Coronavirus disease (COVID-19) was spread in December 2019 and was recognized as a zoonotic disease (Drosten et al., 2017; Andersen et al., 2020). Severe acute respiratory syndrome (SARS) virus was detected in sputum samples in 2003, and advanced stages in fecal samples may have been transmitted to humans by an intermediate host such as bats and civets (Wang and Eaton, 2007; Graham and Baric, 2010). Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) can be transmitted from an unknown carrier to a healthy person who could infect many people. SARS-CoV-2 resulted in pneumonia in Wuhan, China, with various symptoms reported. The disease has developed into a pandemic (Wu C. et al., 2020; Wu D. et al., 2020; Guan et al., 2020). Appropriate methods could treat and control the disease. CRISPR/Cas9 was first recognized as a microbial immune system through which these organisms acquire immunity to invading viruses and plasmids (Garneau et al., 2010). When the invaded foreign DNA enters the bacteria, it is cleaved by cas nuclease enzymes. A portion of the cleaved DNA is then placed between two repeating sequences at the CRISPR site. Here, it is called a spacer (Barrangou and Horvath, 2017; Shmakov et al., 2017). The spacer sequences are used as templates to generate short RNA sequences. These sequences direct the Cas protein to the invasive DNA. Once the Cas protein binds to the invasive DNA, the enzyme cuts the outer DNA sequence into both strands, creating the region of double-strand breaks (DSB) (Jinek et al., 2012; Wei et al., 2015; Brinkman et al., 2018) and the nucleotides at the DSB position change the structure or end codon in the gene. Non-homologous end-joining repair (NHEJ) or homology-directed repair (HDR) systems are induced to edit the genome to cut and cleave external DNA. These deletions and additions lead to a permanent change in the open reading frame (Hsu et al., 2014; Klein et al., 2019).

Some model systems, including mammalian cells, can efficiently cleave any complementary sequence to the gRNA and target and cleave the RNA. Targeted genome editing, often called CRISPR/Cas9, is increasingly recognized as an effective tool in medicine (Bawage et al., 2018; Freije et al., 2019). It can inactivate the SARS-CoV2 virus in mammalian cells by truncating the specific sequence of the virus. Cas proteins appear to help in detection and treatment of viral infections. They are introduced into the viral genome via guide RNAs and destroy it in the target regions (Aman et al., 2018; Xiao et al., 2018; Dolan et al., 2019). However, more regular interaction and collaboration between virology and molecular biology is needed to achieve substantial results in the treatment of SARS-CoV-2 (Rodrí guez-Rodr í guez et al., 2019). In this review, we first introduce general concept of CRISPR/Cas9. Then, we discuss potential challenges for treatment, and finally, we address prospects for CRISPR/Cas9-based antiviral strategies for SARS-CoV-2.

The coronavirus family (CoVs) contains many virus species, and these viruses can cause various diseases in birds, livestock, and humans (Miłek and Blicharz-Domanska, 2018). Members of the family are spherical and have an approximate diameter of 125 nm. The spines protrude from the surface of the virion and give the virus a particular crown-like appearance (Neuman et al., 2006). There are four main structural proteins in coronavirus: Membrane (M), spikes (S), envelope (E), and nucleocapsid (N) proteins (Figure 1A). The spikes bind the virus to the host cell receptor (Beniac et al., 2006; Wrapp et al., 2020). Researchers have discovered human coronavirus receptors, such as angiotensin-converting enzyme 2 (ACE2) for SARS-CoV (Li et al., 2005) and HCoV-NL63 (Wu et al., 2009) or aminopeptidase N (APN) for HCoV-229E (Yeager et al., 1992; Li et al., 2005). ACE2, APN, and DDP4 are ectopeptidase enzymes with different functions expressed on the surface of various cell types, including those of the human respiratory tract. MERS-CoV has been shown to require a dipeptidyl peptidase-4 (DPP-4) surface receptor to enter the host cell. SARS-CoV-2 uses the endogenous enzyme angiotensin-converting enzyme 2 (ACE2) to enter host cells (Raj et al., 2013; Monteil et al., 2020). After binding to the appropriate receptor, a fusion of the virus and cell membrane occurs, and the viral genome is transferred to the host cell’s cytoplasm. In the host cell, the viral products are produced and assembled (Belouzard et al., 2009). After the virus particles are assembled, they are transported to the cell surface by vesicles and released by exocytosis (Fehr and Perlman, 2015). SARS-CoV-2 mainly infects epithelial cells in the lung but can also invade macrophages and dendritic cells (Weinheimer et al., 2012; Zhou et al., 2015). The exact mechanism of lung injury caused by SARS-Cov-2 is still unknown (Zhou et al., 2020). Serologic evidence of SARS-CoV-2 infection was observed in some individuals at animal markets before the disease outbreak. Some animals have also been infected with SARS-CoV-2 viruses isolated from camels, Himalayan palm civets, and raccoon dogs (Azhar et al., 2014; Fung et al., 2020). Further evidence from phylogenetic analysis suggests that SARS-CoV-2 is bat-derived (Lau et al., 2005; Zheng, 2020), as the genetic similarity between SARS-CoV-2 and bat-SARS is greater than 95%. Bat-SARS has the potential to be transmitted to humans. Although the risk of transmission is lower, it is affected by bat SARS in an area (Cook et al., 2021). Although one study has shown that SARS-CoV-2 is not a mosaic (Paraskevis et al., 2020), there is speculation about its heritability. The UK Medicines Agency has approved molnupiravir to treat mild to moderate COVID-19 in people with at least one risk factor for severe disease. Molnupiravir could cut the number of people who need to go to the hospital in half and reduce the number of deaths. However, the supply would not last long if it were given to everyone who is sick because the daily caseload is high. Use of the drug would likely be limited to those at the highest risk for disease complications, such as older adults with heart, lung, or kidney disease, diabetes, or cancer (Wu F. et al., 2020; Mahase, 2021). Vaccination is the most important method of epidemic control. The emergence of numerous SARS-CoV-2 variants that are less prone to disease- and vaccine-induced immunity threatens progress. Despite these ongoing threats, the efficacy of the SARS-CoV-2 vaccine provides a reason for optimism for 2021 (Creech et al., 2021). SARS-CoV-2 neutralizing antibodies in the serum of cured patients can be recovered and reused if SARS-CoV-2 recurs (Zhou and Zhao, 2020). Such antibodies will help protect individuals at high risk.

Host-virus conflict is a dynamic process. Viruses use host factors to complete their life cycle, and the host uses the body’s immune system to fight off the viral infection, so it does not waste energy. The virus must enter the host cell to replicate its genome and complete its life cycle (Sicard et al., 2019) (Figure 1B). Theoretically, antiviral treatments can prevent the virus from entering the host cell or destroy the genetic elements of the virus. Targeting host factors can help the virus become resistant to antiviral drugs (Lin and Gallay, 2013). However, this hypothesis needs further investigation to identify its weaknesses and exploit them after these weaknesses have been addressed. The CRISPR/Cas9 system could be useful because it targets viral nucleic acid and host material quickly and conveniently (Lino et al., 2018). Cas9 is known in CRISPR/Cas systems as a DNA endonuclease directed from a guide RNA (sgRNA) to the target DNA to alter the genome of the target region (Kennedy and Cullen, 2015; Ishino et al., 2018). This genome editing leads to an antiviral status within the host cell. CRISPR/Cas9 was the first system studied in HIV-1 gene therapy research (Xiao et al., 2019). Host cell receptors, CCR5 and CXCR4, help HIV enter the host cell (Wilen et al., 2012; Santos-Costa et al., 2014). Therefore, one of the antiviral candidates to treat HIV is suppressing these receptors. Researchers have successfully suppressed the expression of CCR5 in primary CD4 T cells at an appropriate level using the CRISPR/Cas9 gene-editing system. These cells develop resistance to HIV-1 and do not cause extracellular toxicity (Hou et al., 2015; Li et al., 2015).

Host cell factors such as the Apo-Lipoprotein B Editing Complex (APOBEC3) and Tripartite Motif Containing 5 (TRIM5) have been identified as viral limiters for HIV infection (Bogerd et al., 2015). APOBEC3 is thought to act as an antiviral agent by causing mutations in the viral genome. CRISPR/Cas9-based regulation of the host APOBEC3 factor reduces HIV reporter gene expression and provides antiviral effects (Jern et al., 2009; Bogerd et al., 2015). Two specific amino acids in TRIM5 have made it an actual antiviral agent against HIV-1 infection. This antiviral candidate can induce cleavage of viral capsid proteins, demonstrating its antiviral properties (Sastri and Campbell, 2011; Weatherley et al., 2017). Therefore, TRIM5 may be a suitable target for the CRISPR/Cas9 system. Eliminating microRNA-146 by CRISPR/Cas9 resulted in a significant increase in HIV-1 limiting factors (Teng et al., 2019).

Some host factors are essential for virus replication, assembly, and budding. Therefore, knocking out their genes could be an alternative to preventing HIV-1 infection (Lin and Nagy, 2013; Xu and Nagy, 2015); such gene deletions can be performed using the CRISPR/Cas9 system (Gilani et al., 2019). Interestingly, these gene deletions have no significant effects on host cells (Chen J. S. et al., 2018). These results demonstrate the antiviral potential of CRISPR/Cas9-based therapies, as it is possible to disrupt key host factors essential for HIV infection.

The worldwide pandemic of SARS and CoV-2 poses a significant threat to global public health and societal stability and has become a significant global public health problem. Regrettably, current diagnostic and therapeutic methods to prevent and control SARS-CoV-2 have many limitations. CRISPR/Cas technology has emerged as a potential complement to conventional methods in recent years. Biomedicine has extensively used biological tools based on the CRISPR/Cas systems. They are helpful in pathogen detection, clinical antiviral treatment, and drug and vaccine discovery. Therefore, CRISPR/Cas technology could be promising in preventing and treating SARS-CoV-2 and other emerging infectious diseases.

CRISPR-Cas systems could be used for molecular diagnosis of nucleic acids (Figure 2) (Jia et al., 2020:; Kaminski et al., 2021). CRISPR-Cas-based diagnostic methods have the same sensitivity and specificity as conventional PCR. However, their cost is low because they do not require complex or expensive technology (Ayanoğlu et al., 2020). The application of CRISPR-Cas in molecular diagnostics could transform global diagnostic and healthcare systems (Gootenberg et al., 2017). The Cas proteins used in CRISPR-Cas systems vary depending on the DNA or RNA targeted and the intended applications (Makarova et al., 2015). Following the global spread of the COVID-19 pandemic, rapid and straightforward diagnostic techniques are in high demand. CRISPR-based methods, which have demonstrated superior detection capability in as little as 30–60 min, could overcome this obstacle. In addition, a CRISPR/Cas9-mediated lateral flow nucleic acid assay (CASLFA) has been developed to identify infections using the CRISPR/Cas system (Wang et al., 2020). However, FDA approval is still pending. Regarding “collateral breast activity,” CRISPR-based diagnostic techniques have been developed using the Cas12a or Cas13 nuclease. The Cas12a/Cas13 nuclease, a component of the CRISPR tool, is activated after CRISPR RNA binds to the target bosome (crRNA). When produced, it non-specifically cleaves ssDNA/RNA particles in the vicinity and explicitly acts as a collateral bosome or transbosome. Researchers took advantage of this property to develop fluorescently labeled ssDNA/RNA press reporter probes capable of detecting visible bands in a paper strip via a side-stream assay, enabling the development of a novel nucleic acid-based diagnostic test (Chen S. et al., 2018). Viral RNA targeting crRNA can activate Cas protein, resulting in collateral cleavage of press reporter probes and a helpful band on the paper strip (Chen J. S. et al., 2018). In the newly developed Specific High-sensitivity Enzymatic Reporter Unlocking (SHERLOCK) technology, the activity of the crRNA-Cas13a protein complex is used to recognize RNA molecules and cut collateral RNA near the target RNAs. Metsky et al. have developed a website with CRISPR-Cas13-based assay designs for the detection of 67 diseases, including SARS-CoV-2, Zika virus, and dengue fever, with a choice of single or multiple panels (Metsky et al., 2020). The comprehensive SARS-CoV-2 diagnostic test is based on advanced technology from SHERLOCK. This technique uses fluorescently identified, non-targeted press reporter RNA (Kellner et al., 2019). SHERLOCK test for detecting SARS-CoV-2, which has a sensitivity of 10 copies per microliter and can be fluorescently confirmed, has been validated with counterfeit RNA fragments. This molecular analytical test should be inexpensive and provide rapid results. The DETECTOR is a similar technique used to amplify pathogenic DNA with RPA, and reverse transcription to identify RNA viruses is also used as a SHERLOCK system in this procedure. Cas12a-crRNA identifies the target and activates the Cas12a nuclease, which cleaves fluorescently labeled reporter sDNA without discrimination. In less than an hour, DETECTOR distinguished between human papillomavirus 16 (HPV16) and human papillomavirus 18 (HPV18) in pure DNA from cultured human cells and professional samples (Myhrvold et al., 2018). Broughton et al. diagnosed COVID -19 using two specific crRNAs targeting genes E and N and a discovery series ranging from 70 to 300 copies per microliter of sample material. They used LAMP with reverse transcription instead of RPA-based amplification to identify COVID -19 in less than 30 min. These CRISPR-Cas-based nucleic acid detection methods require independent amplification of nucleic acids. They require human activity, complicating detection and increasing the risk of spreading contamination (Broughton et al., 2020a). Ding et al. developed the AIOD-CRISPR (All-In-One Dual CRISPR-Cas12a) assay method, which enables rapid visual detection of viral nucleic acids with high sensitivity and accuracy. In this article, all materials required for viral nucleic acid detection are incubated in a single pot at 37°C, which simplifies the process and minimizes the risk of contamination. With high sensitivity, SARS-CoV-2 genomic RNA was detected with the AIOD-CRISPR assay (Ding et al., 2020). LED assay uses blue light illumination to image the tubes instead of a paper dipstick with sidestream detection. Scientists in India have identified the Francisella novicida Cas9 orthology (FnCas9) as sensitive to nuclear differences, and the Linked Attire Discovery Assay (FELUDA) for FnCas9 has been developed as a low-cost point-of-care (LCC) assay for identifying SARS-CoV-2 infections in the clinical setting (Azhar et al., 2020).

“Metagenomics” and “qRT-PCR” are two widely used molecular methods for identifying novel viruses (Gu et al., 2019; Corman et al., 2020). Current qRT-PCR-based SARS-CoV-2 diagnostic methods are efficient and accurate for virus detection. Where qRT-PCR technology is not available, the virus can spread globally (Lucia et al., 2020). Finally, the diagnostic accuracy of many molecular methods has not yet been clarified. Although CRISPR/Cas has been a widely used gene-editing strategy since 2013, the simultaneous promiscuous cleavage tasks of a specific collection of Cas nucleases were discovered later and used to detect nucleic acids from artificial insemination (Chen S. et al., 2018; Harrington et al., 2018). Due to its exceptional sensitivity, specificity, and reliability, RNA-directed nucleic acid detection based on CRISPR/Cas nuclease has recently shown significant potential for developing next-generation molecular diagnostic technology (Ding et al., 2020; Lucia et al., 2020). For example, AIOD-CRISPR can detect only 1.2 copies of DNA targets and 4.6 documents of RNA targets in 40 min of incubation without preamplification when detecting SARS-CoV-2 (Lucia et al., 2020). CRISPR-nCoV has the same sensitivity and uniqueness as next-generation metagenomic sequencing (mNGS) in less than 40 min (Hou et al., 2020). Some of the major advantages of this approach over existing techniques such as qRT-PCR are 1) uniformity of signal in a nucleus (e.g., SARS-CoV-2 guide RNAs can be distinguished from SARS-CoV and MERS-CoV at the N₂ site) and 2) integration with low-cost portable reporting sheets and side streptometers. 3) isothermal signal amplification for rapid target detection in the absence of termocycling. CRISPR-based assays are more accessible and convenient than RT-PCR viral RNA identification assays because the CRISPR system does not require bulky instrumentation or complicated processes (Ganbaatar and Liu, 2021). Most CRISPR-Cas-based detection methods require pre-amplifying a specific nucleic acid combination and manual procedures. These modifications will undoubtedly complicate procedures and impose costs on the environment. The following table summarizes the current studies on the diagnosis of SARS-CoV-2.

SARS-CoV-2 is a novel coronavirus of the positive-sense RNA virus family that infects the respiratory tract and causes disease through direct cytotoxic effects and the production of host cytokines (Liu et al., 2020). The life cycle of SARS-CoV-2 is like that of other strongly associated coronaviruses, such as the virus that causes SARS. The virus spreads its RNA genome in the cell, synthesizes the genomic and subgenomic negative sense RNAs used in the viral mRNA, and produces a new copy of the viral positive sense genome (Du et al., 2009; Mocarski et al., 2020). While conventional vaccines recognize viral proteins or viruses by activating the human immune system and limiting viral entry into the cell (Rappuoli, 2018), the CRISPR-based approach is an alternative antiviral strategy to recognize and eliminate the viral genome and mRNAs within the cell. It should be possible to restrict viral replication to specific positives and viral mRNAs while destroying viral genome replication and gene expression templates. Therapeutic applications of CRISPR are on the rise. Technology plays an essential role in exploring potential therapies for various genetic diseases through direct modification of the genome (Straiton, 2019).

Besides DNA targeting Cas9, RNA targeting CRISPR-Cas13 is an antiviral approach against single-stranded RNA viruses such as lymphocytic choriomeningitis virus (LCMV), influenza A virus (IAV), and vesicular stomatitis virus (VSV) in human cells (Freije et al., 2019). Conversely, Stanford College (CA, United States) researchers are working on CRISPR-based therapies for infectious diseases, using a different method and going beyond the human genome. When researchers worked on the flu virus, they followed in the footsteps of many others. They shifted the focus of their gene-targeted antiviral drug to COVID-19 and the pandemic (Abbott et al., 2020a; Abbott et al., 2020b). It was reported that the prophylactic CRISPR antiviral approach in human lung epithelial cells (PAC-MAN) was identified as a potentially helpful new technique to stop viral traits and replication and that the PAC-MAN approach was identified as a type of genetic intervention to target SARS-CoV-2 and potentially all sequenced coronaviruses (Figure 3). Interestingly, a pool of crRNA suppressed about 70% of the reporter signal, demonstrating the potential of CRISPR PAC–MAN technology to degrade viral genetic material. In addition, several crRNAs targeting the entire conserved region of the SARS-RdRP CoV-2 and N-protein genes caused RNA degradation of over 80 and 90%, respectively.

To reprogram CRISPR-Cas13b against the genomic and subgenomic RNAs of SARS-CoV-2, we performed genome-wide computational predictions and screens at single-nucleotide resolution. Cas13b effectors reprogrammed to target accessible segments of spike and nucleocapsid transcripts had silencing efficiencies greater than 98 percent in virus-free animals. Tailored and multiplexed Cas13b CRISPR RNAs (crRNAs) inhibit viral replication in mammalian cells infected with replication-competent SARS-CoV-2, including novel dominant variants. CRISPR-Cas13-based viral suppression strategy is readily adaptable and can be extended to harmful viruses other than SARS-CoV-2 and, therefore, could provide an effective platform for antiviral treatments (Fareh et al., 2021). However, it is critical to identify and study the deleterious effects of using single-guide RNAs (sgRNAs), the CRISPR/Cas system, or PAC-MAN on host physiology. Although the Cas method seems to have a high chance of successfully identifying therapies, it needs further investigation.

However, it is critical to identify and investigate the deleterious effects of using single-guide RNAs (sgRNAs), the CRISPR/Cas system, or PAC-MAN on host physiology. Although the Cas method seems to have a high chance of successfully identifying therapies, it needs further investigation.

CRISPR technology is advancing rapidly. Although recently discovered and new, CRISPR/Cas is a tool with multiple genome engineering capabilities. Because of its ability to edit genomes in such a user-friendly way, it has attracted the attention of biomedical researchers. CRISPR can appropriately solve various viral diseases. In cell-based and animal studies, successful results have been achieved in several human viral infections (Scheufele et al., 2017; Brokowski, 2018; Li et al., 2019). The therapeutic use of CRISPR/Cas to treat human viral diseases has generally gained great importance (Scheufele et al., 2017). However, gaining expertise in the diagnostic and therapeutic use of CRISPR/Cas in viral infections is associated with potential risks. Because this is a new science, we will briefly describe the limitations of CRISPR/Cas. We hope these limitations will be addressed, and an appropriate therapeutic and diagnostic system for SARS-CoV-2 will be developed. There are several legitimate concerns about CRISPR technology’s efficacy and technical limitations. According to research, both target and off-target editing provide limited and partial results, and CRISPR studies in animals and human cells have demonstrated these limitations (Guo and Li, 2015; Peng et al., 2016; Bohaciakova et al., 2017; Zischewski et al., 2017).

Although few studies have shown off-target editing and most studies support CRISPR/Cas, one of the major concerns associated with the CRISPR/Cas system is the possibility of off-target activity and mutant viruses. Viral escape mutations are caused by deletions (indels) at the Cas9 segregation site (Guo and Li, 2015). Using NHEJ repair system, Cas9 causes a mutation and renders the virus ineffective. Typically, NHEJ repair system repairs the damage (Ingram et al., 2019). However, a subset of these mutations can cause the virus to survive and escape, and viruses with such mutations are no longer interested in the original gRNA (De Silva Feelixge et al., 2018). Thus, inappropriate mutations can occur with any virus. If these cells are infected with the mutant virus, they might resist CRISPR/Cas treatment. Therefore, the desired results may not be achieved. Non-mutated virus-infected cells may provide a viral reservoir for disease spread in subsequent disease episodes (Allocati et al., 2016). Therefore, CRISPR/Cas system is risky and not profitable in the antiviral market.

Transferring CRISPR/Cas9 to virus-infected cells is another limitation of this new method. The success of this technology in the clinical setting is necessary to control the most severe viral diseases, including SARS-CoV-2 (Rath et al., 2015). As CRISPR becomes more efficient and sensitive, these concerns may become obsolete. Technology is advancing at an unprecedented pace.

Emerging viruses such as SARS-CoV-2 are responsible for hundreds of thousands of illnesses and deaths worldwide each year. The disease is spreading everywhere and destroying the economies of affected populations. Genome editing strategies to deactivate the viral genome could be a suitable way to treat such diseases. The lack of effective drugs and vaccines may be contributing to so many SARS-CoV-2 samples being collected for rapid diagnosis. Containing and preventing further spread of the virus appears to be critical, and rapid detection of infection in organisms may prevent further spread of the disease. CRISPR/Cas is a solution for treating virus-related diseases with many future applications. Therefore, the CRISPR/Cas9 system could be helpful, especially if some valuable and specific sgRNAs are developed. If the CRISPR/Cas system leads to therapeutic and diagnostic solutions, the financial burden will be reduced because CRISPR/Cas-based therapies will eliminate the need for drugs. In addition, the treated individual will not suffer repeated disease relapses. Therefore, advances in the CRISPR/Cas system and the success of clinical trials in animal models are critical Table 1, Guanghui et al., 2020.