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

Abstract

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.

Recent advances in nanocomposite-based delivery systems for targeted CRISPR/Cas delivery and therapeutic genetic manipulation

“…Thus, the in vivo administration of these systems for clinical gene therapy is awaited. Wu et al 133 reported a broad technique for constructing a dsDNA-RNP fused nanostructure by folding dsDNA with a CRISPR/dCas9/12a system. They fused dCas9 and dCas12a via a stimuli-responsive peptide linker.…”

Cellular Ingestible DNA Nanostructures for Biomedical Applications

“…They fold double‐stranded DNA with a covalently bivalent between dCas9 RNPs and dCas12a RNPs to codeliver the dCas9 and dCas12a systems. [ 89 ]…”

Regulation of Target-activated CRISPR/Cas12a on Surface Binding of Polymer Dots for Sensitive Electrochemiluminescence DNA Analysis

“…CRISPR (clustered regularly interspaced short palindromic repeats) system as a powerful gene editing tool has been considered as an excellent signal amplification strategy for the development of biosensing field. − This system consists of CRISPR-associated proteins (Cas) and related guide CRISPR RNA (crRNA). − Notably, the newly founded CRISPR/Cas12a trans-cleavage activity, namely, Cas12a and its corresponding CRISPR RNA (crRNA; Cas12a/crRNA duplex), can specifically recognize and cleavage specific DNA based on the recognition of protospacer adjacent motif (PAM) sequence, , which endows CRISPR/Cas12a with remarkable signal amplification ability and precise specificity. , A CRISPR/Cas12a-based ratiometric fluorescent sensor has been constructed by digesting TAMRA-labeled DNA with Burkholderia pseudomallei (Bp) DNA activated CRISPR/Cas12a for Bp DNA detection, and human papilloma virus (HPV-16)-activated CRISPR/Cas12a has been used to increase the photocurrent of Nd-doped BiOBr nanosheets for sensitive detection of HPV-16 . The HPV-16 activated CRISPR/Cas12a has also been used to cleave off methylene blue (MB)-labeled ssDNA from electrode surface for electrochemical biosensing of HPV-16 .…”