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Review
.2022 Sep 27;23(19):11400.
doi: 10.3390/ijms231911400.

Insights Gained from RNA Editing Targeted by the CRISPR-Cas13 Family

Affiliations
Review

Insights Gained from RNA Editing Targeted by the CRISPR-Cas13 Family

Li Liu et al. Int J Mol Sci..

Abstract

Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems, especially type II (Cas9) systems, have been widely developed for DNA targeting and formed a set of mature precision gene-editing systems. However, the basic research and application of the CRISPR-Cas system in RNA is still in its early stages. Recently, the discovery of the CRISPR-Cas13 type VI system has provided the possibility for the expansion of RNA targeting technology, which has broad application prospects. Most type VI Cas13 effectors have dinuclease activity that catalyzes pre-crRNA into mature crRNA and produces strong RNA cleavage activity. Cas13 can specifically recognize targeted RNA fragments to activate the Cas13/crRNA complex for collateral cleavage activity. To date, the Cas13X protein is the smallest effector of the Cas13 family, with 775 amino acids, which is a promising platform for RNA targeting due to its lack of protospacer flanking sequence (PFS) restrictions, ease of packaging, and absence of permanent damage. This study highlighted the latest progress in RNA editing targeted by the CRISPR-Cas13 family, and discussed the application of Cas13 in basic research, nucleic acid diagnosis, nucleic acid tracking, and genetic disease treatment. Furthermore, we clarified the structure of the Cas13 protein family and their molecular mechanism, and proposed a future vision of RNA editing targeted by the CRISPR-Cas13 family.

Keywords: CRISPR-Cas VI system; CRISPR/Cas13; Cas13X; Cas13d; RNA cleavage activity.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Mechanism of immunity against virus invaders through CRISPR-Cas systems. The molecular defense process of the CRISPR-Cas system includes three phases: adaptation, maturation, and interference. Adaptation: the host organism captures the nucleotide fragments from invaders and integrates them into the CRISPR array; Maturation: CRISPR array is transcribed into precursor crRNA (pre-crRNA) and further processed into mature crRNA; Interference: crRNA binds to the effector Cas protein to form a complex, and the target invader genome (DNA/RNA) is identified and bound with the help of crRNA, which eventually leads to the degradation of the invader genome (DNA/RNA).
Figure 2
Figure 2
The architecture of CRISPR locus of VI CRISPR-Cas systems and their phylogenetic relationships. The class 2 systems (type VI CRISPR systems) consist of a single, comparatively larger Cas effector protein and CRISPR locus, which can be divided into four subtypes VI-A, VI-B1, VI-B2, VI-C, VI-D, VI-X, and VI-Y. Different colors represent different functional domains in this phylogenetic tree. The HEPN domains of each effector are represented by dark purple squares separated by other structural units. Grey rectangles denote CRISPR direct repeats (DRs), and dark purple squares indicate spacer sequences. The size of each effector and its corresponding conserved domain are indicated as the gene box.
Figure 3
Figure 3
Schematic diagram of DNA and RNA spacers acquisition by CRISPR-Cas systems. (a) Small segments of invasive DNA are assimilated into CRISPR arrays by Cas1 and Cas2 in a canonical spacer acquisition process that allows adaptive immunity in a wide variety of bacteria and archaea. (b) In the type Ⅵ CRISPR systems, an RT fused to Cas1 enables the acquisition of spacer sequences directly from RNA. This process can mediate adaptive immunity against RNA-based parasites [40].
Figure 4
Figure 4
Applications of CRISPR-Cas13 system. (a). Cas13-driven biosensor for disease diagnosis. Cas13-based DNA and RNA detection methods and sample preparation steps are integrated into microfluidic biosensors for rapid pathogen detection; (b). Real-time nucleic acid imaging and tracking of living cells using type VI CRISPR systems. dCas13 proteins fused with fluorescent proteins (FPs) effector and coupling with specific guide RNA can be used for spatiotemporal visualization of the target RNA transcripts in living cells; (c). Antiviral application of type VI CRISPR systems. The type VI CRISPR systems target the genomic region of evolutionarily conserved ssRNA viruses to inhibit viral infection in human animals and plants; (d). CRISPR-Cas13 system-mediated genetic disease treatment.
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