Numerous diseases affect the plant kingdom worldwide, mainly caused by various viruses, and these viruses badly affect crop quality and quantity, which may cause about 50% loss (Fargette et al., 2006; Nicaise, 2014). Specifically for plants, several technologies of pathogen-derived resistance against current plant diseases are available (Baulcombe, 1996; Simón-Mateo and García, 2011; Younis et al., 2014). Furthermore, antiviral gene introduction into crops has a promising capability for engineering resistant crop strains, including R genes (Soosaar et al., 2005). However, these encouraging techniques are confronted with many drawbacks that could limit the usage in plants as reviewed elsewhere (Voinnet, 2005). Therefore, developing new technologies that could interfere with plant viruses is crucial in the near future.

The CRISPR–Cas9 system has been exploited to confer protection against many plant pathogens (Ali et al., 2015, 2016). Additionally, the considerable losses in crops caused by RNA viruses inspired several groups to investigate deployment of CRISPR–Cas13 to interfere with plant viruses (Aman et al., 2018a,b; Mahas et al., 2019; Zhan et al., 2019; Zhang et al., 2019). Upon proofing the concept of using Cas13 effector for targeted RNA degradation (Abudayyeh et al., 2017), Mahfouz’s group was the first to comprehensively harness the prototype CRISPR–Cas13a from Leptotrichia shahii (LshCas13a) to interfere with the GFP-labeled turnip mosaic virus (TuMV-GFP) in planta (Aman et al., 2018a). The results revealed an almost 50% reduction of the replicating TuMV in the Nicotiana benthamiana model (Aman et al., 2018a). Various tested crRNAs directed against multiple locations in the genome of the reporter-expressing virus were assayed. Notably, varying efficiencies in viral CRISPR–Cas13-mediated interference were noticed according to the tested crRNAs (Aman et al., 2018a). The reason behind this variation in viral interference could be the alteration in the secondary RNA structure of crRNA in either transient or stable expression (Abudayyeh et al., 2016; Aman et al., 2018a). Additionally, the processing of Cas13a pre-crRNA arrays into functional units has been validated in plants, which could be harnessed for future multiplexing against various invading viruses (Aman et al., 2018a).

In a follow-up investigation, the same group extends their investigations to corroborate TuMV-GFP viral interference in another plant model using the CRISPR–Cas13 machinery of LshCas13a, Arabidopsis thaliana. Viral interference in stable expression in multiple generations was also verified (Aman et al., 2018b). Most importantly, the interference in A. thaliana revealed the same level of reduction in GFP and quantified RNA, suggesting that LshCas13a might have reduced efficiency in planta (Aman et al., 2018b). These findings lead to a comprehensive screening study to validate and compare various CRISPR–Cas13 orthologs, including Cas13a, Cas13b, and Cas13d, with different localization motifs inside N. benthamiana (Mahas et al., 2019). Leptotrichia wadei (LwaCas13a) was reported to achieve more efficient activity than the prototype LshCas13a. Additionally, the authors demonstrated a higher efficiency in viral interference of Prevotella sp. P5-125 (PspCas13b) than that of Bergeyella zoohelcum (BzCas13b); interestingly, the Cas13d from Ruminococcus flavefaciens XPD3002 (CasRx) achieved the highest record of efficiency of viral interference in plants (Mahas et al., 2019). The tested CRISPR machinery showed an absence of collateral activity in the plant (Mahas et al., 2019). Collectively, they established a novel approach with high specificity and deployment to target plant viruses, which could be formulated to target multiple viruses inside the plant cell using various CRISPR–Cas13 effectors (Mahas et al., 2019).

Having demonstrated CRISPR–Cas13a in the plant field, additional research has harnessed LshCas13a to fend off RNA infection in the potato model. With various correctly designed crRNAs, Potato virus Y (PVY) was targeted in stable transgenic cell lines that were stably expressing LshCas13a (Zhan et al., 2019). The specificity was performed in closely related viruses, PVS, or PVA as controls (Zhan et al., 2019). The CRISPR–Cas13a machinery was verified in monocot plants as well, including rice. A piece of evidence shows that LshCas13a can target the dsRNA genome of the Southern rice black-streaked dwarf virus (SRBSDV), as investigated previously (Zhang et al., 2019). Collectively, Cas13 orthologs can be utilized efficiently in plants to resist a wide variety of invading pathogens, which could provide the best solutions to improve crop quality along with plant breeding or targeted trait engineering, mainly in developing countries.

Similar to RNA targeting and degradation in planta, the Cas13 nucleases were utilized to perturb RNA in the mammalian cell model (Figure 1B). Bawage et al. (2018) have proposed the use of Cas13a as prevention and therapy to halt both vRNA and mRNA of influenza A virus (IAV) and human respiratory syncytial virus model (RSV). Synthetic mRNA was selected to express Leptotrichia buccalis Cas13a (LbuCas13a) instead of using transfecting dsDNA plasmids to avoid genome integration concerns and provide a safe, rapid, and transient action, which will be discussed later in detail. Upon testing of various crRNAs targeting various influenza segments, crRNA targeting the PB1 segment was superior for vRNA and mRNA interference even after 72 h of viral infection, highlighting the prophylactic potential over time (Bawage et al., 2018). The effect of the cleavage Cas13a has no noticeable off-target outcome. Similar findings were validated in previous literature (Abudayyeh et al., 2017; Cox et al., 2017; Bawage et al., 2018; Konermann et al., 2018). Various viral concentrations, timing of viral infection, and cell type showed an equivalent RNA cleavage level, suggesting the treatment possibility upon Cas13 expression. A similar approach was latter applied on the RSV, giving equivalent results (Figure 1B; Bawage et al., 2018).

It has been suggested that Cas13 carries antiviral activity and can be programmed to target various mammalian viruses (Freije et al., 2019). Stepwise investigations were performed to verify the usage of Cas13 orthologs for viral interference. The crRNAs were selected through computational analysis of 396 viral RNAs that correlate with any potential diseases’ progress in humans in a way similar to that adopted for RNA interference (RNAi)-based approaches (von Eije et al., 2008). Two Cas13 orthologs were investigated: LwaCas13a and PspCas13b. Three widely separated virus models have been assayed; lymphocytic choriomeningitis virus (LCMV), vesicular stomatitis virus (VSV), and IAV. LCMV was downregulated 2–14-fold using LwaCas13a. The same conclusions were obtained in IAV and VSV models utilizing PspCas13b even with different MOIs. Additionally, the comparison between Cas13b and shRNA revealed comparable viral interference, suggesting the possibility of successful future use in viral interference (Freije et al., 2019). However, Cas13 can provide superiority in delivering multiple crRNAs using the CRISPR array. The PspCas13b activity has been evaluated using four different crRNAs successfully, highlighting the Cas13 technology for targeting multiple viruses in vivo (Freije et al., 2019). Furthermore, localization of Cas13 expression was reported to be an essential factor for optimal antiviral infectivity. The Cas13b version expressed in the cytosol has significant antiviral activity compared with the nuclear-expressed version, highlighting that the mRNA of the influenza virus was intensively targeted upon the cytoplasmic expression of Cas13b (Freije et al., 2019). Moreover, the supernatant viruses’ sequencing reveals the absence of mutations at the crRNA target sequence after Cas13-mediated activity, highlighting the lower possibility of viral adaptive mutations upon Cas13 expression and activity (Freije et al., 2019).

Recently, upon the prevalence of COVID-19, many scientists raced to develop antiviral approaches to combat the global pandemic. Abbott et al. (2020) have developed a CRISPR–Cas13d-based system as a prophylactic method against SARS-CoV-2 infection, dubbed as the prophylactic antiviral CRISPR in human cells (PAC-MAN) strategy using Cas13d. The Cas13d is usually retrieved from the R. flavefaciens XPD3002 and utilizes a 22-nucleotide crRNA spacer for target RNA degradation (Konermann et al., 2018). Notably, among Cas13 nucleases, the Cas13d ortholog exhibited superiority in its targeted degradation among various tested CRISPR–Cas13 machineries identified so far (Konermann et al., 2018; Mahas et al., 2019). The PAC-MAN approach was assayed against the synthetic RNA of SARS-CoV-2- and IAV-infected lung epithelial cells (Abbott et al., 2020). Both transient and stable cells expressing Cas13d and crRNA were confirmed to degrade the synthetic-labeled RNA fragments in the presence of adequately designed crRNA, which was essential to achieve maximum interference (Abbott et al., 2020). Testing of IAVs using a similar approach was performed by targeting the end of viral segment utilizing a high MOI for infection (Abbott et al., 2020); this approach differs from those targeting the conserved sequence inside viral segments using a low MOI (Freije et al., 2019). However, both exerted a significant viral degradation, testifying to the potential of this approach as an antiviral strategy.

Very recently, the Santangelo group complemented their approach described before (Bawage et al., 2018) for utilizing the mRNA expressing Cas13a for prophylaxis and treatment of COVID-19 and flu diseases (Blanchard et al., 2021). Fascinatingly, they verified their Cas13 methodology using an animal model for the first time for treating viral infections so far. Notably, both cytosolic and nuclear-expressed versions showed a reduction in PB1 transcribed RNA that differs from what was found in Cas13b targeting NP, as stated before (Freije et al., 2019). It appeared that the crRNA against the target sequence was the cause of this notion. Notably, careful selection of RNA target sites and exploration of various crRNAs’ efficiency might be essential to maximize the Cas13 effector’s activity and the success of the Cas13-mediated RNA interference.

In comparison to an established siRNA system against the influenza virus, superior interference of Cas13a was noticed. Very recently, it has been identified that an epigenetic force controls siRNA specificity and potency (Rydzik et al., 2021). The activity was maximized in the presence of a combination of crRNA target PB2 transcripts (Blanchard et al., 2021). Additionally, authors evaluated the interference over time and found that after Cas13a expression, the influenza virus replication was downregulated over 3 days post transfection. Similar viral inhibition was detected in testing SARS-CoV-2-infected VeroE6 cells using crRNA targeting the replicase and nucleocapsid transcripts (Blanchard et al., 2021). The success of viral degradation in cell models encourages them to fetch the Cas13 nuclease performance in animal models. The CRISPR–Cas13 machinery was delivered along with promising guides in a nano-based polymer (poly-beta-amino-esters nano-vehicles based, PBAE) (Patel et al., 2019). Notably, influenza virus reduction was verified in the animal model, suggesting a robust knockdown of influenza in vivo. A similar finding was achieved in hamsters with infection by SARS-CoV-2 (Blanchard et al., 2021). A similar success of using Cas13a was reported against hepatitis disease virus (HCV), suggesting an excellent antiviral activity against chronic diseases (Ashraf et al., 2021).

Vis-à-vis Cas13’s success for an animal viral model, porcine reproductive and respiratory syndrome virus (PRRSV) was verified (Cui et al., 2020). Owing to their maximal RNA knockdown effect, authors decided to use the PspCas13b ortholog compared with an array of Cas13a, Cas13b, and Cas13c effectors (Cox et al., 2017; Cui et al., 2020). RNA cleavage was assayed primarily using a synthetic plasmid expressing ORF7 (nucleocapsid protein) fused with eGFP. They noticed that a transient expression of ORF7 along with a single vector expressing Cas13b and crRNA leads to about 60% RNA degradation (Cui et al., 2020). Furthermore, the generation of an all-in-one system that can express both Cas13b and two crRNAs for multiplexing was confirmed to attain superior RNA interference (Cui et al., 2020). Additionally, they confirmed this finding after stable expression of the all-in-one system in MARC-145 cell, achieving an almost complete degradation of the PRRSV genome (Cui et al., 2020). All these conclusions support the successful use of Cas13 proteins in controlling viral infection in the future, in either prophylactic or treatment conditions, and importantly, in engineering mammalian cells that stably express Cas13 effectors and poly-crRNAs targeting multiple viruses and could provide stable immunity in vitro. This approach could be used as a first step for a future generation of transgenic animals that resist enzootic diseases and for targeted antiviral therapeutic in humans. However, comprehensive investigation of safety-related issues and immunogenicity in vivo are of paramount importance prior to any field applications.

Intracellular protein labeling is usually attained by linking with either fluorescent proteins or dyes. In contrast, intracellular transcript imaging is an ambitious target to study RNA dynamics. Until recently, the fluorescence in situ hybridization technique (FISH) was predominantly used to explain the intracellular RNA dynamics in fixed cell models (Raj et al., 2008; Alonas et al., 2016). The breakthrough, which have performed to understand the prototype Cas13 function, highlighted also that Cas13 could label specific transcripts including ACTB utilizing the catalytically inactive form of Cas13 (dCas13) (Abudayyeh et al., 2017). Inactivation is usually achieved by inducing mutation in the putative catalytic arginine residues within the HEPN domains. RNA-binding assessment has been compared with the standardized method for targeting transcripts in fixed samples, FISH (Abudayyeh et al., 2017). Regardless of the genome nature of the viruses, all use RNA as an intermediate molecule for various functions, including protein translations. Therefore, unraveling the localization, transportation, nuclear exportation of some viruses, assembly, and dynamics of viral RNA is pivotal in underpinning the fundamental role and interaction of viruses with the cellular machinery (Figure 2C). Additionally, this imaging will likely open new frontiers for generations of antivirals.

Recent RNA invasive labeling techniques validate tracking cellular RNA in many organisms, including the bacteriophage coat protein (MS2)-based reporter system, spinach, broccoli, and Cas9 designed for RNA targeting (RCas9) (Briley et al., 2015; Miorin et al., 2016; Nelles et al., 2016; Yang and Chen, 2017; Rauch and Dickinson, 2018; Yang et al., 2019; Liu et al., 2020). Specifically, in virology, the MS2-based system was previously used for tracking HIV in various transcription and assembly processes (Jouvenet et al., 2009; Sardo et al., 2015). Furthermore, multiply labeled tetravalent RNA imaging probes (mTRIP) were used for visualizing RSV (Alonas et al., 2016). Besides, others used fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) for labeling various flaviviruses (Miorin et al., 2016). However, due to the limitations of the intracellular RNA labeling techniques that affect cellular and possibly viral RNA dynamics as noticed in the MS2-based system or short-time tracking process, novel labeling techniques are of paramount importance (Alonas et al., 2016; Yang et al., 2019).

A recent systematic investigation comparing the Cas13 orthologs and the most common method of RNA labeling, the MS2-based labeling, was performed. The study revealed that the Cas13s outperform the labeling efficiency of the MS2-based system and that dPspCas13b was the most efficient ortholog without affecting cellular transcript dynamics (Yang et al., 2019; Han et al., 2020). They also validated using various Cas13 orthologs simultaneously, which could open the way to target more than one transcript as segmented RNA viruses, including influenza (Yang et al., 2019; Ahmed et al., 2020). Additionally, combining the catalytically inactive Cas13 (dCas13) with the dCas9 version could open new avenues for tracking RNA and DNA simultaneously in DNA-replicating viruses, including herpesvirus models (Yang et al., 2019; Bayoumi et al., 2020a,2021).

Similar to the investigations performed for validation using Cas13 effectors for targeted RNA degradation and binding (labeling) (Abudayyeh et al., 2016, 2017). The same group performed further investigations reporting possible usage of Cas13 effectors for targeted editing to the transcriptome as well (Cox et al., 2017). They fused dead (inactive) Cas13 effectors with the adenosine deaminase acting on RNA type 2 (ADAR2). This approach yielded a high specificity of binding and efficiency for editing adenosine to inosine in cellular transcripts. The method for editing is called RNA Editing for Programmable A to I Replacement (REPAIR) (Figure 2C; Cox et al., 2017). This promising approach could alleviate the inherent genetic-based mutations by producing fully functional proteins via editing the intermediate RNA level instead of introducing exogenous functional proteins that usually have aberrant outcomes with enhanced immunogenicity (Qu et al., 2019). The system has high binding editing efficiency with unnoticeable off-target effects all over the transcriptome (Cox et al., 2017; Qu et al., 2019). The Cas13 targeted editing system had a broad spectrum of applications, including in various mammalian cells (Cox et al., 2017; Qu et al., 2019) and yeasts (Jing et al., 2018).

Moreover, the Cas13-based RNA editing approach was recently repurposed to generate epitranscriptome modifications to cellular transcripts via fusing dCas13 effectors with various m6A-related proteins (Li et al., 2020; Wilson et al., 2020; Zhao et al., 2020; Xia et al., 2021). The m6A is an epitranscriptomic mark that controls multiple aspects of viral replication and the outcomes of virus–host interactions. Thus, the manipulation of viruses intracellularly could be achieved through RNA editing as well (Figure 2B; Kennedy et al., 2017; Bayoumi et al., 2020b; Tsai and Cullen, 2020; Bayoumi and Munir, 2021a,b). Given the aforementioned successful application for RNA editing, the Cas13s can be repurposed to induce specific mutations to viral transcripts to improve viral intervention approaches shortly. Similarly, editing of more than one segment utilizing various orthologs can be attained. In this way, we could manipulate viral RNA both genetically and epigenetically. Cas13 effectors were utilized very recently to study circular RNAs driven from cellular transcripts, which could be exploited for use in circRNAs containing viruses shortly (Nahand et al., 2020; Li et al., 2021).

As an obligate intracellular parasite, viruses are dependent on majority of cellular machinery to establish competent replication. Therefore, the identification and characterization of various interactions of viral RNAs with the cellular proteins could revolutionize our understanding of viral RNA function (Girardi et al., 2021). Hence, we can develop potential antiviral ways to block the vRNA interaction with those proteins that have a proviral effect (Schmidt et al., 2021). dCas13 effectors have been verified to block cellular transcript RNA–protein binding sites targeted by crRNA (Yao et al., 2019). In this way, viral RNA binding sites can be blocked to inhibit downstream function. Alternative splicing is another aspect that viruses can benefit from to expand their proteome from the smaller genome. However, maintaining a specific ratio between various protein isoforms is essential for efficient viral replication (Dubois et al., 2014; Artarini et al., 2019). Additionally, Cas13 has been fused efficiently with various splicing factors to drive alteration in exon exclusion and inclusion in the cellular transcriptome (Du et al., 2020; Leclair et al., 2020). Therefore, modulation of viral replication through alteration of alternative splicing could be a tool for studying viral replication kinetics and potential antiviral strategy. Similarly, the viral infection could be modulated by alteration in the polyadenylation state of viral transcripts intracellularly, through binding the Cas13 with polyadenylation factors to drive cleavage and/or polyadenylation accordingly (Pritlove et al., 1998; Anderson et al., 2019). Collectively, Cas13 effectors could enrich our understanding of viral RNA function and the interplay of the virus–host interaction.

Given the aforementioned applications of Cas13 effectors through interaction and inhibition of viral replication directly, controlling the viral infection could be achieved indirectly through repurposing the immune system to engage actively and efficiently, such as rescuing MHC class I/II transcript expression, and modulating the activation or suppression of cytokines accordingly, which could alter specific T lymphocytes to respond to certain antigens rapidly. This approach will greatly improve host innate immunity and subsequently adaptive immunity as previously adopted for enhancing antitumor immune response (Alves et al., 2021). Finally, it should be noted that several recently identified Cas13 proteins have not been investigated yet. Comprehensive investigations of Cas13c effectors are still scarce so far, which could revolutionize our ability to perturb RNA and provide additional clues and support various biotechnological applications, including multiplexing, RNA binding, and RNA-mediated degradation (Shmakov et al., 2017; Palaz et al., 2021). These successful applications collectively prove that CRISPR–Cas systems should be exploited massively for viral diagnosis/degradation, understanding of virus replication in depth, and development of various Cas13-based viral manipulation strategies in the near future.