Programmable System of Cas13-Mediated RNA Modification and Its Biological and Biomedical Applications

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13 has drawn broad interest to control gene expression and cell fate at the RNA level in general. Apart from RNA interference mediated by its endonuclease activity, the nuclease-deactivated form of Cas13 further provides a versatile RNA-guided RNA-targeting platform for manipulating kinds of RNA modifications post-transcriptionally. Chemical modifications modulate various aspects of RNA fate, including translation efficiency, alternative splicing, RNA-protein affinity, RNA-RNA interaction, RNA stability and RNA translocation, which ultimately orchestrate cellular biologic activities. This review summarizes the history of the CRISPR-Cas13 system, fundamental components of RNA modifications and the related physiological and pathological functions. We focus on the development of epi-transcriptional editing toolkits based on catalytically inactive Cas13, including RNA Editing for Programmable A to I Replacement (REPAIR) and xABE (adenosine base editor) for adenosine deamination, RNA Editing for Specific C-to-U Exchange (RESCUE) and xCBE (cytidine base editor) for cytidine deamination and dm⁶ACRISPR, as well as the targeted RNA methylation (TRM) and photoactivatable RNA m⁶A editing system using CRISPR-dCas13 (PAMEC) for m⁶A editing. We further highlight the emerging applications of these useful toolkits in cell biology, disease and imaging. Finally, we discuss the potential limitations, such as off-target editing, low editing efficiency and limitation for AAV delivery, and provide possible optimization strategies.

Keywords: CRISPR; CRISPR-Cas13 system; RNA modification; cell biology; epigenetic editing.

FIGURE 1

Schematic diagram of chemical formula, relative proteins and physiology function of common RNA modifications.

FIGURE 2

Schematic diagram of RNA modification engineering platforms: (A) REPAIR/xABE (A-to-I editing platform): Specifically designed CrRNAs base-pair with RNA targets and recruit dCas13-ADAR2dd to the dsRNA, which deaminates adenosines to inosines in the dsRNA region. C-A mismatch in editing sites enhances the editing specificity. (B) RESCUE/xCBE (C-to-U editing platform): dCas13 is fused to evolutionary ADAR2dd, which performs increased cytidine deamination activity. With the guidance of CrRNA, dCas13-ADAR2dd mediates A-to-U transformation in the dsRNA. A mismatched uridine in the CrRNA opposite the target cytidine enhances the editing reaction. (C) The targeted RNA methylation (TRM) system: dCas13b fusions to modified methyltransferase domains, such as METTL3ΔZF (M3) removing zinc finger RNA binding motif or constructed protein consisted with M3 and METTL3-interacting domain of METTL14 can selectively and efficiently install m⁶A in targeted adenosine. (D) dm⁶ACRISPR system: The targeted demethylation system is consist of a catalytically inactive Cas13b, full-length of demethylase ALKBH5 and gRNA, which enable to demethylate either close or remote m⁶A modifications. (E) Photoactivatable RNA m⁶A editing system using CRISPR-dCas13 (PAMEC): PAMEC harnesses CIBN-CRY2, a robust light-dependent protein–protein interaction system. Under the explosion of blue light, the RNA anchor probe including CIBN and dCas13b and the effector probe containing RNA editing effector and CRY2 tight together and form a functional unit for m⁶A installation and erasure. (F) PAMECR: RNA–protein interaction system, MS2-MCP, is further introduced to primary PAMEC platform, which ensures manipulation of multiple transcripts robustly and simultaneously.

FIGURE 3

Schematic diagram of potential limitations of epigenetic editing platforms.