. Am. Chem. Soc. 2019, 141, 19032; Angew. Chem. Int. Ed. 2019, 58, 14224; Angew. Chem. Int. Ed. 2018, 57, 15486). Focused on this research field, this group firstly assembles chemically-modified nucleic acid nanostructures to controllably load the gene therapeutic drugs for precise gene therapy in vivo, which provides new strategies for development of novel treatment model of diseases. 

In the aspect of nucleic acid self-assembly, nucleic acid nanostructures are usually constructed by complementary base pairing between single-stranded nucleic acids. However, the widely existing functional nucleic acids with double-stranded structure are difficult to be used as structural units for controllable self-assembly. On the basis of previous research, Prof. DING Baoquan’s group collaborates with Prof. WANG Haoyi from Institute of Zoology of Chinese Academy of Sciences (CAS), to report a facile strategy for folding genetically encoded double-stranded DNA by bivalent clustered regularly interspaced short palindromic repeats (CRISPR)/nuclease-dead CRISPR-associated protein (dCas) system for gene expression and regulation. In this 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 ribonucleoproteins (RNP) 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.

Figure 1. Construction of bivalent CRISPR/dCas system for precise folding and controllable release of functional gene to achieve gene expression and regulation. (Image by DING Baoquan et al)

This work was published in J. Am. Chem. Soc. on March 31, 2022 (DOI: 10.1021/ jacs.2c01760). WU Tiantian (a PhD graduate from NCNST) and CAO Yuanwei (a PhD candidate from Institute of Zoology) are the co-first authors. WANG Haoyi from Institute of Zoology, LIU Jianbing from NCNST, and DING Baoquan from NCNST are the co-corresponding authors. The research was supported by the National Natural Science Foundation of China, the Strategic Priority Research Program of CAS, the Key Research Program of Frontier Science, CAS, and so on. The core technology of the above research has been applied for a Chinese invention patent.



DING Baoquan

National Center for Nanoscience and Technology



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