Malaria has been a major global health problem for humans throughout their history and is a leading cause of mortality across many tropical and subtropical countries (Hemingway and Bates, 2003; Cowman et al., 2016; Monroe et al., 2022). Malaria control efforts have been undermined by the decline in the effectiveness of the primary malaria-figurehting tools, increasing resistance to treated-insecticide nets and anti-malarial drugs, and no effective vaccine (Olotu et al., 2013; Haldar et al., 2018; Memvanga and Nkanga, 2021). Spotlights from the 2022 world malaria report by WHO showed that cases of malaria increased from 245 million in 2020 to 247 million in 2021, although malaria-related deaths slightly decreased from 625,000 to 619,000 (World Health Organization, 2022). The etiological agent of malaria, Plasmodium, mainly thrives in human and mosquito hosts. Of all Plasmodium parasites, five species are responsible for global malaria infections in humans: Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale, and Plasmodium knowlesi (Lalremruata et al., 2017; Memvanga and Nkanga, 2021; Sato, 2021).

Microscopic examination of Giemsa-stained peripheral blood smears has over the years been the “gold standard” for the diagnosis of malaria (Oboh et al., 2021; Fitri et al., 2022). However, its usefulness has been confronted with a host of challenges as results generated from microscopy are dependent on the technical know-how of the microscopist, the quality of blood sample, the parasite density, and the subjectivity of results interpretation (Barber et al., 2013; Berzosa et al., 2018; Ospina-Villa et al., 2018), indicating a void in diagnosis and the need to develop new effective tools. The Plasmodium falciparum genome encompasses 22.8 mega-bases with an average gene density of 1 gene per 4,338 base pairs—distributed among a total of 14 chromosomes (Gardner et al., 2002). Almost two-thirds of the proteins coded by these genes are unique to Plasmodium (Gardner et al., 2002) providing peculiar targets for molecular detection. In an attempt to fill the gaps in malaria diagnosis, several molecular diagnostic tools have been established to target various unique genes in Plasmodium. Molecular diagnosis notwithstanding remains a big huddle as the performance of these tools varies depending on the epidemiological setting (Oboh et al., 2018).

Interestingly, 74% of the total genes in Plasmodium remain functionally uncharacterized despite the availability of the parasite’s genome (Tan and Mutwil, 2020). To address this paucity, forward and reverse functional genomic tools have been developed to manipulate the parasites’ genomes to functionally characterize these genes (Zhang et al., 2014; Walker and Lindner, 2019; Cárdenas et al., 2022; Thiam et al., 2022). In particular, RNA-targeting tools hold great potential to understand the biological functions of genes and identify novel anti-malarial targets and effective vaccines (Nisbet et al., 2016; Liu et al., 2019). RNA level manipulation/editing affects the timing and/or level of transcription and alters the stability of gene expression, thereby offering scientists the platform to study the basic cellular functions of mRNAs, and non-coding RNAs (ncRNA) such as small nuclear RNA (snRNA), ribosomal RNA (rRNA), microRNA (miRNA), and piwi interaction RNA (piRNA; Briquet et al., 2022).

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR-associated protein) system is found in archaea and bacteria—responsible for coordinating defense against foreign genetic elements (Mojica et al., 2000; Marraffini, 2015; Moon et al., 2019). The CRISPR-Cas system is a powerful biological tool whose usefulness scientists have been unlocking in the last few decades. It is a double-edged tool that scientists have applied for both diagnosis and gene editing to unravel the function of many genes at the DNA and RNA levels in several organisms (Graham and Root, 2015; Hartenian and Doench, 2015; Zhang D. et al., 2021). Currently, the CRISPR-Cas system is structurally and functionally diverse and divided into 2 classes, 6 types, and more than 30 subtypes. The first class includes type I, type III, and type IV, and the second class includes type II, type V, and type VI (O'Connell, 2019). The small size, atypical hypercompact architecture, and high efficiency of class 2 multi-domain Cas proteins have made them a prime focus of many researchers compared to class I proteins (Cao et al., 2022; Liu and Pei, 2022). Of these, the majority of research in the past decade has focused on CRISPR-Cas9 belonging to the type II category (Atkins et al., 2021), and its properties have been harnessed for gene knockdown, gene editing, epitranscriptomics modification, and sensing of RNA targets (Zhang H. et al., 2021). CRISPR-Cas13 systems—which constitute the type VI group—have been demonstrated to generally lack DNase (Deoxyribonuclease) activity, but are fixated on RNA cleaving, and could offer distinct targeting advantages to RNA. Thus they represent a clinically promising platform capable of efficiently characterizing genes at the RNA level (Gaj, 2021).

In this review, we have summarized the CRISPR-Cas13 system from its discovery, classification, principle of action, and its application in malaria diagnosis. Given that malaria parasites lack relevant interference systems (Baum et al., 2009) thereby limiting the use of endogenous interference-dependent RNA editing systems, we will further discuss the application prospects of Cas13 in Plasmodium at the RNA level.

In 1987, Yoshizumi Ishino of Osaka University in Japan detected regularly spaced direct palindromic repeats downstream of the stop codon of the iap (isoenzymes of alkaline phosphate) genes of E. coli K-12 cells (Ishino et al., 1987). This event provided the blueprint for the discovery of new CRISPR-Cas systems, ushering scientists into a new era of gene editing and genomic engineering. At the time, the relevance of these repeat sequences remained obscure. Mojica and his team detected a similar sequence in phylogenetically distinct organisms: Haloferax mediterranei and Haloferax volcanii of the Archaea domain (Mojica et al., 1993). They later postulated that these repeats are involved in replicon partitioning (Mojica et al., 1995). In 2002, substantial progress was made by Jansen and collaborators resulting in the official naming of this mysterious repeat as CRISPR which is now commonly accepted by the scientific community (Jansen et al., 2002). Soon after, spurred on by his previous findings, a major leap toward the understanding of the function of CRISPR was made by Mojica and his team (Mojica et al., 2005). They pointed out that the spacer sequence in CRISPR had homology with foreign phages or plasmids, and that the virus could not infect cells carrying homologous spacer sequences, but was easy to invade cells without spacer sequences. Against this background, it was posited that CRISPR might participate in the immune function of bacteria and it requires a precise sequence match between the spacer and the target sequence of the virus (Mojica et al., 2005; Lander, 2016). The functionality of the second component of the CRISPR-Cas complex, CRISPR-associated protein (Cas), was computationally demonstrated later by Marakova and collaborators as a composite of the DNA repair machinery (Makarova et al., 2006). In a later study by Horvath, he noticed a palindromic repeat that coded for an endonuclease protein, Cas9, and intimated that the protein cleaves viral DNA and thus grants immunity to the host bacteria (Horvath and Barrangou, 2010). A few years later, in 2012, Doudna and Charpentier’s team discovered the third component of the CRISPR system—the trans-acting crRNA (tracrRNA) which oversees the maturation of precursor crRNA into crRNA and also directs Cas9 cleavage of dsDNA (Jinek et al., 2012). Subsequent development saw the exploitation of the CRISPR-Cas system as a genomic editing tool. In 2013, the gene-editing tool CRISPR-Cas9 was applied in mammalian cells and became popular all over the world, because of its novelty, efficiency, and versatility (Shmakov et al., 2015).

Different from CRISPR-Cas9, which binds and cuts at the DNA level, as shown in Figure 1, in 2016, deeply searching for microbial genome data, it was found that a new CRISPR-Cas system effector protein, named c2c2 (now referred to as Casl3a), could specifically bind and cut RNA under the guidance of crRNA, and regulate the activity of genes at the RNA level (Abudayyeh et al., 2016). This opened up a new avenue for editing targeted RNA. In 2017, through rigorous biochemical experiments and genetic computations, Zhang Feng’s team identified two new Cas13 subtypes, Cas13b and Cas13c (Cox et al., 2017; Smargon et al., 2017). Subsequent investigation revealed that CRISPR-Cas13 systems uniquely lack the third component, tracrRNA (Yan et al., 2019). Going further, they demonstrated that Cas13b could be applied to precisely edit the full length of transcript harboring deleterious mutations in mammalian cells (Cox et al., 2017). In 2018, Konermarm et al. analyzed a prokaryote genome and discovered the CRISPR-Casl3d system, which has the smallest molecular weight of all CRISPR-Casl3 effectors, and was suitable for virus vectors, and had great application potential in vivo (Konermann et al., 2018). Through the exploration of bulk metagenomic data, Hu et al. (2022) identified novel hypercompact Cas13 systems and therein named them Cas13e, Cas13f, Cas13g, Cas13h, and Cas13i. However, their functional relevance remains to be described in future studies.

Since its advent, Cas13 has been widely used in various fields. For example, in 2019, Zhang Feng’s team created a new CRISPR-Cas13 system called CARVER (Cas13-assisted Restriction of Viral Expression and Readout) antiviral, which combines the antiviral activity of Cas13 and its diagnostic capabilities, as a promising system for the diagnosis and treatment of viral infections (Freije et al., 2019). In the same year, Chen Lingling’s group used the CRISPR-Casl3d system for RNA live cell labeling (Yang et al., 2019). In 2020, Gopal Kushawah and others used CRISPR-Cas13d to induce high-efficiency nucleoprotein degradation in animal embryos, providing new conditions for animal antiviral research (Kushawah et al., 2020). More recently, in 2021, Yang Hui’s team identified Cas13X and Cas13Y from high-salinity samples and designed an RNA interference experiment for Cas13X.1 in a mammalian cell line (Xu et al., 2021). During the same period, Zhang Feng’s team discovered “Mini”-Cas13bt, functionalizing Cas13bt through the use of adenosine and cytosine deaminase (Kannan et al., 2022).

Collectively, the catalog of CRISPR-Cas13 systems keeps expanding as novel types and orthologs are being described in current studies. At present, based on phylogeny, this important class of effector proteins is categorized into VI-A (Cas13a), VI-B (Cas13b), VI-C (Cas13c), VI-D (Cas13d; Zhang H. et al., 2021), VI-E to I (Cas13e-Cas13i; Hu et al., 2022), and the latest CRISPR-Cas13 system like Cas13X, Cas13Y (Xu et al., 2021) and Cas13bt (Kannan et al., 2022). Here, the classification and average size of Cas13 proteins, which have been widely used in various fields, were summarized (Figure 2).

As shown in Figure 3, using Cas13a as an example, the CRISPR-Cas13 system mainly consists of crRNA (CRISPR RNA) and CRISPR-related nucleases (Cas1, Cas2, and Cas13). The biology of these components is briefly discussed below.

During bacteria immune surveillance and cleavage of exogenous RNA, CRISPR-Cas13 requires a guide from a matured crRNA (Bayoumi and Munir, 2021; Krohannon et al., 2022). At a glance, this seems a challenge since CRISPR type V1 lacks Cas6 or Cas5d endonuclease activity for processing pre-crRNA into matured crRNA (crRNA biogenesis). Now, it has been shown that Cas13 performs both crRNA biogenesis and RNA cleavage employing two chemically and mechanistically distinct mechanisms for both tasks (East-Seletsky et al., 2016). Using three purified recombinant Cas13 proteins, it was revealed that Cas13 cut at two to five nucleotides upstream of the target pre-crRNA to produce a 60–66 nucleotide-long matured crRNA (Huynh et al., 2020). The resulting matured crRNA contains a single spacer sequence (20–30 nucleotide long) that is complementary to the target RNA sequence and a direct repeat region (Ai et al., 2022).

A generalized molecular action of CRISPR-Cas13 is briefly described in Figures 3B,C. CRISPR-Cas13-mediated cleavage is initiated as pre-crRNA recognizes and binds to the REC leaves of Cas13 to form an intermediate transition state complex. This event induces a conformational change in the conserved residues between Helical-1 and HEPN2 in the NUC region (Zhang et al., 2019). HEPN-2 provides an acid–base catalytic site that catalyzes the processing of pre-crRNA into a matured crRNA (Zhang et al., 2019). Upon complementary pairing between the matured crRNA and target ssRNA, a conformational rearrangement is triggered, as the catalytic HEPN domain of HEPN-2 moves close to the catalytic HEPN domain of HEPN-1 (Zhang et al., 2019). The two HEPN domains combine to form a single catalytically competent active site—which subsequently cleaves the RNA target sequence (Makarova et al., 2017; O'Connell, 2019).

As described earlier, the promiscuous nuclease activity of some Cas13 systems possess a central challenge in their application. This challenge is not essentially negative but opens up new ways for nucleic acid detection if it is carefully harnessed and appropriately applied. This is what the recently developed SHERLOCK platform does. As shown in Figure 4, the SHERLOCK CRISPR tool combines an isothermal nucleic acid amplification technique, and recombinant polymerase amplification technique (RPA), and leverages the indiscriminate endonuclease activity of Cas13 effector protein for clinical diagnosis of diseases (Kellner et al., 2019; Mahas et al., 2021; Mustafa and Makhawi, 2021). While many Cas13 orthologs have been identified and functionally characterized in different bacteria, LwCas13a is the most widely used type in the SHERLOCK applications (Patchsung et al., 2020). Recent advances have seen LwCas13a-based SHERLOCK adopted in different ways (Patchsung et al., 2020). Regardless of the approach, the Cas13-based SHERLOCK platform essentially makes use of: (1) target sequence found in the test sample; (2) T7 RNA polymerase promoter and T7 RNA polymerase for RPA; (3) RNA reporters or sensors sensitive to Cas13 collateral activities; and (4) custom-designed crRNA linked to a programmed Cas13 effector protein for guided detection of target RNA and collateral cleavage of reporters, respectively. The SHERLOCK protocol usually starts with the pre-amplification of the DNA/RNA using RPA primers and the conversion of target dsDNA into ssRNA using the T7 transcriptase enzyme. This is followed by the detection of RNA targets with custom Cas13-crRNA. The custom-designed crRNA is generally composed of a variable spacer region complementary to the target RNA, a constant region that recruits Cas13a, and a T7 promoter sequence that enhances in vitro transcription (Gootenberg et al., 2017; Kellner et al., 2019). Once the spacer of the crRNA aligns with the target RNA, the constant region binds to the Cas13 effector protein resulting in the activation of the off-target HEPN endonuclease activity of Cas13 which consequently cleaves an RNA-tagged reporter to generate a signal for detection (Rajan et al., 2022). To improve signal generation, Yang et al. (2022) engineered a LwCas13a with improved collateral activity by inserting different RNA-binding domains into a unique site within the HEPN domain. The SHERLOCK application has been developed into a cheap and convenient paper-based CRISPR-Cas13-base diagnosing assay capable of detecting and differentiating RNAs of viruses (Zika virus and Dengue virus) and genotyping DNA of bacteria (Escherichia coli and Pseudomonas aeruginosa; Gootenberg et al., 2017; Myhrvold et al., 2018; Manning et al., 2021).

Most eukaryotic organisms possess an evolutionarily conserved mechanism—RNAi, which regulates genes post-transcriptionally and guards against intrinsic and extrinsic threats by degrading the coding regions of target genes. Typical RNAi-related genes such as Dicer and Argonaute have been identified in Plasmodium, but they lack endogenous RNAi machinery, which hinders gene annotation (Mueller et al., 2014). In this regard, non-canonical RNAi systems have been designed to target RNAs in Plasmodium. In 2020, Franziska Hentzschel’s team designed the rodent Plasmodium berghei to express a minimal, unconventional RNAi mechanism that only requires Ago2 (argonaute 2) and a modified short loop RNA, AgoshRNA. The non-canonical AgoshRNA for target genes structurally encompasses a 5 bp hairpin loop (CTTCA) with a sense sequence (having G-U and G-C mismatches initial and terminal codons, respectively) and an antisense sequence attached to either end of the construct. By integrating an AgoshRNA episomally maintained in plasmid into an RNAi-competent Plasmodium, they achieved an efficient gradient gene-knockout of several genes although its application is currently limited to the erythrocytic-stage parasites (Hentzschel et al., 2020).

To circumvent the occurrence of cell death when essential genes are detected for editing/manipulation, conditional manipulating tools such as glms ribozyme system and the Tet operating system have been designed to manipulate the RNA of Plasmodium (Prommana et al., 2013; Briquet et al., 2022). In Tet systems, the promoter of the target gene is replaced with unstable multiple tetracycline operating sites that bind to a transactivator domain for transcription. This system can be turned off or on depending on the presence or absence of anhydrotetracycline (de Koning-Ward et al., 2015). The glms/riboswitch system is designed to integrate an auto-cleaving ribozyme gene into the target gene sequence which upon expression cleaves the UTR region of the target mRNA to induce degradation (Prommana et al., 2013; de Koning-Ward et al., 2015).

This review has summarized the discovery, classification, principle of action, and diagnostic platforms of CRISPR-Cas13 system. Further, it has shown that Cas13-based systems could be prospectively used for single-base editing, and characterizing alternative polyadenylation or alternative splicing in Plasmodium and detecting contaminants in in vitro Plasmodium cultures. The promising CRISPR-Cas13-based tools will be immensely useful for investigating the parasites’ genome.

CZ: conceptualization. YC, EQ, CZ, and LY: writing–original draft. CZ, LY, DS, YC, EQ, SY, JW, YZ, and MC: writing– review and editing. All authors contributed to the article and approved the submitted version.

This work was supported by the National Natural Science Foundation of China (82072304 and 81871671 to LY), grants from Scientific Research of BSKY from Anhui Medical University (XJ201807 to CZ), and the Foundation of Education Department of Anhui province (KJ2021A0213 to CZ).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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