CRISPR-Cas13a system: A novel tool for molecular diagnostics

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR) system is a natural adaptive immune system of prokaryotes. The CRISPR-Cas system is currently divided into two classes and six types: types I, III, and IV in class 1 systems and types II, V, and VI in class 2 systems. Among the CRISPR-Cas type VI systems, the CRISPR/Cas13a system has been the most widely characterized for its application in molecular diagnostics, gene therapy, gene editing, and RNA imaging. Moreover, because of the trans-cleavage activity of Cas13a and the high specificity of its CRISPR RNA, the CRISPR/Cas13a system has enormous potential in the field of molecular diagnostics. Herein, we summarize the applications of the CRISPR/Cas13a system in the detection of pathogens, including viruses, bacteria, parasites, chlamydia, and fungus; biomarkers, such as microRNAs, lncRNAs, and circRNAs; and some non-nucleic acid targets, including proteins, ions, and methyl groups. Meanwhile, we highlight the working principles of some novel Cas13a-based detection methods, including the Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK) and its improved versions, Cas13a-based nucleic acid amplification-free biosensors, and Cas13a-based biosensors for non-nucleic acid target detection. Finally, we focus on some issues that need to be solved and the development prospects of the CRISPR/Cas13a system.

CRISPR/Cas gene editing and delivery systems for cancer therapy

“…CRISPR/Cas13a system consists of Cas13a protein and single crRNA. Cas13a protein has two types of RNAase activity, one for pre-crRNA processing to promote the formation of ribonucleoprotein complexes (RNPs), and the other for cis-cleavage of targeted RNA or trans-cleavage of nonspecific RNA (Zhao, Qiu, et al, 2022). When the complexes of Cas13a and crRNA bind to the targeted single-stranded RNA, the conformational change of the RNP complex is triggered to cleave targeted site and results in the editing and regulation at the RNA level.…”

Cas-based bacterial detection: recent advances and perspectives

“…1d), which tends to cleave at uracil or adenine bases and possesses target-dependent single-stranded RNA trans -cleavage activity. 69 Cas13a also possesses a distinctive feature: once it recognizes and cleaves the RNA target specified by the crRNA sequence, it transitions into an enzymatic “activated” state. In this state, it binds to and cleaves other RNAs, regardless of their homology with the crRNA or the presence of a PFS.…”

The CRISPR/Cas System: A Customizable Toolbox for Molecular Detection

“…Cas13 in type VI targets single-stranded RNA and is the first Cas protein discovered to recognize RNA specifically [ 37 ], with the ability to cleave ssRNA in both cis and trans orientations. Cas13a (C2c2), Cas13b, Cas13c, and Cas13d make up the majority of the Cas13 family [ 38 , 39 , 40 , 41 ]. Further investigation revealed that the system does not require tracrRNA, but rather a protospacer flanking site (PFS) similar to PAM that has an “adjunct cleavage effect” on RNA [ 11 ].…”