CRISPR use in diagnosis and therapy for COVID-19 (2024)

Methods in Microbiology. 2022; 50: 123–150.

PMID: 38013928

Table 1

A summary of different approaches/methods developed using Cas12a system.

3.1.1.1. DNA endonuclease-targeted CRISPR trans reporter (DETECTR)

The DETECTR system has been successfully used to differentiate between human papillomavirus 18 (HPV18) and human papillomavirus 16 (HPV16) from clinical samples as well as in the crude DNA isolated from cultured human cells within an hour (Myhrvold et al., 2018). It simultaneously performs the reverse transcription and isothermal amplification using the loop mediated amplification (RT-LAMP) and specifically recognizes the viral sequence via Cas12, leading to the cleavage of fluorescent and lateral flow reporter ssDNAs (Ramachandran et al., 2020). The main steps of this procedure include, (a) RNA extraction from nasopharyngeal/oropharyngeal swabs in the universal transport media (UTM), (b) Cas12 detection of pre-established coronavirus sequences, and (c) Cleavage of the reporter molecule (confirming virus) (Broughton et al., 2020). It has been demonstrated on predicted SARS-CoV-2 sequences initially and further studied on many clinical samples. This assay takes less than 40min to complete and its limit of detection (LoD) is noted to be 10copies/μL (Broughton et al., 2020). The primers for this test were designed to amplify the E (envelope) gene, having overlaps with the WHO assay (E gene region) and N (nucleoprotein) with US CDC assay (N2 region in the N gene) of SARS-CoV-2. The N1 and N3 regions lack the suitable PAM sites for the Cas12 gRNAs, therefore these were not targeted for amplification (Safari et al., 2021). This test was given EUA (emergency use authorization) by the FDA recently, provided it must only be used at a single centre (https://www.fda.gov/media/139934/download). Further studies on multi-centre comparison using clinical samples (Brandsma et al., 2020) suggested that the DETECTR method was demonstrated to be an efficient and rapid point-of-care (POC) test, with comparable sensitivity and specificity to RT-qPCR. Some limitations of this technique include the carry over contamination, need of nucleic acid extraction, kits and reagents, and requirement of personal protective equipment (Rahimi et al., 2021).

3.1.1.2. All-in-one dual CRISPR-Cas12a (AIOD-CRISPR) assay

The CRISPR-Cas-based nucleic acid detection method usually needs individual nucleic acid pre-amplification and multiple manual operations, that potentially increases the risk of carry-over contaminations due to amplified products shifting. In the AIOD-CRISPR assay, all the ingredients required for the nucleic acid amplification and CRISPR dependent detection are meticulously mixed in a one-pot reaction system, and incubated at a fixed temperature, to eliminate the requirement for individual pre-amplification and transfer of amplified product (Ding, Yin, Li, Lalla, et al., 2020). The target sequence for SARS-CoV-2 in this method includes a 121bpN gene fragment (GenBank accession MT688716.1) and the initiation of dual CRISPR-based nucleic acid detection with high efficiency done by dual crRNAs without protospacer adjacent motif sites (PAM). The primers and crRNA of the AIOD-CRISPR assay were designed by selecting four sites of the target sequence, which were found to be highly conserved using the GISAID's multiple sequence alignment analysis (n=4663) of SARS-CoV-2 genomes (https://www.gisaid.org/epifluapplications/next-hcov-19-app). The projected LOD of AIOD-CRISPR is about 4.6 copies for RNA targets and 1.2 copies for DNA targets in a 40min incubation. This assay has been implemented to identify the genomic RNA of HIV and SARS-CoV-2 with high sensitivity within an hour (Ding, Yin, Li, Lalla, et al., 2020; Zhang et al., 2020). It was corroborated by testing 28 COVID suspected clinical swab samples where its ultra- specificity is demonstrated by detection of HIV-1 with negligible background as compared to the reported real-time RPA (Safari et al., 2021). The results were found to be consistent with that of the RT-qPCR method and is developed for rapid, simple, specific, ultrasensitive, one-pot, and visual detection of SARS-CoV-2. In addition, it replaced the need for a big incubator with a low-cost hand warmer leading to an instrument-free point of diagnostic method of COVID-19 (Ding, Yin, Li, Lalla, et al., 2020). A few limitations of the AIOD-CRISPR assay are the need for nucleic acid extraction, and the need for kits and reagents (Rahimi et al., 2021).

3.1.1.3. CRISPR/Cas12a-NER (naked eye readout)

Advances in diagnostic techniques has led to a more rapid and accurate method to detect SARS-CoV-2 nucleic acid using the CRISPR/Cas12a-NER system, the Cas12a protein, SARS-CoV-2 specific CRISPR RNAs (crRNAs) and a single-stranded DNA (ssDNA) reporter labelled with a quenched green fluorescent molecule. This reporter molecule is cleaved by Cas12a when there is SARS-CoV-2 nucleic acid in the detection system leading to green fluorescence clearly seen with the naked eye under 485nm light (Wang et al., 2020). There are 15 crRNAs designed on four domains of the orf1a, orf1b, N and E genes over the Wuhan-Hu-1 strain (GenBank accession number MN908947) that can distinguish other SARS-related viruses on the basis of single nucleotide polymorphisms (SNPs). Results revealed that 14 crRNAs, except the E-crRNA1, targeting SARS-CoV-2 were validated, and were highly specific. Clinical validation of CRISPR/Cas12a-NER showed that it has 100% agreement with the results of RT-qPCR assays confirming the high performance of this platform (Wang et al., 2020). The portability, simplicity, sensitivity, specificity, no need for special instruments, time-efficiency, and visibility of results with the naked eye are some of the significant advantages of this method. However, the need for nucleic acid extraction with availability of automated extraction equipment, kits and reagents remain unaddressed drawbacks (Rahimi et al., 2021).

3.1.1.4. iSCAN (in vitro specific CRISPR-based assay for nucleic acids detection)

The iSCAN system involves the RT-LAMP coupled with CRISPR/Cas12 for the rapid, specific, accurate, sensitive detection of SARS-CoV2. Its development targeted two regions in N (at the highly conserved 3'end) and E genes wherein the identified primer set efficiently amplify the synthetic virus fragments, but not the controls. The LAMP primers were generated to ensure a robust amplification to suffice the LAMP-based detection i.e., ~200bp amplification products. This approach is specifically suitable for large-scale and early detection of SARS-CoV-2 carriers, allowing the effective isolation of individuals to limit the spread of the virus. Ali et al. validated the detection kit using extracted RNAs from clinical samples of COVID-19 positive patients (Ali et al., 2020). A requirement of only rapid, field deployable, simple equipment is highly advantageous in using iSCAN, as the specific and easy to use RTLAMP and CRISPR/Cas12 takes less than 1h (colorimetric reaction coupled to lateral flow immunochromatography makes easy interpretation of the results). However, the need of kits and reagents remains unsolved (Rahimi et al., 2021).

3.1.1.5. Variant nucleotide guard (VaNGuard) assay

This assay is highly efficient for viral mutations and can be utilized on purified RNA or directly on nasopharyngeal (NP) swab samples (Ooi et al., 2021). It includes three steps, viz. sample preparation, RT-LAMP reaction, and Cas12a based detection via fluorescence or lateral flow assay. The sample preparation requires proteinase K digestion of NP swabs and heat inactivation. The purified RNA or digested NP swab samples are mixed as the templates into RT-LAMP reactions which is followed by an incubation at 65°C for 22min. The enAsCas12a and ssDNA-probes are added after the incubation to lead to a further incubation at 60°C for another 5min.

The end-point fluorescence in this assay may be spotted by a plate reader or a RT-qPCR machine. Otherwise, a lateral flow strip may be implanted into each reaction tube for an equipment-free read-out. (Ooi et al., 2021). This assay is a robust, rapid, sensitive, affordable, specific, convenient point-of-care test for SARS-CoV-2. However, the need for nucleic acid extraction, kits and reagents are among the disadvantages (Rahimi et al., 2021).

Table 2

A summary of different approaches/methods developed using Cas12b system.

3.1.2.1. STOP (SHERLOCK testing in one pot)

Recently, this assay was developed as a simple test for detection of SARS-CoV-2 in about 1h, that is appropriate for point-of-care applications. As compared to RT-qPCR-based SARS-CoV-2 tests, the sensitivity of STOP COVID has the LOD of 100 copies of viral genome/μL. The test results are obtained in 70min with a dipstick, and in 40min with a fluorescence readout. To make the test less complex Zhang et al. developed a simple protocol that does not require the sample extraction as it lyses the viral particles with QuickExtract at room temperature (22°C) or in one-pot at an incubation temperature of 60°C for 10min (Zhang et al., 2020). The Cas12b is from Alicyclobacillus acidiphilus (AapCas12b) that sustains sufficient activity at the same temperature range as LAMP (55–65°C). It is utilized for the detection of the N gene of SARS-CoV-2 in this defined assay. Since the AapCas12 locus lacks a CRISPR array the AapCas12b was integrated with the scaffold of Alicyclobacillus acidoterrestris Cas12b (AacCas12b) as tracrRNA (Trans-activating CRISPR RNA). The validation test on the clinical samples showed this assay successfully diagnosed 12 positive and 5 negative COVID-19 patients, with a minimum 2 of 3 replicates scoring positive in infected persons (Joung et al., 2020). The application of this platform may considerably help “test-trace-isolate” approach, particularly in the low-resource areas (Joung et al., 2020). Its simplicity, suitability for point-of-care (POC) analysis, sensitivity, cost efficiency, handiness of its components, no need of RNA extraction is among the main advantages of this assay whereas its disadvantages are negligible (Rahimi et al., 2021).

3.1.2.2. CASdetect (CRISPR-assisted detection)

Another diagnostic assay based on CRISPR, called Cas12b-mediated DNA detection (CDetection), was developed for the detection of SARS-CoV-2 (Guo et al., 2020). The detection range of the CASdetect system for SARS-CoV-2 pseudovirus was 1×10⁴ copies/mL, without cross-reactivity to other human endemic coronaviruses. Incorporation of the sample treatment protocols and the nucleic acid amplification strategies with CDetection, an integrated viral nucleic acid detection system CASdetect (CRISPR-assisted detection) was developed (Guo et al., 2020). Some of the advantages of this tool are no cross-reactivity, reduced false positive rate, and accuracy whereas disadvantages are the need for nucleic acid extraction, need of kits and reagents (Rahimi et al., 2021).

Table 3

A summary of different approaches/method developed using Cas13 system.

3.1.3.1. Specific high-sensitivity enzymatic reporter unlocking (SHERLOCK)

The SHERLOCK (Gootenberg et al., 2017) approach exploited Cas13a for the detection of RNA molecules. Later, a diagnostic platform based on this method was developed in 2018 (Gootenberg et al., 2018). This tool was employed for the detection of COVID- 19 (Zhang et al., 2020), to develop an improved and specific diagnostic kit for COVID-19 to two targets - one from the S-gene and the other from the Orf1ab gene. The primers and gRNAs (LwaCas13a CRISPR) were developed to detect COVID-19 RNA (not cross reacting with related viral genomes) (Hou et al., 2020). This approach implements a non-targeted reporter RNA tagged to a fluorescent dye for the identification of specific RNA molecules (). A web resource containing CRISPR-Cas13 based assay designs has been developed to identify 67 viruses, including SARS-CoV-2, Zika virus, and dengue virus, capable of selecting single or multiplex panels (Chen et al., 2018). The sensitivity of COVID-19 target sequences using SHERLOCK method is estimated in a range between 10 and 100 copies per microliter of input (20 and 200aM), i.e., LoD was 10–100 viral RNA copies/μL.

The SHERLOCK COVID-19 detection protocol can be completed in 1h involving the following steps:

• (1)

  An isothermal amplification of the sample (25min incubation) with the help of recombinase polymerase amplification (RPA) kit.

• (2)

  Identification of pre-amplified viral RNA with Cas13 (30min incubation)

• (3)

  Read out of the outcome with paper dipstick (2min incubation)

  See Also

  Transcription factors (article) | Khan Academy

SHERLOCK was validated on 154 clinical samples, with 96% and 88% sensitivity for the fluorescence and lateral flow readouts, respectively (Patchsung et al., 2020). Additionally, both the assays had 100% specificity (Patchsung et al., 2020). The advantages of SHERLOCK include amenability to automation and the use of a minimum volume of reagents, rapid, sensitive, and no need for sophisticated equipment (Rahimi et al., 2021).

3.1.3.2. CREST (Cas13-based, rugged, equitable, scalable testing)

To minimize the blockade to COVID-19 diagnostics, a method called CREST (Cas13-based, Rugged, Equitable, Scalable Testing) was devised (Rauch et al., 2021). The CREST platform with Cas13 detection is integrated with a thermal cycling amplification step (PCR), a linear amplification step (transcription), and enzymatic signal amplification via fluorescence detection. It eliminates the 3 main barriers, viz. reagent accessibility, equipment availability, and cost. Therefore, this tool has been developed by harnessing the advantage of widely available enzymes, low-cost thermocyclers (more cost effective than RT-qPCR), and easy-to-use fluorescent visualizers. Moreover, the CREST is equivalent in sensitivity (LOD-10 copies of a target RNA molecule per microlitre) to the reverse transcription quantitative polymerase chain reaction (RT-qPCR) method for COVID-19 testing. It therefore has the potential to pick up positive cases earlier than regular tools (Rauch et al., 2021). The advantages of CREST include that it can be executed from RNA sample to result without any requirement of a dedicated facility, within ~2h, relieve some of the strain on the global supply chain for testing reagents, does not need specialized instrumentation, and requires very little specialized training. A few disadvantages are that, it requires nucleic acid extraction, needs kits and reagents (Rahimi et al., 2021).

3.1.3.3. SHINE (SHERLOCK and HUDSON integration to navigate epidemics)

To eliminate the need of nucleic acid extraction by using heat and chemical reduction, SHERLOCK can be combined with HUDSON (Heating Unextracted Diagnostic Samples to Obliterate Nucleases), for both viral particle lysis and elimination of RNA-degradation (Myhrvold et al., 2018). The combined SHERLOCK and HUDSON can be carried out with minimal infrastructure because only a heating element is required. But, the need to prepare multiple reaction mixtures and handling multiple samples in between are limitations. To address the current limitations of nucleic acid diagnostics, a special tool has been developed, named SHINE (SHERLOCK and HUDSON Integration to Navigate Epidemics) for extraction-free, rapid, and sensitive detection of SARSCoV-2 RNA. It has been shown that SHINE can identify SARS-CoV-2 RNA in HUDSON-treated samples (clinical) with either a paper-based colorimetric readout, or an in-tube fluorescent readout that can be carried out with portable equipment and reduced risk of sample contamination. Its advantages include sensitivity, specificity, single-step reaction, no need for hospitals, laboratories and nucleic acid extraction, whereas its disadvantages are negligible (Rahimi et al., 2021).

3.1.3.4. CARVER (Cas13-assisted restriction of viral expression and readout)

CARVER is an end-to-end platform that uses Cas13 to detect and destroy viral RNA. Hundreds of crRNAs along with the LCMV (lymphocytic choriomeningitis virus) genome were screened to assess how conservation and target RNA nucleotide content influences Cas13’s anti-viral activity (Freije et al., 2019). CARVER was also used to detect RNA viruses such as, influenza A and vesicular stomatitis, providing examples of its potential expanded application for the detection of a broad range of viral nucleotides in disease diagnosis (Freije et al., 2019). Hence, it is quite clear that Cas13 can be utilized to target a wide range of ssRNA viruses and CARVER has immense potential for diverse utility of rapid diagnostic and antiviral drug development.

3.1.4.1. Cas3-operated nucleic acid detection (CONAN)

A combination of RT-LAMP and Cas3-based nucleic acid detection, resulted in an approach known as (CONAN) for COVID-19 diagnosis. Using post RNA isolation from clinical samples, RT-LAMP was carried out for SARS-CoV-2, and the leading amplicons were targeted using Cascade/Cas3 to achieve a fluorescence or lateral flow readout. The lateral flow-based CONAN approach was implemented on 31 clinical samples and showed 90% sensitivity and 95% specificity compared to the RT-qPCR assay. Similar results were reported with the DETECTR method (Broughton et al., 2020). Although, Cas3 was implemented for pathogen recognition for the first time, efficiency was at a level comparable to that of the Cas12a-based detection method, which has been in use for more than 2 years (Chen et al., 2018). Therefore, an even more sensitive SARS-CoV-2 detection method can be created by further optimization of the CONAN method. Advantages of this assay are time and cost efficiency, highly sensitive, and efficient single-base-pair discrimination. Whereas, disadvantages are nucleic acid extraction, and need of kits and reagents (Rahimi et al., 2021).

3.1.5.1. FnCas9 editor linked uniform detection assay (FELUDA)

This class of CRISPR/Cas approaches for nucleotide recognition is dependent on the specific binding and cutting activity of CRISPR/Cas9. Previously, this method was used for the detection of Zika virus. Explicitly, Cas9 from Francisella novicida (FnCas9) was found to be highly specific for both target DNA binding and cleavage (Acharya et al., 2019). Its high specificity helped in developing a FnCas9-based nucleic acid detection method named (FELUDA) (Azhar et al., 2020), which was quickly adapted by these authors for diagnosis of COVID-19. This method used synthetic DNA fragments coding N gene of SARS-CoV-2, demonstrating that this assay can identify SARS-CoV-2 and is capable of distinguishing it from SARS-CoV and H1N1 viral sequences. This method was validated by screening total RNA of COVID-19 patient samples within 45min. It is also claimed that FELUDA was compatible with fluorescence readout and with RT-RPA as a pre-amplification method (Azhar et al., 2020). Thereafter, the FELUDA method was adapted for the lateral flow readout by using the catalytically inactive form of FnCas9 (dFnCas9), FAM-labelled trans-activating CRISPR RNA (tracrRNA), and biotinylated PCR primers (Azhar et al., 2020). A summary of the limits of detection (LOD) and the Cas enzyme used for all the diagnostic methods discussed above is provided in Table 4.

4.1.3.1. Cas13a

Cas13a was originally identified in 2015 by Shmakov and co-workers, as Leptotrichia shahii (LshCas13a) which utilizes a short 24nt long crRNA. Unlike the most widely used CRSIPR/Cas9 system which needs crRNA and trcrRNA (as a single guide RNA (sgRNA)), Cas13a utilizes only a crRNA. The crRNA interacts with the LshCas13a via a Uracil rich stem loop and requires a PFS consisting of A, C or U base pair, located at the 3′ end of the spacer sequence (Shmakov et al., 2015). The Zhang lab at the Broad Institute of MIT and Harvard discovered that some orthologues of Cas13 can be used in mammalian and plant cells (Abudayyeh et al., 2017). Cas13a from Leptotrichia wadei (LwaCas13a) is well characterized and it does not require a PFS at the target site, making it more flexible for therapeutic applications. However, it requires stabilization by a monomeric superfolder green fluorescent protein (GFP) for effective RNA knockdown in mammalian cells (Cox et al., 2017). Moreover, the LwaCas13a does not display collateral cleavage activity in eukaryotic cells. For Cas13a, the processing of pre-crRNA into mature crRNA is not required, as pre-crRNAs themselves can acts as a guide for the cleavage of the ssRNA target sequence ().

Blanchard and colleagues demonstrated that the specific activity of Cas13a to knock down endogenous genes can be utilized for treatment of respiratory viral infections including SARS-CoV-2 infections (Blanchard et al., 2021). They performed a prophylactic as well as a therapeutic study in VeroE6 cells by targeting the conserved regions (Nucleocapsid and replicase) of SARS-CoV-2, using synthetic mRNA to express LbuCas13a (from Leptotrichia buccalis) and demonstrated that SARS-CoV-2 was inhibited. They also demonstrated an in-vivo mitigation of the virus in a Syrian hamster model by using an inhalation-based apparatus to deliver Cas13mRNA along with crRNA. In another study, with the help of bioinformatics tools, crRNAs against the SARS-CoV-2 receptor binding domain (RBD) were identified and their editing efficiency was evaluated in human epithelial type II (AT2) cells and human hepatocarcinoma cells (HepG2). They designed CRISPR/Cas13a (LwaCas13a) based technology against SARS-CoV-2 for targeting and cleaving its RNA and found that crRNA6 was a potential candidate (Wang et al., 2021). Apart from the cleavage of SARS-CoV-2 RNA (a strategy considered useful in early infection), they also used the same strategy for reducing viral proteins (more appropriate during late infections) using the dCas13a-crRNA6. DeadCas13a (dCas13a) is a catalytically inactive version of Cas13a, generated by causing R474A and R1046A mutations in the HEPN domains, which only binds with the target RNA sequence and does not cleave it, thus regulating the transcription of SARS-CoV-2 genes (Abudayyeh et al., 2017).

4.1.3.2. Cas13b

Like the Cas13a subtype, CRISPR loci encode a single effector protein, Cas13b which contains two predicted HEPN domains at its N and C- termini. The CRISPR/Cas13b system cleaves ssRNA using HEPN domains and also exhibits collateral RNase activity. Apart from HEPN domains there is no sequence similarity between 13a and 13b. Unlike Cas13a, Cas1 and Cas2 are absent in the Cas13b system and it can process its own CRISPR array. The Cas13b system has two variants denoted as VI-B1 and VI-B2 with their accessory proteins named as Csx27 and Csx28 respectively. In the VI-B1 system, the Csx27 causes repression of Cas13b whereas Csx28 enhances Cas13b activity. It has been shown that along with several factors, the presence of secondary structure in the RNA target impacts the activity of Cas13b enzymes (Smargon et al., 2017). A research group from Thailand showed that CRISPR/Cas13b system effectively abrogated the porcine reproductive and respiratory syndrome virus (PRRSV), by simultaneously targeting ORF5 and ORF7 genes ().

The antiviral potential of Cas13 systems (both 13a and 13b) have been evaluated in 3 different ssRNA viruses [lymphocytic choriomeningitis virus, (LCMV), Influenza A virus (IAV) and vesicular stomatitis virus (VSV)] in cell culture by Freije et al. (2019). They carried out a computational analysis followed by experimental validation of viral genome sequences to create a repository of antiviral crRNAs. They used Cas13a (LwaCas13a) and Cas13b (PspCas13b) from L. wadei and Prevotella sp. P5–125, respectively, to demonstrate the generalizability of the Cas13 system. Their study demonstrated that Cas13 can efficiently reduce the viral RNA levels in mammalian cells, confirming the potent antiviral activity of Cas13 against different ssRNA viruses (Freije et al., 2019). In comparison to the Cas13a system, the CRISPR/Cas13b system was found to be more robust and its activity can be upregulated or downregulated depending on the accessory proteins encoded by its loci (Cox et al., 2017). Considering that SARS-CoV-2 also possess a ssRNA viral genome, the same principles can be applied to generate anti SARS-CoV-2 therapeutics.

4.1.3.3. Cas13d

For working with this system, guide RNA is made up of a 30nt direct repeat with a 22nt spacer (Fig. 1). Spacer length of 22nt is desirable as below this length the cleavage activity is significantly dropped. In mammalian cells, the collateral cleavage activity of Cas13d is absent. Also, the target cleavage does not depend on the flanking sequence requirements but the cleavage pattern varies with the target. For example, Eubacterium siraeum/EsCas13d prefers uracil bases in the target region above all other bases. The study was conducted to find out an active Cas13d orthologue in eukaryotic cells and revealed that an engineered variant of Cas13d Ruminococcus flavefaciens strain XPD3002 (Rfx) Cas13d (CasRx) can be developed into a flexible tool for programmable ssRNA targeting in mammalian cells. They compared the small hairpin RNA (shRNA) interference, CRISPR subtype VI-A/VI-B and Cas9 mediated transcriptional inhibition (CRISPRi) with CasRx and found that CasRx outperformed with 96% knockdown as compared to 65% knockdown by shRNA, approximately 70% by CRISPRi and 53% by CRISPRi; suggesting CasRx was the most efficient RNA regulating method. Another study by Yan and co-workers, characterized E. siraeum (EsCas13d) and Ruminococcus sp. (RspCas13d) orthologues of Cas13d enzyme (Yan et al., 2018). They also showed that Cas13d associated accessory proteins have a WYL domain, because the target activity of RspCas13d was increased, indicating that this particular domain regulates the activity of Cas13d (Yan et al., 2018). Recently, a research group from Stanford University developed a potential CRISPR/Cas13d-based pan-coronavirus inhibition strategy. They named it prophylactic antiviral CRISPR in human cells (PAC-MAN) and showed that it could cleave the SARS-CoV-2 sequences that were effective as novel antiviral therapy against COVID-19 (Abbott et al., 2020). Abbott and colleagues used CasRx and synthesized 20 crRNAs targeting RNA-dependent RNA polymerase (RdRp) and Nucleocapsid (N) genes, each. They chose a Cas13d expressing human lung epithelial cell line (A549) to transduce the pool of crRNAs and found that the targeting RdRp and N gene repressed a reporter by 86% and 71% respectively. It was also demonstrated that the SARS-CoV-2 inhibition was quite sensitive to different crRNA concentrations and lowering the Cas13d expression has little effect on the inhibitory activity of the Cas13d system (Abbott et al., 2020). They also suggested that CRISPR/Cas13d can be used as an antiviral strategy, both prophylactically and therapeutically (Fig. 2).

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Fig. 1

Schematics for SARS-Cov-2 detection using CRISPR/Cas platform. The RNA is extracted from the patient using conventional RNA extraction method. For Cas12 based detection the RNA is amplified into dsDNA whereas for Cas13 based detection the amplified DNA is transcribed into ssRNA. The collateral activity of both Cas12 and Cas13 is used for the detection of SARS-CoV-2.

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Fig. 2

(A) CRISPR/Cas13d array showing HEPN domains. (B) Schematic of CRISPR/Cas13d mechanism as an antiviral strategy. Cas13d shown as a green cloud and guide RNA (DR and spacer) together forms a Cas13d:crRNA complex required for viral RNA degradation. DR: Direct repeat.

Consolidating the studies on antiviral capabilities of subtypes of CRISPR/Cas13 systems, Cox and co-workers detected that among the different orthologues of Cas13a, b and c; Cas13b (PspCas13b) was the most specific and efficient for RNA knockdown in mammalian cells (Cox et al., 2017). LwaCas13a had two major issues; first it must be stabilized with monomeric super-folded GFP and secondly, the average RNA knockdown efficiency was around 50% whereas PspCas13b provided better efficiency as compared to LwaCas13a with 62.9% knockdown. Although these systems can be reprogrammed to target ssRNA, it is difficult to pack them into adeno-associated vectors (AAV) for in-vivo delivery due to their large size. In comparison to Cas13a (1250 aa), Cas13b (1150 aa), and Cas13c (1120 aa), Cas13d effector has an average length of 930 aa (the smallest class2 CRISPR effector) (Cox et al., 2017; Shmakov et al., 2015; Smargon et al., 2017). Overall, the CRISPR/Cas13d system seems to be the most practical system for a therapeutic approach to target the SARS-CoV-2 genome. The small but robust CRISPR/Cas13d system seems to be the latest technology for the RNA engineering toolbox.

This claim may be further supported by the following statements.

• i.

  The size of Cas13d (approx. 930aa) is 17–26% smaller than other class2 type VI- CRISPR/Cas subtypes (Cas13a-c); which makes it suitable for packaging in low- capacity vectors, like AAV; making it particularly suitable for delivery in primary cells and in-vivo applications.

• ii.

  It lacks any sequence constraints for flanking sequences i.e., Cas13d does not require the presence of PFS. This property makes it possible to target theoretically any ssRNA sequence.

• iii.

  Modular activity of WYL1 for CRSISPR/Cas13d system: CRISPR/Cas13d locus consists of an accessory protein with the WYL domain (named for three amino acids conserved in the original domain) (Yan et al., 2018). Co-occurrence of Cas13d with accessory proteins having a WYL-domain increases the targeted cleavage, implying such proteins act as regulators for Cas13d activity.

• iv.

  Comparison between Cas13a/b effectors and Cas13d effector (CasRx) as RNA regulating systems, showed knockdown of approximately 70% by Cas13a/b enzymes whereas 96% by CasRx.

Therefore, the strong catalytic activity, high specificity, and small size of the Cas13d protein makes it the best choice for targeting the SARS-CoV-2 genome (Abbott et al., 2020). Studies showed that the highly conserved regions of SARS-CoV-2 [(Abbott et al., 2020) nucleocapsid (N) which protects the viral genome and (Abudayyeh et al., 2017) RNA-dependent RNA polymerase (RdRp) which catalyses the transcription of all viral mRNAs] can become potent targets of CRISPR/Cas13d (Chan et al., 2020), thus disabling virus production and function (Abbott et al., 2020).