CRISPR/Cas System: An Effective Tool Against Pathogenic Diseases

Coronaviruses are involved in respiratory diseases containing RNA as genetic material [67]. Therapies, drugs, and vaccines discovered to cure coronavirus focus on the inhibition of virus entry in cells [87]. Many diagnostic techniques are also developed but they have low accuracy & efficiency. CRISPR system on the other hand identifies and degrades the genome of virus and diagnoses the virus with higher efficiency. COVID-19 can be detected via CRISPR based technologies including: SHERLOCK, DETECTR and CREST [32]. CRISPR system also provides therapeutic approaches for COVID-19 targeting different regions of Coronavirus genome.

The cells which contain ACE2 on their surface are highly targeted by SARS-CoV-2. ACE2 high expression is on kidney, lungs, and heart cells surface [28]. ACE2 helps in maintaining blood pressure [98]. It can be made target for preventing entry of SARS-CoV-2 into host cell but in such a way that its function is not disturbed. For this purpose, only its binding domain can be edited by use of CRISPR.

In-silico study, Pedro Tanaka et al. evaluated the impact of replacing particular residues of amino acid in ACE2 alpha-helix, which are important in SARS-CoV-2 viral binding, using techniques for protein modeling. To interact with the virus, they created a CRISPR/Cas9 model system that destabilizes a portion of the ACE2 protein (N-terminal alpha-helix amino acid residues Phe28, Lys31 and Tyr41). The destabilizing effect of the edited alterations preserved the helicoidally structure at the ACE2 side while weakening interface connection between SARS-CoV-2 S protein and ACE2 alphahelix [104]. Most important amino acid residues for ACE2 catalytic action, Arg273, His505, His345, Arg169, Arg514, and Tyr515, are preserved while also maintaining the structural shape of this system [44].

PAC-MAN is a therapy for treating coronavirus based on CRISPR technology developed by Abbott et al. [1, 122]. PAC-MAN reorients Cas13d system capacity to cut RNA of coronavirus in humans and thus inhibiting its life cycle. This CRISPR/Cas13d system is extracted from Ruminococcus flavefaciens XPD3002 [60]. Cas13d calls up crRNAs containing 22 nucleotides-spacer seq. crRNA helps the Cas13d proteins in degrading the target viral genome [11, 39, 54]. Cas13d PAC-MAN strategy minimizes the replication and expression of coronavirus by its action of cleaving SARS-CoV-2 RNA genome. It thus results in degradation of viral mRNAs that can produce viral essential proteins [94] (Fig. 2).

Fig. 2.

Abbott et al. reported two conserved regions in SARS-CoV-2 important for its functioning and replication: Nucleocapsid and RdRP genes [1]. They target these genes and constructed crRNA library. They tested 40 designed crRNAs on human lung epithelial A549 cells. For this purpose, proteins of SARS-CoV-2 were tagged with florescent reporter genes and then transfected them to lung epithelial cells under study along with crRNA and Cas13d. They reported that many crRNA effectively worked in reducing the formation of mRNA from RdRP and nucleocapsid genes with 85% and 70% reduced GFP signal, respectively [1].

Coronavirus NSP1 to NSP16 proteins can be targeted by CRISPR/Cas13 in treatment. Polyprotein encoding ORF1a/b plays role in pathogenesis of SARS-CoV-2 could be a target for CRISPR. gRNA have been designed for targeting the regions of ORF1a/b coding peptides [1, 79].

In treating the COVID-19 pandemic, the suggested CRISPR-based antiviral medicines show considerable promise. However, the justification for the effective use of antiviral CRISPR therapeutics in humans is solely based on an in vitro trial. However, the main obstacle preventing clinical use is the transport of CRISPR components into patients. Even though current delivery techniques like polymer-based, liposomal, and adeno-associated virus (AAV) vectors seem doable, they are only somewhat effective [111]. Engineering Cas13 nuclease so that it can adhere to viral particles and deliver CRISPR components along with virus into host cell is one possible method to achieve selective and effective delivery. Once virus, that possesses its own shredding machinery (CRISPR/Cas), reaches cell, the CRISPR system can quickly target viral RNA and cleave it before Viral RNA is replicated or translated. Ironically, the virus itself may be employed to help distribute information selectively and effectively [76].

ABACAS is therapeutic strategy against virus in which Cas13 fuse with specific AB (antibody) of S protein of SARS-CoV-2 helping in delivery of Cas13 in infected cells. Detection of SARS-CoV-2 S-protein could be made by using ABACAS fused antibody that helps in delivery of virus along with Cas13 in cells being infected [56, 125]. RNA of virus is degraded when it is exposed to ABACAS in infected cell (Fig. 3). In this way off-tissue effects are reduced by this approach due to target delivery by virus. CR3022, LY-COV555 and REGN-COV2 are ideal for ABACAS development because they bind SARS-CoV-2 S protein tightly. The efficiency of CR3022 was tested on 293F cell lines by Joyce et al. [56].

Fig. 3.

SHERLOCK (Specific High-Sensitivity Enzymatic Reporter unLOCKing) is FDA approved detection method [40] introduced to detect COVID-19 with sensitivity of 10–100 copies/μl of input [128]. This technique is based on CRISPR Cas13 of type VI. It includes combined action of RT-RPA and Cas13a nuclease activity [41]. crRNA-Cas13a complex bind & then cleave specific target (RNA) along with non-target fluorescent reporter coupled RNA. As a result of cleavage florescence is produced that enables real time & rapid virus detection [119].

CREST (Cas13-based, rugged, equitable, scalable testing), developed by Rauch et al. is used for SARS-CoV-2 detection. This involves utilizing Cas13 detection along with PCR, transcription, and then final detection of positive results by using fluorescence detector. It targets viral RNA with LOD 10 copies/μl [88].

Cas12 based DETECTR (DNA endonuclease-targeted CRISPR trans reporter) detects SARS-CoV-2. It also uses RNA sample of patient. Its principle is based on RT-LAMP [82], that amplifies RNA in sample extracted from oropharyngeal or nasopharyngeal. Reporter molecule cleaves from the target coronavirus sequence followed by its recognition by Cas12. The target is then cleaved by Cas12. The overall procedure takes half an hour for completion. In this time, RT-LAMP reaction takes 20 minutes at 62°C temperature followed by Cas12 detection requiring 10 min at 37°C and final visualized or detection of virus on dipstick. Detection of Nucleoprotein and Envelope protein genes is required for positive test output to confirm infection caused by SARS-CoV-2. Detection limit for DETECTR is 10 copies/μl [10].

HAART is used for treating AIDS inhibiting replication of Human Immunodeficiency Virus [96]. Highly active antiretroviral therapy (HAART) is medical regimen used for treatment and control of human immunodeficiency virus type 1 (HIV-1). It contains a variety of drugs from the antiretroviral groups [33]. Although latent viral reservoirs can be reduced in number by highly active antiretroviral therapy (HAART), they cannot be completely eliminated in HIV-1/AIDS patients. With the potential to treat HIV-1/AIDS, the CRISPR/Cas9 system has just been developed. To diminish HIV-1 infection and eradicate the provirus, it could be used to target cellular co-factors or the HIV-1 genome. It can also be employed to promote transcriptional activation of latent virus in latent viral reservoirs for eradication. [71, 51]. CRISPR technology is being used for inactivating viral genome by gRNAs with specific viral genes or LTR promoter site as a target [131].

To test the effectiveness of lentivirus-mediated intravenous delivery of CRISPR in editing HIV-1 genome from circulating peripheral blood mononuclear cell engrafts, Bella et al. used the NRG mouse model, which has multiple immunodeficiencies that are primarily caused by the absence of T, B, and NK cells [84]. Targeted area of the viral genome, which was located inside the HIV-1 LTR, was excised from human peripheral blood mononuclear cells (PBMCs) placed in NRG mice spleens via lentivirus-mediated delivery of a multiplex of guide RNAs and Cas9 endonuclease. After being treated with lentiviral vectors producing Cas9 and gRNAs, Sanger sequence analysis of the viral DNA confirmed the editing and removal of the proviral DNA fragment from the viral genome at the expected places [6].

To eliminate host cell permeability to be infected by HIV-1, its receptors are targeted by CRISPR. Cocchi et al. reported that the viral entry can be prohibited by blocking or altering the receptors responsible for HIV-1 entry into host cell that are CD4 receptor and two co-receptors: CXCR4 & CCR5. We cannot alter CD4 receptor because it plays a necessary role in immune system but co-receptors can be edited. CRISPR/Cas9 has been widely used in disrupting CCR5 and CXCR4 expression. Cho et al. reported that CRISPR/Cas9 could be used to silence CCR5 receptor gene in HEK-293 T-cells by transfecting sgRNAs and Cas9 [20]. Biti et al. stated that if a person has missing homozygous 32 base pair in gene of CCR5 (CCR5132) shows a natural resistance against R5-tropic HIV-1 [8]. When CCR5 is being inactivated by CRISPR the viral envelope is susceptible to mutation that will result in shift of the usage of viral receptor from CCR5 to CXCR4 [34, 109].

Li et al. disrupted CCR5 expression by combining sgRNAs that target the fourth exon of CCR5 with adenovirus-delivered CRISPR/Cas9. In TZM-BL cells, two particular sgRNAs can generate more than 60% cutting efficiency. The outcomes were also confirmed in human T cell lines and Chinese hamster ovary (CHO) cell lines. Finally, they delivered the CRISPR/Cas9 system to human CD4+ T cells using a chimeric Ad5/F35 adenovirus vector in order to mute CCR5 expression, which protected cells from HIV-1 infection with high efficiency and little off-target consequences [64]. Meanwhile, numerous studies have documented CRISPR/Cas9-induced CXCR4 gene disruption. With the use of two sgRNAs that selectively target conserved CXCR4 regions and CRISPR/Cas9 supplied by lentivirus, Hou et al. successfully disrupted CXCR4 expression in human CD4+ T cells and rhesus macaque CD4+ T cells [101]. The CXCR4 [48] co-receptor or other replication factors of virus can be targeted by CRISPR strategy [52, 83].

Ebina et al. used CRISPR technology for HIV-1 infection prevention for the first time in 2013. They used CRISPR for setting viral LTRs as target and thus suppressing the gene expression in cell line of immortalized T lymphocytes [31]. They used CRISPR/Cas9 to inhibit Human Immunodeficiency Virus-1 provirus replication and transcription. For this purpose, they targeted the NF-κB binding cassettes in TAR sequence of R region and LTR sequence of U3 region [31].

Hu et al. worked to remove genome of HIV-1 by using CRISPR/Cas9 technology in 2014. Cas9-gRNA was used by Hu et al. for targeting the conserved spots in HIV-1 LTR U3 region with no off-target editing and small geno-toxicity. They inactivated the replication and gene expression of virus in cell lines that were latently infected by HIV-1. The studied cell lines include microglial, pro-monocytic and T cell line [50].

In the past few years, CRISPR/Cas9 technology has been widely used in HIV/AIDS research employing experimental, laboratory-adapted HIV-1 strains due to its simple, high-efficiency, and limited off-target effect properties [30]. CRISPR/Cas9 systems are used for effective elimination of HIV-1 proviruses by targeting the viral genomic regions like: LTR region and several genes and proteins (Rev, Pol, Env, and Gag). Some of the studies based on the targeted viral regions for the elimination of HIV-1 by targeted delivery of CRISPR system via transfection and lentiviruses are explained in Table I.

CRISPR/Cas9 strategy was used to target provirus LTR in infected (HIV-1) T cell lines and then treating it with TNF-α [131]. Sequencing showed that target sites were mutated that causes the lowered replication and expression of virus [131].

When various sites of Human Immunodeficiency Virus-1 genome is targeted then deletion and interference of un-integrated part of proviral genome can be increased by using CRISPR technology [66]. When using two sgRNAs together for targeting various genomic regions of HIV-1, the escape and replication of virus can be prevented [62].

Wang G et al. reported that CRISPR editing by sgRNA causes the target site mutations in HIV-1 provirus resulting in its inactivation [126]. Although sgRNA causes inactivation of viral genes but some viruses escape from being cleaved by sgRNA. These viruses can be made less infectious by using CRISPR/Cas9 approach [62]. In presence of specific stimuli, the resting HIV-1 infected cells will become activated and undergo replication and starts infecting the neighboring cells. This will cause the re-formation of latent reservoirs and thus these reservoirs are the most important challenge for potent cure of AIDS.

The mutations caused by CRISPR/Cas9 differs from that of antiviral drugs [110]. Targets of CRISPR system in viral genome, which were poorly, conserved showed insertions or deletions by action of Cas9 while substitutions were observed in highly conserved regions. gRNA directs the Cas9 to target site on HIV DNA. Upon recognition Cas9 introduces dsDNA break at distance of three nucleotides from PAM. HIV DNA will then be repaired by host cell DNA repair mechanisms that results in mutations: nucleotide deletion, insertion, or substitutions at the site where Cas9 has cleaved. These mutations can either activate or inactivate the viral replication. Sometimes these mutations result in preventing recognition of HIV DNA by gRNA and virus will escape from the treatment [65].

Herpes viruses cause many diseases and some of them lack symptoms. For treating the Herpesvirus-based diseases treatments involve using nucleoside analogs. These analogs can limit the replication of virus by restricting the activity of polymerase [75] but due to negligible effectiveness against latent viruses [22] they are to be replaced by some effective treatment strategies. Along with these issues’ viruses develop nucleoside analog resistance, such as against ganciclovir and acyclovir [22]. CRISPR/Cas9 is effective in providing protection to cell against infection and removing latent virus from the infected cell. This technology is also easy to use. It also allows the formation of mutant herpes virus to treat existing ones [93, 121]

CRISPR/Cas9 is used in studying the role of different factors involved in controlling viral progression. Human antiviral factor IFI-16 binds DNA of virus and facilitates the antiviral response [27]. IFI-16 plays role in regulating induction and transcription of IFN-β. Diner BA et al and Johnson KE et al used CRISPR/Cas9 for determining IFI-16 role in HSV infection control. They generated IFI-16 knockouts [27, 55]. They reported that IFI-16 lowers gene expression of HSV-1 and prevent the attachment of transcription factors with promoters of HSV-1. Thus, ultimate result is the inhibition of viral replication and it might have played role in modification of histone [27, 55].

Besides editing the viral genome and constructing mutants of virus, CRISPR provides therapeutic approaches too to kill HSV within host body. anti-HSV gRNAs for targeting viral genome can cause change in specific genes making it non-functional. PC Roehm et al. and Van Diemen et al. reported in their studies that anti-HSV-1 sgRNAs can restrict the replication of virus in both human & non-human cell lines [90, 108]. PC Roehm et al. performed tests on human oligodendroglioma cells to check the effectiveness of anti-ICP0 sgRNAs that were designed by them against HSV-1. ICP0 is a viral protein responsible for viral replication and gene expression. They reported that these gRNAs had effectively reduced the viral replication lowering the load of HSV-1 to ~ 10-folds [90].

Van Diemen et al. used CRISPR/Cas9 lentiviruses for inhibition of HSV-1 replication in cells of human lung fibroblast (MRC5). When one gRNA was used for inhibition of viral replication, viral titer lowered up to 4-log but resistant virus outgrowth was still present. Mutations were displayed at target site of gRNA by variants that results in making these variants resistant to be edited by CRISPR/Cas9. But if two gRNAs are used instead of one, multiple sites of viral genes can be targeted. In this way the chances of virus to become resistant is reduced resulting in complete removal of viral particles [107].

When the genome of HSV-1 is in its resting state in MRC5 infected cells, Van Diemen et al. reported that CRISPR/Cas9 was unable to target their genome while it targeted the genomes of viruses that produced from the re-activated HSV-1. They concluded that Cas9 activity might be interfered by resting genome of HSV-1 [107]. Latent genomes of HSV-1 are in inactivated state having close association with nucleosome [80]; nucleosome presence inhibits the binding and functioning of Cas9 at target site [19, 47].

Herpes viruses enter the cell and its dsDNA serve as target for CRISPR/Cas9. sgRNA induces viral gene disruption while multiple gRNAs result in viral genome fragmentation. If repair template is present, then mutation is inserted in herpes virus (Fig. 4).

Fig. 4.

Replication of herpes virus in lytic and latent infection is reduced by using CRISPR/Cas9 as reported by Diemen et al [107]. They determined efficiency of CRISPR against HSV, EBV, and HCMV. They designed sgRNAs for targeting EBV latent and lytic infection. Viral miR-BART6, smiR-BART5, and miR-BART16 were set as target of designed sgRNAs for models with latent infections. Transduction of gastric carcinoma cells latently infected by EBV was made to check the efficacy of CRISPR/Cas9 based sgRNAs. Viral EBNA1 and EBV Ori were set as target of designed sgRNAs for models with latent infections. In this way they successfully edited and downregulated the miRNAs, antigens, and Ori under study. In the same way, gene editing by CRISPR showed viral infections suppression [107].

Wang and Quake designed sgRNAs for targeting specific genomic regions of EBV that are responsible for transformation, structure, and latency of virus. They used this designed CRISPR/Cas9 strategy to treat EBV latently infected cells derived from patients. They successfully removed genome of EBV and decreased load of virus in quarter and half of cell respectively [115].

EBV genome EBNA1, W repeats and OriP regions were targeted by Yuen et al. sRNAs were designed by them. Copy number of EBV genome was reduced up to 50%. EBV latent infected cells were not affected by this suppression but it produced effect in other infected cells [126].