Advances in the application of CRISPR-Cas technology in rapid detection of pathogen nucleic acid

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) are widely used as gene editing tools in biology, microbiology, and other fields. CRISPR is composed of highly conserved repetitive sequences and spacer sequences in tandem. The spacer sequence has homology with foreign nucleic acids such as viruses and plasmids; Cas effector proteins have endonucleases, and become a hotspot in the field of molecular diagnosis because they recognize and cut specific DNA or RNA sequences. Researchers have developed many diagnostic platforms with high sensitivity, high specificity, and low cost by using Cas proteins (Cas9, Cas12, Cas13, Cas14, etc.) in combination with signal amplification and transformation technologies (fluorescence method, lateral flow technology, etc.), providing a new way for rapid detection of pathogen nucleic acid. This paper introduces the biological mechanism and classification of CRISPR-Cas technology, summarizes the existing rapid detection technology for pathogen nucleic acid based on the trans cleavage activity of Cas, describes its characteristics, functions, and application scenarios, and prospects the future application of this technology.

Keywords: CRISPR-Cas; Cas12; Cas13; Cas14; Cas9; pathogen nucleic acid; rapid detection.

FIGURE 1

Adaptive immune response in the CRISPR-Cas system. This figure was drawn by Figdraw. (A) In the adaption stage, some short fragments of viruses, plasmids and other foreign nucleic acids are integrated into CRISPR repeats to form spacer sequences. (B) In the expression stage, CRISPR sequences were processed into crRNA containing spacer and repeat sequences, which were combined with CRISPR-associated effector proteins to form complexes. (C): In the interference stage, the complex performs specific cleavage and inactivation of foreign nucleic acids complementary with crRNA sequences, so as to protect itself from viruses, plasmids and other invasion.

FIGURE 2

Basic principles of CRISPR-mediated nucleic acid detection. This figure was drawn by Figdraw. (A) RNA-guided target recognition system, which specifically recognizes and cleavages the target sequence containing PAM sites for detection by RNA-guided effector proteins. (B) Incidental cleavage system triggered by targeted recognition, after specifically recognizing and cleaving the target sequence, incidental cleavage of the surrounding labeled single-stranded nucleic acid is performed to generate detection signals for detection.