Therapeutic Strategies of Clustered Regularly Interspaced Palindromic Repeats-Cas Systems for Different Viral Infections

Abstract

The clustered regularly interspaced palindromic repeats (CRISPR)/Cas systems have rapidly transitioned from intriguing prokaryotic defense systems to an efficient therapeutic tool. This cutting-edge technology is currently revolutionizing to combat hostile viruses because of its reproducibility, high potency, ease of use, limited off-target activity, and development of quick immune response against viruses. CRISPR gene editing technology eliminates the virus by breaking the DNA that ultimately halts viral replication. This review summarizes the advancements that have been made in the use of CRISPR-Cas technology in viral therapeutics.

Introduction

The clustered regularly interspaced palindromic repeats (CRISPR)-CRISPR-associated (Cas) system is an adaptive defense network to kill plasmids, phages, and all other invading mobile genetic elements (3). CRISPR-Cas system facilitates bacteria and archaea to memorize former encounter with infectious agents because of integration of short fragments of invading foreign DNA into an array of CRISPR within the host chromosome (29). The presence of series of repeating 20–50 bp nucleotides interspaced by specific spacer sequences of same length in the CRISPR loci matches the nonself DNA and helps in identification and elimination of plasmids and cognate viruses during ensuing attacks.

Stages involved in CRISPR-Cas system are: First, foreign DNA spacers are inserted in CRISPR repeat cassettes by Cas1 and Cas2. Second, transcription of entire CRISPR array into pre-crRNA occurs followed by processing of pre-crRNA into short crRNAs that are incorporated inside interference complex containing one or several Cas proteins. Third, these interference complexes target invading genetic elements (2).

There are three different types (I, II, and III) and ten subtypes of CRISPR-Cas systems on the basis of cluster of Cas genes and its corresponding CRISPR array, typified conserved signature protein (Cas3, Cas9, and Cas10 in type I, type II, and type III, respectively), and composition of the CRISPR ribonucleoprotein (crRNP) complexes and their target cleavage mechanisms (7,27). All types require a seed sequence. DNA targeting by type I and type II CRISPR-Cas systems depends on protospacer adjacent motifs or short conserved sequences (28,32). The type II CRISPR/Cas system is constituted of crRNAs, two short RNAs, and DNA endonuclease Cas9. It is also known as CRISPR-Cas9 system because of its constituents: human codon-optimized Cas9 (hCas9) nuclease and guide RNA (gRNA) with sufficient potential to treat viral infections.

Cas9 has been utilized in a number of biological systems where it catalyzes DNA cleavage, but recently a unique form of Cas9 has been reported that is responsible for targeting endogenous mRNA in Francisella novicida. Comparison of different forms of Cas9 has been shown in Table 1.

Table 1.

Different Orthologs of Cas9

Orthologs	FnCas9	SpCas9	SaCas9
Organism	Francisella novicida	Streptococcus pyogenes	Staphylococcus aureus
Length	1,629 amino acids	1,368 amino acids	1,053 amino acids
PAM recognition sites	50-NGG-30	50-NGG-30	50-NNGRRT-30
Cleavage	Catalyzes cleavage of both target DNA and RNA	Catalyzes cleavage of target DNA	Catalyzes cleavage of target DNA
Type of cleavage	Two types of cleavage crRNA:tracrRNA-dependent DNA cleavage, scaRNA (small CRISPR/Cas-associated RNA):tracrRNA-dependent cleavage	crRNA:tracrRNA-dependent DNA cleavage	crRNA:tracrRNA-dependent DNA cleavage

CRISPR, clustered regularly interspaced palindromic repeats; PAM, protospacer adjacent motif.

The utility of CRISPR-Cas technology in antiviral therapeutics

CRISPR-Cas technology is emerging as a promising antiviral strategy in humans because of its known adaptive antiviral immune response in bacteria. The simple formulation of CRISPR-Cas has made it a useful tool in antiviral therapeutics because it requires only mature crRNA, transactivating crRNA (tracrRNA), and Cas9 endonuclease for proper functioning. To achieve immunity against viruses, Cas9 must cleave viral DNA or viral replication gene dispensable in the human host. The coexpression of Cas9 and single guide RNA (sgRNA) makes Cas9-sgRNA complex, which forms complementary base pairing with its target using sgRNA because of which Cas9 exerts its nuclease activity on the target molecule.

CRISPR-Cas9 allows multiplexing and is known as an efficient tool for mutational inactivation of viral infections because of simultaneous use of multiple sgRNAs for target cleavage at multiple loci. DNA viruses or viruses that exist in DNA form during their life cycle are the appropriate direct targets for elimination using CRISPR/Cas technology. The replication of virus should occur in the discrete region of body that is accessible to vectors expressing a virus-specific Cas9/sgRNA complex. The expression of CRISPR transcripts is necessary to mount successful defense because of their ability to guide the interference machinery to invasive targets leading to obstruction of invasion. The current status of research regarding the use of CRISPR/Cas technology in treatment of different viruses is discussed below. Table 2 summarizes the application of CRISPR-Cas technology in viral therapeutics.

Table 2.

The Uses of CRISPR-Cas Systems Against Different Viral Infections

Virus	System	Genotype	Major targets	Results	References
HIV	HEK293 cells	HIV-1	Viral genome near the DSBs	CRISPR/Cas9 system disrupts latently integrated viral genome	Liao et al. (24)
	HEK293 cells	HIV-1	LTR-R region		Liao et al. (24)
	JLat10.6 cell line	HIV-1	T10 (second exon of Rev)	Efficient mutation and deactivation HIV-1 proviral DNA	Zhu et al. (61)
	T cells	HIV-1	LTR	Reduced expression of HIV-1	Ebina et al. (12)
	T cells, promonocytes, microglia	HIV-1	LTR	Provirus excision	Hu et al. (18)
HCV	Huh-7.5 cells transfected with vectors encoding FnCas9/rgRNA and later infected by HCV (HCVcc) recombinant virus having gene for Renilla luciferase	2a	3′ UTR (essential for viral replication) and 3′ UTR (which is essential for not only replication of RNA but also for translation of viral protein.)	By immunostaining for the viral protein, there were reduced level of E2 glycoprotein.	Bi et al. (5)
HCV		2a		Expression of viral proteins was also checked by luciferase production.	Bi et al. (5)
HPV	HeLa, 293T, and SiHa cells transfected with Cas9/sgRNA	HPV-16	E6, E7 ORF	Induction of P53 and Rb occurred, which resulted in cell cycle arrest and eventually into cell death.	Kennedy et al. (21)
	HeLa, 293T, and SiHa cells transfected with Cas9/sgRNA	HPV-18	E6, E7 ORF		Kennedy et al. (21)
	SiHa and CaSki cells	HPV-16	E6	Elevated apoptotic rates in HPV16-positive SiHa and CaSki cells when transfected with gRNA-2/Cas9 and gRNA-3/Cas9	Yu et al.(53)
	SiHa and CaSki cells	HPV-16	E6	In both cell lines, there was reduced level of E6 protein, whereas expression level of p53 was increased	Yu et al.(53)
	SiHa cell line	HPV-16	E6, E7 promoters	Accumulation of p53 and p21 protein occurred, but the proliferation of cervical cancer cells in vitro was greatly reduced.	Zhen et al. (58)
HBV	HepG2.2.15 hepatoblastoma cell line		S, C, X, and P ORF	Reduced in viral DNA and cccDNA level	Ramanan et al.(35)
	HepG2.2.15 hepatoblastoma cell line			Viral gene expression and replication were dramatically reduced	Ramanan et al.(35)
	Huh7 cell line		P1, S1, XCp, and PS2 ORFs	HBsAg level reduced in serum	Lin et al. (25)
	HBV hydrodynamics-mouse model		P1, S1, XCp, and PS2 ORFs	HBsAg level reduced in serum	Lin et al. (25)
Herpesviruses	Gastric carcinoma cell line SNU719	EBV	EBNA1 binding sites or EBNA1 protein-coding sequence	50–60% loss of latent EBV from cells	Yuen et al. (55); van Diemen et al. (43)
	Raji
	C661-1 cells
			miRNAs
			LMP1
			BART promoter	Reduced viral load, miRNA expression, and apoptosis
	MRC5 human lung fibroblast cells were infected with HSV1 followed by introduction of Anti-HSV-1 gRNAs and subsequent superinfection with HCMV	HSV1	UL52 #2	Complete inhibition of replication of both herpesviruses was observed	van Diemen et al. (43)
		HCMV	UL8 #2		van Diemen et al. (43)
Pseudorabies virus	PK15 cells		PRV EP0 gene	Disruption of virus resulted in decrease in viral growth	Xu et al. (51)
Pseudorabies virus	HeLa cells		PRV UL50 gene	Disruption of virus resulted in decrease in viral growth	Xu et al. (51)
JCV	Human oligodendroglioma cell line TC620		N-terminal region of T-antigen	Suppression of viral replication	Wollebo et al. (50)
JCV	SVG-A cell line		N-terminal region of T-antigen	Suppression of viral replication	Wollebo et al. (50)

cccDNA, covalently closed circular DNA; DSBs, DNA double-strand breaks; EBV, Epstein–Barr virus; LTR, long terminal repeat; HCV, hepatitis C virus; HBV, Hepatitis B virus; HIV, human immunodeficiency virus; HPV, human papillomavirus; HSV, herpes simplex virus; JCV, John Cunningham virus; PRV, pseudorabies virus; UTR, untranslated region.

Human immunodeficiency virus

Since last 2 decades, human immunodeficiency virus (HIV) epidemic has grown rapidly with 35 million death toll. The annual global bioburden of HIV in year 2015 was 36.7 million (49). The development of highly active antiretroviral therapy reduces plasma viremia and inhibits HIV replication in host cells, but does not clear the virus completely. The clearance of HIV is a challenge because of latent viral reservoir that persists in the body for long time due to integration of proviral DNA into cellular genome.

Recent development of CRISPR/Cas9 may serve as a new avenue toward the treatment of HIV by inactivating integrated latent HIV-1 DNA. CRISPR/Cas9 showed sufficient potential to target, deactivate, and mutate HIV-1 proviral DNA in the latent JLat10.6 cell line. Twentyfold decrease in HIV-1 gene expression and virus production was observed at T10 site within the second exon of Rev gene of HIV-1 genome that was targeted by CRISPR/Cas9 system (61). The excision of integrated viral genome can be mediated by combination of Cas9 nuclease with one or more small gRNAs. Fusion of modified nuclease deficient Cas9 with transcription activating domains may trigger activation of proviral gene expression for purging of the latent reservoirs. Cas9/sgRNA complex effectively blocks de novo infection along with destruction of integrated HIV-1 proviruses. CRISPR/Cas9 technology offers hope for elimination of HIV by blocking integration and progressive infection (37).

Accumulating evidence suggests that (CRISPR)-associated endonuclease Cas9 plays an important role in gRNA-directed, sequence-specific cleavage of HIV proviral DNA in infected cells. Cas9 and antiviral gRNAs harness T cells that lead to profound inhibition of HIV-1 (45). Cas9/sgRNA combination suppresses viral replication but Cas9/sgRNA-induced mutations mediate viral escape (47). Single mutations not only inhibit replication of virus but also cause an unusual resistance. To increase the effectivity of antiviral aspect of CRISPR/Cas9, multiple viral DNA regions must be targeted.

Another study showed decrease in replication of HIV in HEK 293A cell line. Liao KS. and his team also adapted the CRISPR/Cas9 system to human cells for intracellular defense against foreign DNA and viruses. CRISPR/Cas9 system disrupted latently integrated viral genome and provided long-term adaptive defense against new viral expression, replication, and infection in human cells. HIV provirus is flanked by long terminal repeat (LTR) sequences, and CRISPR/Cas9 had the ability to cleave at both ends of virus because CRISPR/Cas9 targeted the LTR regions. Single LTR “footprint” within the genome provides a reference to identify the position of the HIV proviral DNA that is formed as a result of DNA repair of excised region between cleavage sites. Promoter activity is lost because of gRNAs that target two or more sites within the 5′ LTR and deactivates provirus (24, 38).

Human papillomavirus

Human papillomaviruses (HPVs) are well-recognized causative agents of human cancer, including cervical, anogenital, and oral cancers. In most cases, high risk strains such as HPV16 and HPV18 (oncogenic HPVs) are present in HPV linked cancer. Globally, HPV causes more than half of sexually transmitted diseases and cancer in females, and about 5% males are also infected (48). All HPVs are nonenveloped, circular dsDNA viruses with genome size 8 kb pairs (59).

In development of cancer and controlling viral functions during the viral life cycle, HPV oncoproteins, E6, E7, and E5, play a significant role. E6 and E7 oncoproteins cause the tumorigenesis by blocking the functioning of p53 and Rb gene, but other cellular proteins are also involved. For the development of cancers E5 has an auxiliary role (30). Kennedy et al. (2014) used well-established HPV-18 positive cervical carcinoma cell line HeLa and HPV-16 positive cell line SiHa and launched bacterial Cas-9 and E6- or E7-specific single guide RNAs (sgRNAs) in these cell lines. As a result, HPV genome undergoes cleavage due to stimulation of P53 or pRB. Inactivation of deletion and insertion mutation in E6 or E7 genes lead to cell death and cell cycle arrest (20).

Hu et al. (2014) for the first time observed apoptosis and growth inhibition in HPV-positive SiHa and CaSki cells with HPV16-E7 specific single guided CRISPR/Cas system which disrupted HPV 16-E7 DNA at specific sites. But these mechanisms were not happening in HPV negative C33A and HEK 293 cells using this system (19). Zhen et al. (2014) proved that CRISPR/Cas9 had targeted the promoter of HPV16 E6/E7 and E6, E7 transcript. Actually, they transduced a CRISPR/Cas9 into cervical HPV-16-positive cell line SiHa which caused assembly of P53 and p21 protein. As a result, decreased cell proliferation in vitro and suppression of tumorigenesis in nude mice were observed (58). These evidences support the idea of E6- and/or E7-specific CRISPR/Cas as an effective tool to treat and eliminate HPV-induced cancers.

Hepatitis C Virus

Hepatitis C virus (HCV) is a contagious liver disease caused by HCV, with severity ranging from a mild illness to lethal lifelong illness. The prevalence of HCV has been estimated by surveys from different countries and it is reported that its prevalence is higher than hepatitis B virus (HBV), reaching up to level of 10% (31). It is a transmissible disease, and major routes of transmission are unsafe injections, major/minor surgeries, unsafe blood transfusions, barbers, and dentals surgeries. There is no effective licensed vaccine against HCV. Currently directing acting antiviral (DAA) drugs are in practice for HCV treatment, but recently high relapse rate was observed in patients treated with DAA (17,44).

Recently Price et al. demonstrated that HCV having positive-sense single stranded RNA can be inactivated in eukaryotic cells using Cas9 from Francisella novicida (FnCas9). They reported that human hepatocarcinoma cell lines (Huh-7.5) transfected with vectors encoding FnCas9/RNA and RNA targeting guide RNA (rgRNA) combination and coinfected with cell culture derived infectious HCV (HCVcc) recombinant virus (genotype 2a) showed reduced expression of viral proteins. rgRNA had ssRNA targeting sequence that was complementary to highly conserved 3′ untranslated region (UTR) (essential for viral RNA replication) and 5′ UTR (which is essential for not only replication of RNA but also for translation of viral protein.).

Expression studies for viral proteins were done by immunostaining or by quantifying luciferase production (34). Cas9 endonuclease has also been harnessed for getting cellular clones that are genetically safeguarded against HCV infection by genome editing. Using CRISPR/Cas9 system, small hairpin microRNA (shmiRNA) (a microRNA which acts as anti-HCV) has also been integrated into miR-122/hcr locus in liver cancer cells (39).

Hepatitis B virus

HBV is a chronic disease affecting more than 350 million people globally. This infection persists chronically in hepatic cells and progresses toward hepatofibrosis, cirrhosis, and finally hepatocellular carcinoma. About 60% of liver cancer cases are due to chronic HBV infection (15). Therefore, a deeper and more comprehensive research is needed for host-virus interactions and new effective targets for drug discovery.

HBV belongs to Hepadnaviridae family and possesses refractory DNA intermediates in its life cycle. In the infected hepatocytes, HBV exists as an episomal covalently closed circular DNA (cccDNA), which carries utmost importance in its life cycle because it acts as a template for all transcripts to be formed. Similarly, the genome also persists as chromosomally integrated structure (41). This persistence precludes the complete eradication of viral genome with both interferon-based treatment, as well as recently developed interferon-free DAA regimens. Moreover, these therapies need to be taken for indefinite time to hinder relapse rate. Therefore, a targeted nuclease therapy is desired to take over the smart surviving strategy of HBV, such as transcription activator-like effector nuclease, zinc finger nuclease, and CRISPR/Cas9 system (6,14). Among these, CRISPR/Cas9 system is the proficient and most novel technique.

The HBV genome is composed of four open reading frames that encode for seven proteins. There are several reported cases of HBV genome, targeted with CRISPR/Cas technique both in vitro and in vivo, each with different success level. These experiments have shown substantial decline in HBV production with Cas9/sgRNA Huh7 cell line cotransfected with HBV expression plasma. A recent CRISPR knockout study made in 2015 transduced HepG2.2.15 cells with Cas9 and sgRNAs against controls with and nuclease-free Cas9 and sgRNAs. The results revealed a great depletion of cccDNA and relaxed circular forms in cells with Cas9/sgRNA transduction (25,35).

On the same grounds, another quantitative analysis on CRISPR/Cas targeting HBV proved successful with HBV infected HepG2 system to prompt inhibition of HBcAg expression, when combined with CRISPR/Cas9. The results were determined by immunofluorescence with HBcAg antibody (38). Likewise, Zhen et al., cotransfected HepG2.2.15 cells with Cas9 expression plasmid and gRNA expression plasmid and exhibited marked inhibition of HBV antigens in cell culture (56).

Herpesviruses

Herpesviruses are DNA viruses responsible for significant morbidity and mortality across the globe. About 100% of adult population carries herpesviruses. Most common herpesviruses that are posing serious threats to humans are: herpes simplex virus (HSV1), HSV2, Epstein–Barr virus (EBV), and HCMV.

HSV1 and HSV2

HSV type 1 is responsible for genital herpes, herpes simplex keratitis, and cold sores, whereas HSV2 causes genital herpes. Herpesviruses can be a potential target of CRISPR/Cas9. HSV1 and HSV2 genomes are found as circular, nonreplicating DNA episomes in latently infected neuron cell. These dsDNA molecules are cleaved, destructed, or inactivated by CRISPR/Cas possibly due to AAV-mediated delivery of SpCas9 or SaCas9, together with multiple sgRNAs specific for HSV1 or HSV2 to shatter or mutationally inactivate the viral genome (1,8).

A recent study published in PLoS Pathogen exhibited that CRISPR/Cas9 targeting of essential herpesvirus genes efficiently abrogated replication of virus in human cells. The replication of HSV1 was completely impaired from human cells by simultaneously targeting it with two gRNAs. The same technique can be efficiently used to clear EBV from the latently infected EBV-transformed human tumor cells (43).

Epstein–Barr virus

EBV is another commonly found herpesvirus that infects at least 95% of general population. EBV causes infectious mononucleosis and it becomes latent in our body but reactivates afterward. EBV is associated with malignant conditions such as nasopharyngeal carcinoma, gastric cancer, B cell lymphomas, and Hodgkin's disease (9,10). Another study revealed the efficient editing of individual sites within the EBV genome. The finding of the study further confirmed the restoration of apoptosis pathway as well as significant decrease in decline and proliferation of EBV in cells derived from a patient with EBV associated Burkitt's lymphoma after being treated with CRISPR/Cas9 vector targeted to the viral genome using gRNAs specific for EBNA1, LMP1, EBNA-3C, and other genes (46). Genome editing of EBV in human cells using CRISPR/Cas9 system has also been studied.

Researchers used two guide RNAs that deleted 558 bp in the promoter region of BART transcript which encodes viral microRNAs. Targeted editing of EBV genome using CRISPR/Cas9 was found to be efficient because it was successfully achieved in several human epithelial cell lines latently infected with EBV, including nasopharyngeal carcinoma C666-1 cells which led to reduction in loss of activity and expression of BART miRNA (55).

Pseudorabies virus

Pseudorabies virus (PRV) is also herpesvirus that causes infection in swine. It possesses variety of nonessential genes that can easily be altered with foreign genes. Combination of PRV genome and Cas9/sgRNAs was introduced into PK15 cells. CRISPR-Cas9 system halted viral growth by disrupting PRV EP0 and UL50 genes (51). PRV attenuation using CRISPR-Cas9 technology has also been reported recently. Yan-dong tang et al., first developed gE/gI/TK gene-inactivated HeN1 PRV strain and then analyzed the attenuation of PRV in the mice and demonstrated that modified PRV was fully attenuated and induced immune protection in response to a parental PRV challenge (42). The data indicate that CRISPR/Cas9 system is a potent therapeutic strategy against human herpesviruses (36,43).

CRISPR/Cas9 and Cre/Lox system based vaccine against PRV has also been studied. CRISPR/Cas9 system was used to substitute the marker gene at virulent genes of the newly isolated strain followed by an excision done by Cre/Lox system for vaccine safety concern. This is the first successful vaccine based on gene edit technologies and has shown protective efficacy in pigs (23).

John Cunningham virus

Polyomavirus John Cunningham virus (JCV) is responsible for fatal demyelinating disease progressive multifocal leukoencephalopathy in 70–90% of humans. It is dsDNA virus with genome size 1.5 kb and generates two different proteins during early and late phase of infection cycle. CRISPR-Cas system inhibited replication of virus by inducing mutations in viral genome and inactivating gene encoding T-antigen. Cas9 and gRNAs expression targets DNA sequences that encode N-terminal region of T-antigen and induced mutations that ultimately lead to suppression of viral replication in permissive cells because of obstruction in expression and function of the viral protein. Gene editing technology is an efficient therapeutic technique for elimination of virus (50).

Future prospects

CRISPR/Cas technology is being used in screening of cellular factors involved in cell transformation and nonessential viral cofactors. CRISPR-based screen that identifies genes essential for West-Nile virus induced cell death has been developed (26,40,52,57,60). Currently, rapid evolution in CRISPR/Cas9 system has made this technology safe for human use and provides an effective therapeutic solution against genetic infectious diseases. CRISPR-Cas have brought scientists one step closer to development of designer babies that can be used to alter future generations either by removing or editing faulty genes (22,33,52).

Accumulating evidence suggest the efficient use of CRISPR/Cas system in development of engineered crops and insects (4,11,13,16). CRISPR-Cas9 system is also emerging as an efficient viral editing method for the creation of new oncolytic viruses that are antitumor viruses. Mutations in the target region of AdV and HSV1, as well as HSV virus with enhanced green fluorescent protein and viral vectors with or without RFP (red fluorescent protein), have also been successfully created by CRISPR-Cas9 system. Therefore, this genome editing system generates mutations in large viral DNA genomes (54).

Moreover, scientific community must overcome challenges such as off-target mutations in the genome, delivery of editing systems to target cell types, unpredictable effects to the future generations, and high cost of this germ-line editing technology. Some viruses especially flaviviruses Zika virus (ZIKV) and Dengue virus (DENV) are reaching new heights in their potential to threaten global health. CRISPR/Cas9 can prevent the transmission of these viruses by engineering mosquitoes resistant to infection by Zika, malaria, or dengue.

Conclusion

Explosive rise in viruses that infect millions of people worldwide has been observed during last decade. CRISPR-Cas technology is emerging as a novel genome editing system that shows great promise in control of viral infections because of robust viral suppression ability and absence of effective antiviral therapy. This feasibility of eradication of viruses has been efficaciously applied to cell lines, but application in clinical setting with infected individuals is under investigation.

Footnotes

Authors' Contributions

B.W., S.U., K.S., and H.A.A. conceived the study and performed data collection and drafted the article for publication. M.I. and A.A. critically reviewed the article. All the authors read and approved the final article.

Acknowledgment

No funding was received from any source for the present study.

Author Disclosure Statement

No competing financial interests exist.

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