Nucleic acid-assisted CRISPR-Cas systems for advanced biosensing and bioimaging

Highlights

• Recent progress in nucleic acid-assisted CRISPR biosensing and bioimaging are reviewed.

• Strategies for efficiently integrating nucleic acids with CRISPR-Cas systems are summarized.

• Challenges and prospects of future directions are discussed.

Abstract

Nucleic acid molecules possess many superior properties, including high designability and structural predictability, making them attractive biosensing and bioimaging tools. The CRISPR-Cas systems can recognize and cleave nucleic acid targets with high programmability and flexibility, and have been engineered as a multifunctional bimolecular toolbox. Target recognition by CRISPR-Cas systems strictly follows the Watson-Crick base pairing principle, providing a natural interface for coupling with nucleic acids. Such integration has advanced the biosensing and bioimaging methodologies, holding great promise in basic biochemical research and clinical diagnosis. Here, we summarized the latest research progress in integrating nucleic acids with CRISPR-Cas systems for advanced biosensing and bioimaging, including nucleic acid amplification and DNA circuit-coupled CRISPR methods for molecular diagnostics, functional nucleic acid-regulated CRISPR assays for biochemical analysis, and functional nucleic acid-mediated CRISPR bioimaging. We also discussed the challenges and prospects of nucleic acid-assisted CRISPR-Cas systems in biosensing and bioimaging.

Introduction

Biosensing and bioimaging techniques provide the technical means to monitor the key biomolecules involved in physiological and pathological processes in living organisms, holding great value in basic biochemical research, drug screening, and early disease diagnosis and treatment [1]. Over the past decade, the CRISPR-Cas systems, a major category of bacteria adaptive defense systems, have attracted intensive research in gene editing and regulation [[2], [3], [4]], molecular diagnosis [5,6], and genetic locus imaging [7,8]. The RNA-guided recognition and cleavage of target nucleic acids by the CRISPR-Cas systems hold the advantages of programmability and flexibility, and have been engineered into a multifunctional bimolecular toolbox [9,10]. Currently, the CRISPR-Cas systems have been repurposed as a revolutionary tool in gene editing and regulation, allowing researchers to perform biotechnology, bioengineering, and biomedical studies in an unprecedentedly simple and efficient way [4,11,12]. Additionally, the strict molecular recognition and trans-cleavage-mediated signal amplification capacities of CRISPR-Cas systems have also aroused considerable research interest in biosensing and bioimaging in recent years. Many CRISPR-based methods have been proposed for the sensitive sensing of diverse analytes in vitro and in living cells [10,13,14].

Nucleic acids are known as the genetic information-carrying biomolecules in every organism. Given the precise Watson-Crick base pairing principle, nucleic acid sequences can be designed with defined secondary structures in a programmable and predictable manner [15]. With the rapid progress in nucleic acid nanotechnology and chemical synthesis of oligonucleotides, nucleic acids have been exploited as versatile functional materials that can perform challenging technological tasks, from molecular diagnosis and bioimaging to biocomputing [15,16]. For instance, a small DNA segment can be amplified into thousands to millions of copies by polymerase chain reaction (PCR) or isothermal amplification techniques, representing a powerful signal amplification strategy. Nucleic acid circuits built on dynamic nucleic acid technology are able to execute diverse functions, such as signal conversion and logical operation [17]. With the use of systematic evolution of ligands by exponential enrichment (SELEX), aptamers and DNAzymes can be screened with molecular recognition ability and catalytic activity, respectively [18]. Moreover, a few RNA aptamers that can bind and activate the fluorescence of organic dyes with high turn-on ratios, has emerged as a new class of bioimaging probes [19].

In the CRISPR-Cas systems, target nucleic acid recognition and binding by the ribonucleoproteins also strictly follow the Watson-Crick base pairing principle, which naturally exists the interfaces to couple with nucleic acids. The inclusion of comprises nucleic acids has brought new inspirations into CRISPR-Cas systems, boosting their widespread applications in biosensing and bioimaging. In this review, we summarized recent research progress on the integration of nucleic acids with CRISPR-Cas systems for advanced biosensing and bioimaging (Fig. 1) and discussed the challenges and opportunities in this field.