The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) system is widely used as a genome-editing tool in various organisms, including plants, to elucidate the fundamental understanding of gene function, disease diagnostics, and crop improvement. Among the CRISPR/Cas systems, Cas9 is one of the widely used nucleases for DNA modifications, but manipulation of RNA at the post-transcriptional level is limited. The recently identified type VI CRISPR/Cas systems provide a platform for precise RNA manipulation without permanent changes to the genome. Several studies reported efficient application of Cas13 in RNA studies, such as viral interference, RNA knockdown, and RNA detection in various organisms. Cas13 was also used to produce virus resistance in plants, as most plant viruses are RNA viruses. However, the application of CRISPR/Cas13 to studies of plant RNA biology is still in its infancy.

Overview of CRISPR/Cas13 Systems

A single multifunctional Cas13 effector protein contains two higher eukaryotes and prokaryotes nucleotide-binding domains (HEPN) that provide RNase activity. The Cas13 protein, when associated with crRNA, forms an RNA-guided RNA targeting complex to recognize and cleave ssRNA targets. Based on Cas13 phylogeny, features, and functional characterization, this system is further classified into six subtypes: VI-A (Cas13a, C2c2), VI-B (Cas13b, C2c4), VI-C (Cas13c, C2c7), VI-D (Cas13d), and recently, VI-X (Cas13X) and VI-Y (Cas13Y). All Cas13 proteins possess two enzymatically distinct RNase activities, which include processing pre-crRNA into mature functional crRNA and the degradation of target RNA by the HEPN domains. The location of these HEPN domains differs based on the type of Cas13 proteins. In Cas13a, 13c, and 13d, the HEPN domains are present at the center and C terminus, whereas in Cas13b, Cas13X, and Y, they are located at the N-terminus and C-terminus of the proteins. The HEPN domains of Cas13 proteins can cleave not only the desired target, but also exhibit a non-specific collateral cleavage activity resulting in the degradation of the RNA near the Cas13 complex. The length of the crRNA or the spacer sequence varies (24-30 nt) with the type of Cas13. Of all the Cas13 enzymes, Cas13c is the least functionally characterized.

Fig. 1 Summary of CRISPR/Cas13 classification. (Kavuri, N. R., et al, 2022)

Applications of CRISPR/Cas13 Systems in Plants

Type VI CRISPR/Cas systems provide various applications in various organisms through different RNA technologies such as RNA interference, RNA detection, RNA editing, and RNA targeting. Cas13 has been used in plants to target RNA viruses, with very few studies on endogenous plant RNA. However, the recent applications of Cas13 in transcriptome studies of humans, animals, and pathogens are opening possibilities for its application in plants. Here are the different CRISPR / Cas13-based RNA technologies and their current plant applications:

• RNA Interference against Viruses

RNA interference (RNAi) is an innate antiviral immunity mechanism that has been successfully used to combat various plant viruses. The CRISPR/Cas13 is a promising tool for engineering plant immunity against a broad range of RNA viruses that constitute a majority of plant viruses. Although similar to RNAi technology, Cas13 can be highly specific, resulting in fewer off-targets and high knockdown efficiency. Viral RNA interference has successfully demonstrated using LshCas13a against the Turnip Mosaic Virus (TuMV) RNA genome, targeting its helper component proteinase (HC-Pro) and coat protein (CP) sequences in Nicotiana benthamiana and Arabidopsis thaliana, respectively. Subsequently, CRISPR/Cas13 has been applied to target RNA viruses such as the potato virus Y (PVY), tobacco mosaic virus (TMV), southern rice black-streaked dwarf virus (SRBSDV), and rice stripe mosaic virus (RSMV) in various plants.

Fig. 2 Pictorial representation of CRISPR/Cas13-based RNA technologies. (Kavuri, N. R., et al, 2022)

• RNA Targeting/Knockdown

The CRISPR/Cas13 system can target any single-stranded RNA for degradation without altering DNA. Accordingly, in addition to RNA interference against viruses, several Cas13 variants have been successfully applied to target endogenous RNA transcripts of various organisms, including LwaCas13a with efficient mRNA knockdown in human cancer cells, RfxCas13d in mRNA knockdown of zebrafish embryos, porcine cell, and parthenogenetic embryos. In plants, mRNA knockdown using LwaCas13a was successfully performed in rice protoplasts targeting three different endogenous transcripts, such as 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), hydroxycinnamoyl transferase (HCT), and phytoene desaturase (PDS), resulting in more than 50% knockdown efficiency in 48 h.

• RNA Editing

RNA editing involves the post-transcriptional editing of RNA in a site-specific manner. RNA editing tools such as RNA Editing for Programmable Adenosine to Inosine (A to I) Replacement (REPAIR) and RNA Editing for Specific Cytosine to Uracil (C to U) Exchange (RESCUE) have been engineered and successfully applied in insects and mammals. The ADAR2 deaminase domain provides the base editing from A to I (REPAIR) and C to U (with RNA cytosine deaminase from evolved ADAR2) (RESCUE) without cleaving the RNA transcript. Base editing of C to T and A to G of the nuclear genome was previously demonstrated in plants using cytidine base editors (CBEs) and adenine base editors (ABEs), respectively, using a Cas9 nickase. These techniques open doors for CRISPR/Cas13 base editors for potential editing in plants at the RNA level.

• Modulation of Alternative Splicing

Alternative splicing is a process regulating gene expression in which exons of a gene are spliced to form multiple alternative transcripts. The CRISPR/Cas13 system can regulate RNA splicing pathways by introducing point mutations into the 5′ and 3′ ends of a splice donor site (GU) and a splice acceptor site (AG), respectively, resulting in mis-splicing and losing targeted splice variants. In plants, the potential use of CRISPR/Cas13 to modulate alternative splicing in serine/arginine-rich (SR) family proteins has been described to analyze the role of splice isoforms in stress responses. Nevertheless, experimental studies on CRISPR/Cas13 to manipulate alternative splicing in plants are yet to be demonstrated. The studies mentioned above provide feasibility for potentially modulating alternative splicing in plant disease-susceptible genes to achieve crop improvement.

• RNA Tracking and Nucleic Acid Detection

The target RNA recognition and binding abilities of CRISPR/Cas13 provide means for its application in various functional and diagnostic studies. This platform could be used in other agricultural contexts, such as detecting and quantifying genes, and can also be leveraged for the early detection of plant pathogens or pests, enabling rapid responses by farmers to reduce the use of pesticides or herbicides.

Prospective Directions of CRISPR/Cas13 RNA Targeting Systems in Plants

CRISPR/Cas technology has become a powerful genome-editing tool due to its applications in various fields for genetic alterations, disease diagnostics, and crop improvement. The recent discovery of CRISPR/Cas13 RNA targeting systems aids in the advancement of existing RNA technologies and has become a promising tool applicable to various organisms. However, the recent studies on CRISPR/Cas13 systems in plants have mainly focused on viral RNA interference. Cas13-mediated knockdown of endogenous mRNA has been demonstrated in a few studies, targeting known genes as a proof of concept that can be applied to target the mRNA of disease-susceptible plant genes or study the function of unknown genes through transient knockdown without disrupting the DNA sequence.

In addition, targeting non-coding RNA such as long non-coding RNA, microRNA, and circular RNA in plants using CRISPR/Cas13 can become a promising tool to study their role in plants. Previously, targeting non-coding RNA in plants was performed using CRISPR/Cas9 at the DNA level to find their roles in plant growth, development, and stress responses. Similarly, CRISPR/Cas13 can also be employed to target non-coding RNA in plants at the RNA level, thereby promoting plant resistance without altering the genome. In the recent past, base editing in plants was performed using an impaired Cas9 (nCas9 or dCas9) to make single base alterations (C to T or T to A) in the genome. Similarly, RNA base editing can be performed through REPAIR and RESCUE tools that use dCas13 fused with deaminase enzymes causing A to G and C to U conversions. However, questions were raised addressing the challenges in the expression stability of CRISPR base editors and in identifying the phenotypic variations caused by base changes in RNA transcript in recent reviews on base editing using CRISPR/Cas13 in plants. Therefore, more research on RNA editing in plants using CRISPR/Cas13 is needed for its application in future crop improvement.