CRISPR‐Cas Biochemistry and CRISPR‐Based Molecular Diagnostics

Abstract

Polymerase chain reaction (PCR)-based nucleic acid testing has played a critical role in disease diagnostics, pathogen surveillance, and many more. However, this method requires a long turnaround time, expensive equipment, and trained personnel, limiting its widespread availability and diagnostic capacity. On the other hand, the clustered regularly interspaced short palindromic repeats (CRISPR) technology has recently demonstrated capability for nucleic acid detection with high sensitivity and specificity. CRISPR-mediated biosensing holds great promise for revolutionizing nucleic acid testing procedures and developing point-of-care diagnostics. This review focuses on recent developments in both fundamental CRISPR biochemistry and CRISPR-based nucleic acid detection techniques. Four ongoing research hotspots in molecular diagnostics-target preamplification-free detection, microRNA (miRNA) testing, non-nucleic-acid detection, and SARS-CoV-2 detection-are also covered.

Pneumatic nano-sieve for CRISPR-based detection of drug-resistant bacteria

“…26,27 Moreover, Cas12a exhibits collateral activity, referred to as trans-cleavage, which allows it to non-specifically cleave neighboring single-stranded DNAs (ssDNA) following target binding. 28 To exploit this feature for detection purposes, ssDNA can be labeled with a fluorophore-quencher, and upon Cas12a activation through target binding, the cleavage of ssDNA generates an increase in fluorescence signal. 29 CRISPR-Cas12a system operates at 37 1C, making it more suitable for POC detection than traditional PCR methods.…”

CRISPR-cas technology: A key approach for SARS-CoV-2 detection

“…Essentially, CRISPR-Cas systems can be programmed to recognize specific nucleic acid sequences, such as those found in viruses or cancer cells. When the system encounters the target sequence, it triggers a response that can be detected and used for diagnosis (Weng et al, 2023). In recent years, the potential applications of CRISPR-Cas technology have expanded to include the diagnosis and treatment of various diseases (Dhainaut et al, 2022;Katti et al, 2022;Chavez et al, 2023;Musunuru, 2023;Wang and Doudna, 2023), including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).…”

Employment of the Phage Cocktail as a Species-Specific Recognition Agent for Wide-Spectrum Detection of Bacterial Strains

“…pneumoniae mainly depend on bacterial culture, which show deal reliability. However, the operations are cumbersome and time-consuming. , Nucleic acid-based molecular biology diagnostic techniques and ELISA-based immunological methods can effectively and sensitively detect pathogenic bacteria and significantly shorten the detection time but require laboratory precision instruments and well-trained personnels. − In recent years, a variety of biosensors employing some specific molecular recognition agents to conjugate bacteria have been developed for detecting pathogens. , Antibodies have long been one of the most frequently employed molecular recognition agents for the development of biosensors. , Nevertheless, the preparation of antibodies demands immunization of animals and construction of mammalian cell expression systems, which are laborious, time-consuming, and costly. − Accordingly, it is necessary to acquire bacterial recognition agents with better accessibility and lower cost.…”