Genetics and genomics of SARS-CoV-2: A review of the literature with the special focus on genetic diversity and SARS-CoV-2 genome detection.

The outbreak of 2019-novel coronavirus disease (COVID-19), caused by SARS-CoV-2, started in late 2019; in a short time, it has spread rapidly all over the world. Although some possible antiviral and anti-inflammatory medications are available, thousands of people are dying daily. Well-understanding of the SARS-CoV-2 genome is not only essential for the development of new treatments/vaccines, but it also can be used for improving the sensitivity and specificity of current approaches for virus detection. Accordingly, we reviewed the most critical findings related to the genetics of the SARS-CoV-2, with a specific focus on genetic diversity and reported mutations, molecular-based diagnosis assays, using interfering RNA technology for the treatment of patients, and genetic-related vaccination strategies. Additionally, considering the unanswered questions or uncertainties in these regards, different topics were discussed.

An overview of the SARS-CoV-2

In the late December 2019, a novel virus called Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), also known as 2019 novel coronavirus (2019-nCoV), was reported with an unidentified source [1]. The genomic sequence of this newly emerged virus is highly similar to that of severe acute respiratory syndrome coronavirus (SARS-CoV) with a 79.6% sequence identity [2]. The SARS-CoV-2 causes coronavirus disease 2019 (COVID-19), a respiratory tract infection, with a clinical spectrum ranging from asymptomatic to acute respiratory distress syndrome. On March 11, 2020, COVID-19 was declared as a global pandemic and the sixth public health emergency worldwide by the World Health Organization (WHO). During the recent months, some antivirals (e.g., remdesivir) and anti-inflammatory medications (e.g., tocilizumab) showed acceptable outcomes to viral clearance and amelioration of patients' symptoms [3,4,184]. Additionally, following the initial release of some researches for coronavirus vaccine, the spectrum of hope moves from cure to hope for prevention. As of September 19, 2020, 27 vaccines are in Phase 1, 15 in Phase 2, nine in Phase 3, and five have been approved for limited use [5,6]. However, no approval has been granted by the United States Food and Drug Administration (FDA) so far. Understanding the genetics of SARS-CoV-2 may open some new avenues for the development of more effective and safer targeted therapies and even new generations of vaccines for this highly mutable coronavirus. In this review, we tried to cover the most critical and up-to-date findings related to the genetics of SARS-CoV-2 to be used for developing the current strategies of diagnosis, and treatment platforms.

The origin and genomic structure of SARS-CoV-2

Firstly, the genome sequence of SARS-CoV-2 was released in GenBank on January 11, 2020 (accession no. MN908947.3) [7]. Based on the sequence alignment and evolutionary tree analysis, SARS-CoV-2 is now considered as the newest member of the lineage B of genus Betacoronavirus (β-CoV) in the family of Coronaviridae of the order Nidovirales [1]. The initial comparative genomic analysis illustrated that SARS-CoV-2 has almost 79% and 50% sequence identity with SARS-CoV and the Middle East respiratory syndrome (MERS), respectively [2,8]. According to phylogenetic analysis, SARS-CoV-2 is more similar to SARS-CoV than MERS-CoV. It is worthy to note that based on homology modeling studies, it has been shown that SARS-CoV-2 shares 96.2% homology with the BatCoV RaTG13, a bat coronavirus from Rhinolophus affinis [2].

SARS-CoV-2 virus belongs to the B lineage of the β-coronaviruses (β-CoVs); this family comprises an enveloped, non-segmented, positive-sense single-stranded RNA virus genome, with a 5′ cap structure and 3′ poly-A tail, allowing to perform as an mRNA for translation of the replicase polyproteins [9]. Based on the data of whole-genome analyses, the genome of β-CoVs encodes several non-structural and four structural proteins, including spike (S), envelope (E), membrane (M), and nucleocapsid (N) [10,11]. Among the known RNA viruses, coronaviruses, which are single-stranded and positive-sense RNA viruses have the largest genome between other RNA viruses, with the GC content ranging from 32% to 43% [12,13]. The SARS-CoV-2 genomic sequences exhibit a diverse length from 29.8 kb to 29.9 kb with 12 open reading frames (ORFs) encoding 27 proteins [14,15]. The genomic organization includes 5′- leader sequence- ORF1/ab- S- ORF3a- E- M- ORF6a- ORF7a- ORF7b- ORF8- N- ORF10-3′ from left to right and lacks the hemagglutinin-esterase gene which is detected in some β-CoVs [16]. A series of transcription regulatory sequences (TRS) is situated at the junction between each of these ORFs as well as at the 5′ end of the genomic RNA downstream of the leader sequence of β-CoVs [17]. About two-thirds of SARS-CoV-2's RNA comprises ORF1a/b region, which with 16 non-structural proteins (nsp1-16) is considered as the largest ORF. The remaining one-third of the genome near the 3′-terminus contains ORFs encode structural and accessory proteins [10].

Fig. 1 shows a schema of the SARS-CoV-2 genome along with the most critical reported diversities in its different parts, as well as the general structure of the SARS-CoV-2.

Fig. 1

Schematic presentation of structure and genome organization of SARS-CoV-2 based on reference sequence (EPI_ISL_412026). (A) The virion is covered by the spike (S) proteins as well as the membrane (M) and envelope (E) proteins are placed among the S proteins in the virus envelope. The genomic RNA is surrounded by phosphorylated nucleocapsid (N) proteins inside phospholipid bilayers. (B) The SARS-CoV-2 genome (29903 nucleotides) comprises of the 5′ UTR, ORF1a/b encoding 16 nsps for replication, four genes that encode structural proteins including S, E, M, and N proteins, six accessory genes that encode six accessory proteins such as ORF3a, ORF6, ORF7a, ORF7b, ORF8, and ORF10, as well as the 3′ UTR. The location of the seventeen high-frequency mutations and co-mutations reported in the literature are shown on the genome by vertical red lines and circles with similar color, respectively. Abbreviations: SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; 5′ UTR, 5′ untranslated region; OFR, open reading frame; nsp, non-structural protein. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Genetic diversity of SARS-CoV-2

Similar to the other RNA viruses, genetic diversity in SARS-CoV-2 is critical for its fitness, survival, and probably its pathogenesis. In a study performed on the origin of SARS-CoV-2, it was shown that random mutations and recombination are two main sources for genetic diversity in this virus [18,19]. In this regard, nine putative recombinant patterns were identified in the SARS-CoV-2 genome, including six critical recombination regions in the S gene, and one in each of RdRp, nsp13, and ORF3a [19]. Additionally, the results of the genomic analyses suggest that the receptor binding motif in SARS-CoV-2 could possibly arise from the occurrence of a recombination between the strains of coronavirus found in the pangolin and RaTG13 [20,21]. According to data extracted from the public database of the Global Initiative on Sharing All Influenza Data (GISAID) as of September 2020, SARS-CoV-2 virus has a mutation rate of about 8 × 10−4 nucleotides/genome per year [22]. Although this rate is too much for an RNA virus [[23], [24], [25]]. Analyzing 220 genomic sequences from the GISAID database revealed that the number and the occurrence of mutations are significantly higher in Europe, and North America as compared to Asia; this suggests probably different mutation patterns [26]. The distribution of SARS-CoV-2 mutation patterns shows a difference in time, geography, and age, but the similarity in gender [27]. However, according to a recent study on SARS-CoV-2 mutations in the United States, genome samples isolated from women patients have shown a higher mutation rate compared to those isolated from men. Likewise, a female-dominated pattern was seen in mutation 27964C>T (S24L) on the ORF8 protein [28].

Considering the role of mutation as one of the most important mechanism of evolution in RNA viruses [29], several studies have been conducted so far to detect genomic variations of SARS-CoV-2, which resulted in finding rich genetic variations of different types, including missense, synonymous, insertion, deletion, and non-coding mutations [14,27,[30], [31], [32], [33], [34]]. Reportedly, missense and synonymous mutations were the most frequent type of mutations along the SARS-CoV-2 genome [27,35]. Of more than thousands of known variants with different frequencies, 17 high-frequency mutations have been reported in the literature, summarized in Table 1 and located on the SARS-CoV-2 genome in Fig. 1.

Table 1

High-frequency mutations reported in the literature.

Mutation type	Nucleotide change	Amino acid change	Gene/location	References
Non-coding	241C>T	NA	5′ UTR	[14,27,31,34,42,179,180]
Synonymous mutation	3037C>T	F105F	ORF1ab (nsp3)	[26,27,31,34,42,179,180]
Missense	1059C>T	T85I	ORF1ab (nsp2)	[28,34,41,50]
Synonymous mutation	8782C>T	S75S	ORF1ab (nsp4)	[14,26,27,[30], [31], [32],42]
Missense	11083G>T	L37F	ORF1ab (nsp6)	[8,27,28,31,32,42]
Missense	14408C>T	P323L	ORF1ab (nsp12)	[[26], [27], [28],31,34,42,179]
Missense	17747C>T	P504L	ORF1ab (nsp13)	[26,27,31,42]
Missense	17858A>G	Y541C	ORF1ab (nsp13)	[26,27,31,42]
Synonymous mutation	18060C>T	L6L	ORF1ab (nsp14)	[8,26,27,[30], [31], [32],42]
Missense	23403A>G	D614G	S	[8,[26], [27], [28],31,34,42,179]
Missense	25563G>T	Q57H	ORF3a	[28,34,36]
Missense	26144G>T	G251V	ORF3a	[14,27,[30], [31], [32],42]
Missense	28144T>C	L84S	ORF8	[8,27,28,[30], [31], [32],42]
Missense	28881G>A	R203K	N	[26,27,31,34,42,179]
Synonymous mutation	28882G>A	R203R	N	[27,31,34,42,179]
Missense	28883G>C	G204R	N	[27,34,42,179]
Non-coding	29742G>T	NA	3′ UTR	[41,181]

NA: not applicable

The genomic analysis of the SARS-CoV-2 in several studies has reported mutations in various genes, including ORF1ab, ORF3a, ORF6, ORF7, ORF8, ORF10, S, M, E, and N [32,[35], [36], [37]]. Among them, nsp1, nsp2 nsp3, nsp12, and nsp15 of ORF1ab, S, as well as ORF8 genes were reported to have remarkably more mutations than other genes [14,32,35,38]. Furthermore, two insertions with unknown effects were detected on ORF1ab [8]. Based on the critical role of hydrophobicity and charge of amino acid residues in the understanding of the protein functionalities, in a study on detecting homoplasic sites in the SARS-CoV-2, hydrophobic changes (hydrophilic → hydrophobic mutations) on ORF1ab and N genes were significantly found more than hydrophilic mutations. Also, neutral hydrophobic changes occurred more on the S protein [39]. Reportedly, the results of mutational profiles analysis have demonstrated a strong mutation bias towards U in the SARS-CoV-2 genome. Improved immunogenicity, selection for higher expression, and increased mRNA stability could be considered possible mechanisms for this bias [40].

Among the known mutations, 241C>T on 5′ untranslated region (UTR), 3037C>T on nsp3, 14408C>Ton nsp12, and 23403A>G on S are considered to be the most common mutations [33,41]. Furthermore, non-coding mutations are placed on 5′ UTR, 3′ UTR, or intergenic regions and may impact SARS-CoV-2 packaging and titers [8,32,42]. According to the results of several studies, different deletions (in-frame or frameshift) were found in various regions of SARS-CoV-2 genomes, but no mutations have been reported on the M gene so far. These nucleotide deletions may play some roles in virulence and pathogenesis by affecting the third structure and the function of viral proteins, as well as an altered innate immune response [8,30,32,[43], [44], [45], [46], [47], [48]].

According to Shannon entropy, Ceraolo and Giorgi have reported two hypervariable hotspots at positions 8789 and 28151. Both positions are characterized by the presence of either base C or U. Position 8789 is located in the area of the polyprotein gene and leads to a synonymous variation with no phenotypic differences between different strains. On the other hand, position 28151 on ORF8 generates a Ser/Leu replacement in amino acid 84, which seems to be non-conserved in even other coronaviridae [49]. Surprisingly, in a recent study of Feng Wen et al., no genomic hotspot in the SARS-CoV-2 population was reported. They integrated all entirely identical sequences into one because the separate calculation of each mutation in the identical sequences causes a sharp increase in Shannon entropy, and also to omit biases in collection time and location in Shannon entropy analysis [38]. In a recent study, ten hotspot mutations including D614G (23403A>G) on S, L84S (28144T>C) on ORF8, S5932F on nsp14, M5865V on nsp13, L37F (10818G>C) on nsp6, T85I (1059C>T) of nsp2, Q57H (25563G>T) on ORF3a, G251V (26144G>T) on ORF3a, R203K (28881G>A) on N, as well as G204R (28883G>C) on N with a frequency of over 0.10 were reported in SARS-CoV-2 genomes [35].

Due to the role of more mutable regions in viral replication, transmission, and the induced immune responses accordingly, further surveys required to determine the effects of these mutations in the outbreak of SARS-CoV-2 [30,38,43]. Compared to the genome sequence of SARS-CoV-2 isolated from Asia, coinciding mutation 14408C >T (P323L) on nsp12 with more spot mutations was shown in European viral genomes [26]. Reportedly, co-mutations 241C>T (in 5′ UTR) with 3037C>T (F105F), 23403A>G (D614G), and 28144T>C (L84S), as well as 8782C>T (S75S) with 28144T>C (L84S) and 18060C>T>C (L6L), were found. Also, the leader sequence mutation 241C >T tends to coincide with three mutations including 3037C > T (F105F), 14408C >T(P323L), and 23403A>G (D614G) in the virus isolates extracted from European population with a high COVID-19 infection rate reports, thus, these four co-mutations may have a pivotal role in raising virus transmission [31]. In a recent study, the co-occurrence of 8782C > T, 29095C > T, and 28144T > C variants were observed in various samples. Moreover, both single-base substitutions (8782C > T and 29095C > T) are synonymous variants; in contrast, 28144T > C caused a nonsynonymous substitution, where lysine was replaced with serine (L84S) on ORF8 [14].

Since publishing complete SARS-CoV-2 sequences, various studies have been conducted to categorize globally circulating SARS-CoV-2 population, based on mutation profiles [41,[50], [51], [52]]. For instance, a recent study on the genomic distribution of SARS-CoV-2 mutations has grouped detected mutations into five distinct clades, G (241C>T, 3037C>T, 14408C>T, and 23403A>G), GH(241C>T, 3037C>T, 14408C>T, 23403A>G, and 25563G>T), GR (241C>T, 3037C>T, 14408C>T, 23403A>G, and 28881GGG>AAC), S (8782C>T and 28144T>C), and V (11083G>T and 26144G>T), each clade being identified by a specific set of mutations. Considerably, clades allow tracking of the SARS-CoV-2 genetic diversity and prevalence over time [33].

There is presently little evidence on the clinical and molecular effects of mutations detected in the SARS-CoV-2 genome. The results of several preliminary studies suggested a strong link between raising the case fatality rate with the proportion of viruses bearing mutation 23403A > G (D614G), a mutation outside the receptor-binding domain (RBD), over a time period in different regions, as well as the association of this variant with viral infectivity and greater transmissibility [[53], [54], [55], [56], [57], [58]]. Using in silico methods, the effect of mapped mutations R408I, L455Y, F486L, Q493N, Q498Y, N501T on RBD, and A930V, D936Y on heptad repeat 1 (HR1) domain identified deleterious and cause instability of spike glycoprotein [59]. Furthermore, the substitution of proline to leucine (P323L) caused by the 14408C>T mutation on RdRp appears to increase mutation rates. The results of studies have suggested that this mutation leads to rigidify the RdRp protein structure and subsequently change interactions of RdRp with other components of the replication or transcription machinery [26,[60], [61], [62]]. In addition to P323L, A97V and A185V mutations on RdRp have also been reported to alter the secondary structure of the protein [62]. According to a cohort study, COVID-19 patients with a 382-nucleotide deletion on ORF8 have reportedly illustrated a milder infection compared with patients infected with wild-type virus only [63].

Molecular diagnostic techniques for the SASR-CoV-2

Since the first release of genetic information of SARS-CoV-2, various molecular methods including reverse transcription-polymerase chain reaction (RT-PCR), reverse transcription loop-mediated isothermal amplification (RT-LAMP), real-time RT-LAMP, recombinase polymerase amplification (RPA), high-throughput sequencing of the whole genome, and clustered regularly interspersed short palindromic repeats/CRISPR associated (CRISPR/Cas)-based approach have been employed for genetically detection of SARS-CoV-2 [64].

Real-time RT-PCR

Real-time RT-PCR, which has been recommended by WHO could be applied as the effective, straightforward, and gold standard method for the detection of SARS-CoV-2 genome in respiratory secretions, serum, stool, sputum, or ocular secretions, [[65], [66], [67]] although a study indicated that sampling by nasopharyngeal swab is associated with more accurate results than other types of samples [68]. Generally, RT-PCR has been performed as a one-step or two-step format. In the one-step procedure, which is also preferred to identify SARS-CoV-2, a single tube is used for the whole RT-PCR reaction. This method has been considered in SARS-CoV-2 detection due to the possibility of quick start-up as well as reducing the possibility of contamination and pipetting technique errors [69,70].

However, the possibility of false-negative results is considered to be one of the major challenges in real-time RT-PCR test, [71] which has been reported to range between 21% and 67% [[72], [73], [74], [75], [76]]. The most important suggested underlying causes include thermal inactivation, storage time and temperature of specimen preservation, inadequate viral material, laboratory error, and test sample transfer limitation [77,78]. It was also suggested that inappropriate timing of sample collection and deficiency in sampling technique, especially of nasopharyngeal swabs are other reasons for false-negative results of real-time RT-PCR [79].

Furthermore, mutations in the primer- and probe-target regions caused by the rapid evolution of the SARS-CoV-2 genome, infection routes, issues related to sampling (timing and method), and co-infection with other viruses would probably have specific effects on RT-PCR test accuracy [71,72,80]. According to the need to test asymptomatic patients or test before quarantine release as well as the high false-negative rate of RT-PCR for detection of SARS-CoV-2, digital, and digital-droplet PCR methods have received attention due to their high sensitivity. In a study, digital PCR has demonstrated 96.3% accuracy for SARS-CoV-2 detection from pharyngeal swab samples and identified four patients' samples, which were considered negative based on the RT-PCR test [81]. It was shown that digital-droplet PCR capability for SARS-CoV-2 detection with lower minimum detection range is 500 times more sensitive than RT-PCR [82]. The use of the latest laboratory standards, quality sampling at different times from multiple sample types, and selection of RT-PCR kits with the lowest rate of reported false-negative results can effectively prevent the occurrence of false-negative results. Moreover, considering real-time RT-PCR results along with clinical features, especially computed tomography imaging, can be impactful in the timely diagnosis of SARS-CoV-2 infection and COVID-19 management [83,84]. According to the association between the quality of specimens and satisfactory RT-PCR results, evaluating cellular content and considering an internal reference such as RPP30 has been suggested. Indeed, the reliability of the SARS-CoV-2 RNA test and detection of false-negative results could be assessed using the cycle threshold cutoff values of RPP30 RT-PCR [85].

Until now, myriads of real-time RT-PCR kits with different qualities and methods of optimization have been introduced for SARS-CoV-2 detection by clinical laboratories and companies around the world [84,86,87]. Also, many SARS-CoV-2 primers and probes were designed and published by WHO as well as many laboratories worldwide. Employed primers and probes address different SARS-CoV-2 genomic regions target, including ORF1ab or ORF8 regions as well as E, N, S, and RdRP genes [[88], [89], [90], [91], [92]]. Additionally, a two-target system with one universally primer for detection of diverse coronaviruses and a second primer set for the only identification of SARS-CoV-2 can be employed in RT-PCR assay [1,91,93].

Loop-mediated isothermal amplification (RT-LAMP)

As another diagnostic technique, RT-LAMP is considered as a rapid, effective, and one-step nucleic acid amplification method used to identify different viruses including influenza virus, MERS-CoV, Ebola virus, Zika virus, yellow fever virus, and West Nile virus [[94], [95], [96], [97]]. This method is performed at a constant temperature using DNA polymerase and four to six specific primers to detect six to eight sequences of the target gene in the only 60 min [98]. Recently, RT-LAMP has been applied as an alternative test for SARS-CoV-2 detection [[99], [100], [101], [102]]. Compared with the RT-PCR method, the RT-LAMP assay is faster, easier, more cost-effective as well as less time-consuming for diagnosing COVID-19 [103]. Besides, the sensitivity of this assay has been reported to be similar to the RT-PCR assay [93]. Importantly, the RT-LAMP method can be used as a point-of-care diagnostic test [101]. Dao Thi et al. recently tested a two-color RT-LAMP assay by several hundred clinical SARS-CoV-2 samples. Their results showed the colorimetric RT-LAMP could detect patients with a high or moderate SARS-CoV-2 viral load. Additionally, they developed direct swab–to–RT-LAMP assay, a simplified version of RT-LAMP without the need for prior RNA isolation [104]. In different studies, in order to use RT-LAMP assay to diagnose SARS-CoV-2 in patients, several primer sets have been designed that target the ORF1ab, nsp3, S, and N genes/regions for optimization of the assay, which are summarized in supplementary Table S1.

Recombinase polymerase amplification

Recombinase Polymerase Amplification (RPA) is another sensitive isothermal alternative to the PCR, which has been considered due to its high amplification speed and performance at a low constant temperature [105]. El-Tholoth et al. using a 2-stage dubbed rapid amplification, a combination of LAMP and RPA techniques, provided a method 100-fold more sensitive than LAMP and RT-PCR in mimic patient samples for SARS-CoV-2 detection [99].

Metagenomic next-generation sequencing

As another alternative approach, metagenomic next-generation sequencing (mNGS) allows unbiased detection of pathogenic genomes for epidemiological aims, as well as identification of co-infections that may lead to increase morbidity and mortality in emerging infectious diseases [106]. Additionally, this offers an opportunity to identify unknown pathogens with sudden onset in a particular area, similar to the Ebola virus in West Africa [107]. In recent studies, mNGS has been reported as a feasible and efficient approach for detecting a wide range of pathogens, especially the SARS-CoV-2 in clinical samples [108,109]. Moreover, the amplicon-based metagenomic sequencing approach which is a combination of amplicon-based sequencing and metagenomic sequencing has been utilized to recognize the genome sequence of SARS-CoV-2 and the other microbiome from nasopharyngeal swabs of patients with COVID-19 [108].

CRISPR/Cas systems

CRISPR/Cas systems are recently characterized as rapid and versatile platforms for nucleic acid detection [109]. Further, the analysis of the results of CRISPR-based methods can be done using fluorescent, lateral flow strips, or agarose gels in less than an hour [110,111]. Compared to the PCR strategies, CRISPR-Cas diagnostic tools are significantly more sensitive and less costly than PCR-based approaches [112]. Additionally, in a study on the performance of the three technology platforms including mNGS, RT-PCR, and CRISPR in identifying the SARS-CoV-2, results indicated a great sensitivity and specificity of CRISPR-based assay for SARS-CoV-2 detection in clinical specimens with a shorter turn-around time than other methods [113]. Since the beginning of the COVID-19 outbreak, several CRISPR-Cas-based platforms have been developed to detect the SARS-CoV-2, which are compared in Table 2 . For instance, using a combination of CRISPR–Cas12-based assay and LAMP, Broughton et al. were able to reduce the SARS-CoV-2 detection time to around 30 min, while maintaining a relatively low sensitivity (10 copies of RNA per μL) [114]. In contrast, All-In-One Dual CRISPR-Cas12a (AIOD-CRISPR) platform was able to detect 4.6 copies of SARS-CoV-2 RNA per μL of input in about 1 h [115].

Table 2

SARS-CoV-2 detection methods based on CRISPR/Cas.

Platform	Sensitivity	Time	Equipment required	Cas protein	Gene or reign detected	Pre-amplification technique	Positive control	References
DETECTR	10–100 copies of RNA per μL	30–40  min	Eppendorf tubes with reagents, heat blocks or water bath (37 °C and 62 °C), nuclease-free water, pipettes, and tips as well as lateral flow strips.	Cas12a	E gene and N gene (N2 region)	RT-LAMP	human RNase P gene	[114]
SHERLOCK	10–100 copies of RNA per μL	about 60  min	37°C and 42°C water bath and Microcentrifuge for spinning down 1.5 mL test tubes	Cas13	S gene and ORF1ab gene	qRT-PCR	Synthetic virus fragment	[111]
AIOD-CRISPR	4.6 copies of SARS-CoV-2 RNA per μL of input	40 min	37°C heat block, LED blue light illuminator or UV light illuminator	Cas12a	N gene	RT-RPA	human RPP30 gene.	[115]
CREST	10 copies of RNA per μL	Not mentioned, likely about 1–2 h	Mini PCR machines by DIY-Bio, P51 cardboard fluorescence visualizer with 9V battery	Cas13	N1, N2, and N3 regions in N gene	qRT-PCR	Synthetic, T7 RNA polymerase gene	[182]
FELUDA	110 femtomolar	about 1 h	PCR machines/Heat blocks, Lateral flow strips, Agarose gel equipment, Fluorescent emission reader	FnCas9	nsp8 and N gene	RT-PCR/RPA	human ACTB	[110]
CASdetec	5 copies of RNA per μL	40–60 min	42°C heat, fluorescence microplate reader, ssDNA-fluorescently quenched (FQ) reporter	Cas12b	RdRp	RT-RAAa	NA	[183]

Recombinase-aided amplification (RAA).

Multiplex diagnostic approaches

In contrast to the singleplex reaction, in the multiplex diagnostic methods, more than one viral gene can be detected in a short time, which makes these methods acceptable for routine diagnostic tests. Furthermore, the multiplex approach has some advantages including increased sample throughput, as well as a reduced amount of sample required and turnaround time than a singleplex diagnostic approach [116]. Until now, several multiplex assays including FTD21 kit [117], GeXP assay [118], Qiagen ResPlex II V2.0 kit [119], and FilmArray multiplex PCR system [120] have been developed for the detection of respiratory viruses as well as some common human coronaviruses (HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1) in particular. According to the operation assessment results of AusDiagnostics respiratory multiplex tandem PCR assay including SARS-CoV-2, this assay has shown high specificity (98.4%) and considerable concordance with the reference laboratory for the identification of SARS-CoV-2 by the ORF1ab gene [121]. The QIAstat-Dx Respiratory SARS-CoV-2 panel (QIAstat-SARS) has also been indicated the ability to detect 1000 copies per milliliter with 100% sensitivity and 93% specification by targeting the E and ORF1ab genes from SARS-CoV-2. Moreover, the latest multiplex platform for pathogen detection was developed, called Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (CARMEN). This method, in combination with Cas13 created CARMEN-Cas13 technology, which can be used for highly efficient virus detection. Through using CARMEN-Cas13 assay clinicians are able to track and diagnose all 169 human-associated viruses, as well as SARS-CoV-2 [122].

Point-of-care tests

There are some approaches with the aim of moving nucleic acid testing from a laboratory-based setup to the point of care testing in the clinic [88]. For example, a point-of-care system using a smartphone-based LAMP and panel of equine respiratory infectious diseases, as a model for human pathogens like SARS-CoV-2 has recently developed for finding live virus from the nasal swab [123]. Likewise, hybridization chain reactions (HCRs) were suggested as screening strategies for the prevention of widespread infection, including SARS-CoV-2. Based on the HCR designer, SARS-CoV-2 nucleocapsid phosphoprotein gene and human RNase P have been recently targeted for performing HCR reactions on complementary DNA of SARS–CoV-2, which has been shown to be useful for COVID-19 screening [124]. In addition, HCR technology features including the lack of thermal cycler or enzymes need for amplification, as well as the possibility of testing at room temperature, have made HCR technology as a suitable method for quick detection of some viruses such as the hepatitis B and influenza A [125].

Using RNA interference as a weapon for combating SARS-CoV-2: possible challenges and opportunities

RNA interference (RNAi) is a molecular process in which small noncoding RNAs (of endogenous or exogenous origin) match with the target mRNA in a sequence-specific manner and silence its expression [126]. During the past decade, based on the therapeutic potential of RNAi and their abilities to limit gene expression, many of them have been investigated and employed for medical application. RNAi-based technology has a simple and unique design and can be simultaneously used against multiple strains of a virus, making it a tractable and viable option to battle and treat severe respiratory viral infections [127]. Several features, containing thermodynamic characteristics, small interfering RNA (siRNA) structure, percentage of GC content, approachability of the target site, nucleotides at siRNA termini, and selection of highly conservative sequences in the targeted virus genome affect the design of siRNAs with high antiviral activity [[128], [129], [130]]. It has been found that different positive- and negative-strand of RNA viruses induce mammalian antiviral RNAi activity. On the other hand, as a countermeasure, virus-encoded suppressors of RNAi (VSRs) are produced in diverse families of plant, insect, and mammalian viruses to oppose the RNAi pathway at various steps [131,132].

From the chronological perspective, targeting viral RNAs have been used for the suppression of the replication in numerous viruses, including human immunodeficiency virus (HIV), human papilloma virus (HPV), Hepatitis A virus, Hepatitis B virus, Influenza A and B viruses, respiratory syncytial virus (RSV), SARS-CoV, and Adenoviruses [[133], [134], [135], [136], [137], [138], [139], [140]]. Some siRNA-based antivirals have been examined in clinical trials such as ALN-RSV01 to target respiratory syncytial virus nucleocapsid gene (phase II) [141], NucB1000 against four separate targets (Pre-C, Pre-S1, Pre-S2, and X) of chronic hepatitis B virus (phase I) [142], SPC3649 for blocking miR-122 and its interaction with hepatitis C virus RNA (phase II) [143], pHIV7-shI-TAR-CCR5RZ for targeting several human immunodeficiency virus genes (Tat, Tar, and CCR5) (phase I) [144], and TKM-Ebola against some transcripts (L, VP24, and VP35) of Ebola virus (phase I) [145].

In CAS content collection, more than seventy patents reveal the use of RNAi, siRNA molecules, antisense oligonucleotides, RNA aptamers, ribozymes, and microRNA inhibitors in treating SARS-CoV, a close relative of the SARS-CoV-2 [146]. These patents have presented medicines for treatment and inhibition of the SARS, as well as supplied various information about siRNAs against SARS-CoV and techniques for their delivery [147]. Moreover, several studies supported that siRNAs are able to prevent gene expression and the replication of SARS-CoV by targeting some genes or the leader sequence of this virus in cultured cells [[148], [149], [150], [151]]. For example, in 2005, Shi et al. exhibited short interfering RNAs as molecules targeting the open reading frames of the E, M, and N genes of SARS-CoV, which blocked the replication of this human Coronavirus in Vero-E6 cells [152]. In another study, two siRNAs targeted the SARS-CoV genome at S and nsp12 regions, which led to the depletion of fever caused by the virus as well as SARS-CoV viral loads. Likewise, it reduced acute diffuse alveoli damage in Rhesus macaques [140]. Furthermore, the previous study reported the N protein of SARS-CoV as a VSR in mammalian cells by a cellular reversal-of-silencing assay regarding viral infection [153].

The use of siRNA-based therapeutics for COVID-19

In order to target the RNA genome of SARS-CoV-2 and as the first step in the production of antiviral siRNAs, Chen et al. [154] suggested nine potential siRNA targets with 21–25 nucleotides in length within ORF1ab, S, ORF3a, M, and N regions of the SARS-CoV-2 genome and detected those regions as conserved areas. In another study, considering siRNA as a potential therapy for COVID-19, nsp5, and nsp12 were introduced to be targeted using siRNA based therapeutics [147]. Besides, in a study related to the interaction between antiviral RNAi immunity and SARS-CoV-2 has been reported structural protein N of the SARS-CoV-2 as an indicator of viral suppressor of RNAi (VSR) activity, which is a challenge in developing siRNA therapeutic and a factor which opposes the antiviral activity of RNAi in different steps [155].

A recent study found that host cell entry of SARS-CoV-2 is contingent on ACE2, as well as the transmembrane protease, serine 2 (TMPRSS2) produced by the host cell is needed to cleave and activate spike protein in the invasive process of the SARS-CoV-2 [156]. Therefore, siRNA targeting TMPRSS2 and ACE2 can be considered as therapeutic options for blocking SARS-CoV-2 infections, as shown in Fig. 2 . Regarding the strategy of inhibiting viral entry for antiviral treatment, Lu et al [157]. reported a reduction in the replication of SARS-CoV in the ACE2-silenced cells using siRNA technology. However, there is a concern that the selective elimination of ACE2 in vulnerable organs to SARS-CoV-2 may have unanticipated outcomes because ACE2 has critical roles in a variety of pathological and physiological processes [158]. Moreover, siRNA targeting of TMPRSS2 has formerly indicated a significant decrease in SARS-CoV entry into Calu-3 cells [159]. Fig. 2 briefly shows the use of RNAi as a preventive or therapeutic agent against SARS-CoV-2.

Fig. 2

Antiviral applications of RNAi against SARS-CoV-2. Generally, RNAi can either be induced by transfection of chemically synthesized siRNAs or the intracellular expression of double-stranded siRNA precursors stably or temporary. After transcription of pri-miRNA from the corresponding miRNA gene by RNA Pol II/III, and then processed by Drosha to construct pre-miRNA in the nucleus, double-stranded siRNA precursors (shRNA, lhRNA or pre-miRNA) are exported to the cytoplasm and processed into mature siRNAs using Dicer. Subsequently, the antisense strand of the siRNAs/miRNAs load into the RISC. The complex can finally cleave the target mRNA. The siRNA-based strategies against the SARS-CoV-2 can either be directed against the SARS-CoV-2 itself or against the ACE2 receptor or TMPRSS2, whose silencing will inhibit virus entry into the cell. Following the interaction of the SARS-CoV-2 spike protein with the human ACE2 receptor after spike protein activation by TMPRSS2, the virus is endocytosed. Then, the virion releases its RNA for translation into proteins by the cell’s machinery. Abbreviations: RNAi, RNA interference; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; siRNA, small interfering RNA; pri-miRNA, primary miRNA; RNA Pol II/III, RNA polymerase II/III; pre-miRNA, precursor-miRNA; shRNA, short hairpin RNA; lhRNA, long hairpin RNA; RISC, RNA-induced silencing complex; ACE2, angiotensin-converting enzyme-2; TMPRSS2, transmembrane protease serine-2.

miRNAs as the potential therapeutic option for SARS-CoV-2

In viral infections, many known human miRNAs appear to play an important role in regulating immune responses and interfering with functions of viral genes including reproduction, translation, and expression by targeting viral genes. Consequently, miRNA targeting can be considered as an antiviral therapy approach [160]. In a recent computational study, a list of predicted possible host genes targeted by viral miRNAs and viral genes targeted by cellular miRNAs was presented by computational analysis to understand the basic mechanisms of SARS-CoV-2 infection [161]. Additionally, in a recent study, six antiviral host- miRNAs comprising hsa-let-7a, hsa-miR101, hsa-miR126, hsa-miR23b, hsa-miR378, and hsa-miR98 were predicted to act on nsps, S, and N genes of SARS-CoV2 by cleaving their target sites or translation inhibition. The association between these miRNAs and several viruses, including Hepatitis C virus, Herpes simplex virus 1, and Enterovirus 71 has already been identified [44]. Moreover, in a current study on the prediction of miRNAs in SARS-CoV-2 genomes, seven miRNAs, including miR-8066, miR-5197-3p, miR-3611, miR-3934-3p, miR-1307-3p, miR-3691-3p, and miR-1468-5p were introduced that were related to viral pathogenesis and host response using KEGG pathway analysis [162]. In another in silico prediction-based study, the SARS-CoV-2 genome was searched for finding virus-encoded miRNA seed sponges that could bind to human miRNA seed sites and subsequently disrupt the interaction of host miRNA with their native targets. Nearly 80 human miRNAs that can interact with the SARS-CoV-2 genes have been identified. According to the literature, these miRNAs are related to pulmonary and cardiac disorders [163].

Circular RNAs

Circular RNAs (circRNAs) is a novel class of non-coding RNAs with pivotal regulatory roles in human cells. Zhang et al. articulated that MERS-CoV infection leads to considerable changes in the expression of many host cell circRNAs, miRNAs, and mRNAs. Moreover, they reported a significant decrease in viral load by knocking down specific DE circRNAs in MERS-CoV infection. Thus, findings proposed that circRNAs may act as potential antiviral targets [164].

Regarding viral derived miRNA, according to a machine learning based miRNA prediction analysis, PANTHER gene function analysis results represented the ability of viral derived miRNA candidates for targeting various human genes involved in crucial cellular processes including transcription, metabolism, defense system and several signaling pathways such as Wnt and EGFR signalings. Furtheremore, many known human miRNAs appeared to be able to target viral genes involved in viral life cycle such as S, M, N, E proteins and ORF1ab, ORF3a, ORF8, ORF7a and ORF10. Regarding the importance of miRNA-based therapies, comprehending mode of actions of miRNAs and their possible roles during SARS-CoV-2 infections could create new opportunities for the development and improvement of new therapeutics [165].

Nucleotide-based technology for vaccine development against SARS-CoV-2

Among the vaccinations under evaluation, there are three major groups of DNA vaccines, RNA vaccines, and Epitope-based vaccines, which could be categorized into the nucleotide-based vaccines.

DNA vaccine

The past few years have witnessed a surge in the development of highly efficacious delivery materials for nucleic acids with some remarkable results. Following viral sequencing technology, nucleic acid therapeutics have emerged as promising alternatives to conventional vaccine approaches. Regarding previous strains of coronavirus, some studies had been performed to develop DNA-vaccines [166,167]. One of the most successful DNA-based vaccines for MERS is INO-4700, which led to durable neutralizing antibodies and T cell immune responses through targeting MERS-CoV S protein [167]. As another effort with a similar strategy, Smith et al. [168] generated a DNA vaccine, INO-4800, for targeting SARS-CoV-2 S protein. The American company INOVIO started a phase I/IIa trial NCT04336410, NCT0444778, in April, and July 2020 articulating that INO-4800 could recruit humoral and T cell responses in mice. They assessed the biodistribution of SARS-CoV-2 targeting antibodies to the lungs to validate INO-4800 as a capable potential SARS-CoV-2 vaccine candidate in mice and pigs, which resulted in the detection of anti-SARS-CoV-2 specific antibody in the lungs following immunization with INO-4800. In another achievement related to DNA vaccines for SARS-CoV-2, Jingyou et al. [169] developed a series of DNA vaccines, which recruited humoral and cellular immune responses in Rhesus Macaques. The titer of developed neutralizing antibodies was as high as the naturally antibody developed following with SARS-CoV-2. In addition to the INO-4800, Indian vaccine-maker Zydus Cadila began testing a DNA-based vaccine in July, becoming the second company in India to enter the Covid-19 vaccine race after Bharat Biotech, and recently they launched a Phase 2 trial on Aug. 6. Similarly, The Korean company Genexine, as well as the Japanese biotechnology company AnGes started testing the safety of a DNA-based vaccine in June. Furthermore, fifteen other DNA vaccines are in pre-clinical and clinical trial evaluations. The list of such COVID-19 candidate vaccines in both clinical and pre-clinical phases brought in a new report published by WHO in June 2020 [170,171].

RNA vaccine

RNA-based therapies and vaccines have remarkable potential for the treatment and prevention of a wide range of diseases [172]. RNA vaccines offer a promising alternative to conventional vaccine approaches because of their high potency, capacity for rapid development, safe administration, and low-cost production. However, their application has been restricted until recently by the instability and inefficient in vivo delivery of mRNA [173]. Gradually, some techniques served to reduce toxicity and improve the translation of the mRNA. Various modifications including the incorporation of modified nucleosides (particularly modified uridine), optimization of coding sequences, and stringent purification of in vitro transcribed mRNA by high-performance liquid chromatography have been applied to remove double-stranded RNA contaminants [174]. Given that the 5' and 3' UTRs of mRNA can significantly influence the rate of translation and half-life of the transcript, optimization of the UTRs is of paramount value in the design of mRNA vaccines [173].

As of September 2020, the phase III, (NCT04470427) clinical trial of a novel lipid nanoparticle-encapsulated mRNA-based vaccine, mRNA-1273, encoding the S protein of SARS-CoV-2, began in the United States by the Moderna Inc. [175] The German company BioNTech has recently entered into collaborations with Pfizer, based in New York, and the Chinese drug maker Fosun Pharma to develop their mRNA vaccine. They found that one version, called BNT162b2, produced significantly fewer side effects, such as fevers and fatigue, and so they chose it to move into Phase 2/3 trials. On September 12, the companies announced the launch of a Phase 2/3 trial with 43,000 volunteers in the United States and other countries including Argentina, Brazil, and Germany [171].

In addition to these two RNA-based vaccines, which are entered into the clinical trials, there are at least sixteen other ongoing studies in pre-clinical evaluation (based on WHO's report on September, 2020). In addition to the aforementioned active clinical trials, there is a competition between those in pre-clinical for entering the clinic. Imperial College London, which has developed a ”self-amplifying” RNA vaccine with the aim of boosting the production of a viral protein to stimulate the immune system. They begun Phase I/II trials on June 15, and have partnered with Morningside Ventures to manufacture and distribute the vaccine through a new company called VacEquity Global Health. Also, CureVac is working on a similar vaccine and launched a Phase II trial of its mRNA vaccine in August [171].

Epitope-based vaccine

In addition to the DNA and RNA-based vaccines, epitope-based vaccines are other approaches, which somehow rely on the genetic aspect of SARS-CoV-2. In the new era of medicine, the immunoinformatics approaches have been desperately used to provide putative epitopes using a genome database. Recently, a group of computational scientists obtained immunogenic epitopes for all critical proteins of the virus. This list is composed of top-ranked cytotoxic T cell helper and epitopes common across MHC alleles, covering all predominant human leukocyte antigen (HLA) supertypes in population [176]. By immunogenicity predictive models, researchers provide the immunogenicity of detected peptides and their binding potential to HLA alleles [177].

Currently, a total of 63 peptides with a high immunogenicity potential have been identified for SARS-CoV-2. Identification of a ranked list of immunogenic peptides shows that they can be used as potential targets for SARS-CoV-2 vaccine development, and it accelerates the development pipeline. A detailed screen of candidate peptides based on comparison with immunogenic peptides was used using deposited data in the immune epitope database, which resulted in a de novo prediction from SARS-CoV-2-associated 9-mer peptides [177]. It has been shown that these peptides bind various HLA alleles (both class I and class II), although with a higher tendency towards HLA-A:02:01, and can cause activation of effector T cells. In another study to provide a fast-immunogenic profile of these epitopes, linear B-cell epitopes, along with their sequence, position, and length, were introduced for SARS-CoV-2. In a recent study, 13 Major Histocompatibility Complex-(MHC) I and 3 MHC-II epitopes were recognized to have antigenic properties [178]. These epitopes molecularly dock on toll-like receptor-5 to get binding affinity and are usually linked to specific linkers to build vaccine components. Reportedly, a list of 22 SARS-CoV-2 peptides has been revealed, which have a higher predicted immunogenicity score than their target peptides. Further predictive models and algorithms for the characterization of immunogenic peptides, as well as several in vitro and in vivo validations, are required for the most efficient vaccine development [177].

Concluding remarks

Currently, the genome of SARS-CoV-2 has been comprehensively studied. Without a doubt, our understanding of this regard caused molecular-based diagnosis, some targeted therapies, and vaccine developments. Focusing on targeting the SARS-CoV-2 genome via RNAi technology could lead to emerging effective and safe treatments, which probably could prevent disease severity and decrease COVID-19 associated mortality. Although many efforts have been made to reveal the behavior of SARS-CoV-2, based on its genetics, there are still some concerns regarding emerging variation/mutations. These could significantly affect targeted therapies and, more importantly, vaccine development. Moving forward, we must recognize that we have had a new major coronavirus epidemic every decade in the twenty-first century SARS in the 2000s, MERS in the 2010s, and now COVID-19. It is, therefore, a global security priority to advance coronavirus vaccines and to identify international funding support resources, as well as other data regarding their development, manufacture, and storage. This recent outbreak should be considered as a sine qua non to alert the international research community to both react and prepare for the next coronavirus transmission to mammals. We urgently need to manufacture a pan-coronavirus vaccine, and it is something that appears feasible if sufficient resources are provided in an appropriate time. Besides, further molecular research of SARS-CoV-2 is required to develop more sensitive genetic-based detection methods to facilitate the diagnosis of COVID-19. In addition, the development of broad-spectrum antiviral drugs and vaccines based on the genetic profile of both virus and host cells are highly recommended.

The following are the supplementary data related to this article.

Table S1

RT-LAMP reported primers for SARS-CoV-2 detection.

Declaration of competing interest

None.