Recent advances in the use of the CRISPR-Cas system for the detection of infectious pathogens

Abstract

Infectious diseases cause great economic loss and individual and even social anguish. Existing detection methods lack sensitivity and specificity, have a poor turnaround time, and are dependent on expensive equipment. In recent years, the clustered regularly interspaced short palindromic repeats (CRISPR)‍-CRISPR-associated protein (Cas) system has been widely used in the detection of pathogens that cause infectious diseases owing to its high specificity, sensitivity, and speed, and good accessibility. In this review, we discuss the discovery and development of the CRISPR-Cas system, summarize related analysis and interpretation methods, and discuss the existing applications of CRISPR-based detection of infectious pathogens using Cas proteins. We conclude the challenges and prospects of the CRISPR-Cas system in the detection of pathogens.

Keywords: CRISPR-Cas; Diagnosis; Disease detection; Infectious disease; Pathogen.

Fig. 1. Schematic diagram of the structure, working principle, and discovery of the CRISPR-Cas system. (a) Composition and structure of the CRISPR system. (b) cis-cleavage and trans-cleavage of ssDNA and RNA by the Cas12, Cas13, and Cas14 proteins for the generation of fluorescence signals. (c) Timeline of CRISPR-Cas system development. CRISPR: clustered regularly interspaced short palindromic repeats; Cas: CRISPR-associated protein; ssRNA: single-stranded RNA; ssDNA: single-stranded DNA; dsDNA: double-stranded DNA; FDA: the United States Food and Drug Administration.

Fig. 2. Classification of signal detection platforms. (a) ssDNA/RNA is labeled with the fluorescent dye FAM (F) and quenching group (Q), and a fluorescence detection device is used to read the results. (b) F and biotin are used to mark ssDNA/RNA, and a test strip is used to read the results. (c) The turbidity caused by liquid-liquid separation in the test tube can be observed with the naked eye. (d) Nanoparticles are used to observe color changes in the test tube and read the results. (e) Detection results are obtained through the use of electrochemiluminescence and an electrode plate. (f) The results are read using a microfluidic chip. (g) F and Q double-labeled ssDNA/RNA is used, and a mobile phone device is used to read the results. ssDNA: single-stranded DNA; FAM: carboxyfluorescein; MB: methylene blue.