The CRISPR/Cas9 genome editing methodology as a weapon against human viruses

Abstract

Viruses are a therapeutic challenge since their life cycles occur within the host cells and often utilize cellular proteins and hence it is harder to identify therapeutic targets compared to bacteria, which have their own cellular metabolism that is quite different from the host and often present unique targets such as enzymes, etc. Nevertheless, viral proteins may present useful targets for therapy, e.g., small molecule inhibitors of viral polymerases, or prevention, e.g., viral coat proteins for vaccination. However, some viruses may enter an inactive state of persistence or latency where no or very few viral proteins are produced. Thus, reagents that are specifically able to target nucleotide sequences within viral genomes would be a useful addition to the antiviral armamentarium. Such a reagent is the clustered regulatory interspaced short palindromic repeat (CRISPR)-associated 9 (Cas9) system, which is powerful, specific, and highly versatile and provides unprecedented control over genome editing. Here, we will discuss how CRISPR/Cas9 has been used against human viruses and future prospects for novel therapeutic approaches.

INTRODUCTION

Recent years have seen the rapid development of novel genetic-engineering technologies with a number of important clinical applications including the treatment of genetic diseases, cancers and viral infections. New classes of reagents have been developed that are specifically able to target nucleotide sequences within viral genomes and represent important additions to available antiviral strategies. These fall into three main categories. Zinc-finger nucleases (ZFN) are fusion proteins of the nonspecific cleavage domain of the FokI restriction endonuclease with custom-designed Cys₂-His₂ zinc-finger proteins (Gaj et a, 2012; Kim and Chandrasegaran, 1994), which cause sequence-specific DNA double-strand breaks (DSBs). DSBs are repaired by error-prone process nonhomologous end joining (NHEJ) to yield small alterations at targeted genomic loci. If two DSBs are generated, NHEJ will yield large fragment deletion at the targeted genome. Another class of reagents is known as the transcription activator-like effector nuclease (TALEN) system and are also FokI fusion proteins but the targeting domain is derived from the TAL effector proteins secreted by Xanthomonas bacteria (Wright et al., 2014). The third and most powerful class is the clustered regulatory interspaced short palindromic repeat (CRISPR)-associated 9 (Cas9), which provides unprecedented control over genome editing (Gaj et al., 2013; Mali et al., 2013b; Doudna and Charpentier, 2014; Hsu et al., 2014) and this review will focus on the use of the CRISPR/Cas9 system against human viruses.

The CRISPR/Cas9 system is simple and easy to use, adaptable, flexible to different targets and continues to improve rapidly (Ran et al., 2013b). The CRISPR/Cas system evolved in bacteria and archaea as a defense mechanism to cope with virus attack (Bhaya et al., 2011). CRISPR loci and Cas proteins identified on sequenced genomes indicate that they are present in ~90% of archaeal and ~50% of bacterial genomes or their resident plasmids. Exciting breakthroughs have allowed this adaptive immune system to be developed into a very adaptable and precise genetic engineering tool, which uses a short guide RNA (gRNA) to direct degradation of specific nucleic acids. There are two distinct components to the CRISPR/Cas9 system: a guide RNA (gRNA) and an endonuclease (Cas9). When the gRNA and Cas9 are co-expressed in cells, the DNA target sequence can be modified or disrupted. The gRNA is designed to contain a 20 base-pair guide sequence, which recruits the gRNA/Cas9 complex to its target by Watson-Crick base-pairing. In addition to the complementary guide sequence, successful binding of Cas9 to the target and subsequent endonuclease disruption require the correct Protospacer Adjacent Motif (PAM) trinucleotide sequence immediately following the target sequence. Cas9 cuts both strands of DNA causing a DSB, which lies 3–4 nucleotides upstream of the PAM sequence. DSBs may be repaired by NHEJ DNA repair pathway, which is error-prone and often results in the generation of inserts/deletions (InDels) at the DSB site that can lead to frameshifts and/or premature stop codons. This can effectively disrupt the open reading frame (ORF) of the target gene. Since Cas9 functions as a general endonuclease, all that is needed is a small gRNA that can be chemically synthesized, in vitro transcripted or cellularly expressed to provide a highly specific gene-targeting tool. This ease of use has allowed it to be used in thousands of applications in the last two years (Doudna and Charpentier, 2014; Hsu et al., 2014; Kennedy and Cullen, 2015; Khalili et al., 2015). The operation of the CRISPR/Cas9 is shown diagrammatically in Figure 1.

Figure 1. Diagrammatic representation of mutagenesis of viral DNA by CRISPR/Cas9.

Viral DNA is shown indicating endonucleolytic cleavage by CRISPR/Cas9 at a target sequence adjacent to a protospacer adjacent motif (PAM) sequence. This is followed by nonhomologous end-joining (NHEJ), which introduces insertion/deletion mutations (InDel).

In addition to its versatility and simplicity, the Cas9 system is remarkable that it achieves a high degree of specificity and provides almost exclusive on-target cleavage given the large size of the human genome. Off-target cleavage can be assayed by either the SURVEYOR assay, which detects mismatched nucleotide pairs resulting from NHEJ, or whole genome sequencing. Thus, whole-genome sequencing at high coverage to assess the degree of off-target cleavage has revealed this to be a very rare event (Hu et al., 2014a; Veres et al., 2014; Yang et al., 2014) and the Cas9 system has proved to be remarkably specific. However, the Cas9 system continues to be modified and improved with respect to its genome editing efficiency and reduction of potential off-target effects. These include developing a “paired Cas9 nickase” strategy, which increases the site specificity DSB induction and can reduce off-target activity by 50- to 1,500-fold in cell lines (Mali et al., 2013a; Ran et la, 2013a; Cho et al., 2014; Shen et al., 2014). Another approach is to fuse a catalytically dead Cas9 to the FokI restriction endonuclease to create an RNA-guided nuclease with enhanced specificity (Guilinger et al., 2014; Tsai et al., 2014).

In principal, eradication of viruses from cells by CRISPR/Cas9 should be applicable to any DNA virus or RNA virus that has a DNA intermediate in its life cycle (Doudna and Charpentier, 2014; Hsu et al., 2014; Kennedy and Cullen, 2015; Khalili et al., 2015). This system has been used recently as a weapon against viruses that cause human diseases and future possibilities.

APPLICATION OF CRISPR/CAS9 TO SPECIFIC HUMAN VIRUSES

Human Papillomaviruses HPV16 and HPV18

Human papillomaviruses (HPVs) are an established etiological agent of human cancer and the high risk or oncogenic human papillomaviruses, HPV16 and HPV18, are responsible for the majority of HPV-associated cancers (White et al., 2014). Worldwide, HPV infections account for more than half of all infection-linked cancers in females, but less than 5% in males. The HPV genome is a linear double-stranded DNA of 7–8 Kbp (Danos et al., 1982) with about ten 10 ORFs and a long control region (LCR), which regulates epithelial-specific transcription. HPV transmission occurs by mucosal contact and by skin to skin contact and is facilitated by microabrasions. Anogenital HPV infections are transmitted by sexual contact. HPV is the major cause of cervical carcinoma and is also involved in other anogenital cancers (White et al., 2014). The oncogenic properties of high-risk HPVs involve the viral proteins E6 and E7 (Munger et al., 2004; Hamid et al., 2009). Cellular p53, the major cellular tumor-suppressor protein, is a target of E6, which binds and degrades p53 (Beaudenon and Huibregtse, 2008) while E7 complexes with proteins in the retinoblastoma family of tumor suppressors that regulate the cell cycle, pRb, p107 and p130 resulting in phosphorylation and release of the E2F transcription factors promoting cell cycle progression (Felsani et al., 2006). Thus the E6 and E7 genes are prime targets for therapeutic intervention in HPV-associated malignant disease.

Kennedy et al (2014) introduced Cas9 and E6- or E7-specific gRNAs into HeLa and SiHa cervical carcinoma cell lines which contain integrated HPV18 or HPV16 respectively. Inactivating deletion and insertion mutations were induced in E6 or E7 resulting in activation of p53 or pRb, resulting in cell cycle arrest and subsequently cell death (Kennedy et al., 2014). Hu et al (2014b) reported that the CRISPR/Cas system using an HPV16-E7-specific gRNA could disrupt HPV16-E7 DNA at specific sites, resulting in the induction of apoptosis and growth inhibition in HPV-positive SiHa and CaSki cells, but not in HPV negative C33A and HEK293 cells. Zhen et al (2014) established CRISPR/Cas9 targeting promoter of HPV16 E6/E7 and targeting E6, E7 transcribed regions. Transduction into SiHa cells showed that CRISPR/Cas9 targeting the promoter, as well as targeting E6 and E7 genes resulted in accumulation of p53 and p21 protein (cyclin-dependent kinase inhibitor 1, a cell cycle inhibitor protein which promotes growth arrest), and reduced cell proliferation in vitro and dramatic inhibition of tumorigenesis in nude mice (Zhen et al., 2014). Thus the CRISPR/Cas9 approach has proven effective when used on HPV-transformed cell lines in culture and has the potential to be developed as an effective therapy for HPV-associated tumors in the clinic.

Hepatitis B virus (HBV)

HBV was recognized in the 1940s as a bloodborne infectious disease causing jaundice and the HBV virus was identified in 1970 (Dane et al., 1970). HBV causes acute and chronic liver infections, which can lead to liver failure, cirrhosis and hepatocellular carcinoma (HCC). It is a hepadnavirus with a small, circular, partially double-stranded DNA genome (Robinson et al.,1974). Of people who are infected with HBV, ~1% develop fulminant hepatitis B, which is associated with liver failure requiring liver transplantation and ~5% develop chronic infections, which may lead to chronic hepatitis, cirrhosis and HCC (Zur Hausen, 2006). Chronic hepatitis B involves persistence of HBV DNA in an episomal form known as the covalently closed circular DNA (cccDNA), which is a hurdle for eradication of the virus. The DNA genome of HBV is small and encodes only a few proteins including HBV Protein X (HBx) and HBV surface antigen (HBsAg), which exert multiple pleiotropic effects that are important in transformation but are not well understood. The HBV genome may integrate into the host genome and this can occur at many different sites and may causes disruption of key genes that regulate proliferative signaling, which is thought to be an important mechanism in the development of HCC in HBV-infected individuals. HBx is oncogenic and can transform rodent hepatocytes and NIH 3T3 cells (Kew, 2011). HBx may act by activating signal transduction pathways involved in the regulation of cell proliferation such as ERKs, SAPKs and p38 protein kinase (Diao et al., 2001). It has also been reported to bind to p53 (Feitelson et al., 1993).

Curing chronic HBV infection will require the specific eradication of the persistent HBV cccDNA from infected cells. Lin et al (2014) designed eight gRNAs against HBV and showed that the CRISPR/Cas9 system significantly reduced the production of HBV core and HBsAg proteins in the Huh-7 hepatocyte-derived cellular carcinoma cell cells transfected with an HBV-expression vector. Further this system could cleave intrahepatic HBV genome-containing plasmid and facilitate its clearance in vivo in a mouse model resulting in a reduction in serum HBsAg level (Lin et al., 2014). Thus, CRISPR/Cas9 system could disrupt the HBV templates both in vitro and in vivo and may have a potential in eradicating persistent HBV infection. In another study, Seeger and Sohn (2014) tested in HepG2 hepatoma cells expressing the HBV receptor with different HBV-specific gRNAs and found inhibition of HBV infections up to eightfold, which was due to mutations and deletions in the cccDNA caused by Cas9 cleavage and repair by NHEJ. Kennedy et al. (2015) used lentiviral transduction of Cas9 and HBV-specific gRNAs and showed effective inhibition of HBV DNA production for in vitro models of both chronic and de novo HBV infection. Total HBV viral DNA levels were reduced by up to ~1000-fold while cccDNA levels were reduced by up to ~10-fold and the majority of the residual viral DNA was mutationally inactivated. In the most recent study of HBV and CRISPR, Zhen et al. (2015) targeted the HBsAg and HBx-encoding region of HBV, both in a cell culture system and in vivo. HBsAg levels in the cultures media of cells and in the sera of mice were reduced as shown by ELISA. The HBV DNA levels and HBsAg expression in mouse livers were reduced as shown by qPCR and immunohistochemistry respectively. In conclusion, studies from several labs have shown the potential of CRISPR/Cas9 to treat HBV-associated diseases.

Epstein-Barr virus (EBV)

EBV is a DNA virus of the herpesvirus family that causes Burkitt’s lymphoma and nasopharyngeal carcinoma (Pagano, 1999). EBV infections are very common with more than 90% of people becoming infected by their twenties (Henle et al., 1969). EBV has a large, 168–184 Kbp linear double-stranded DNA genome with 85 genes, terminal repeat regions and an internal repeat region (Baer et al., 1984). During latency the genome circularizes to form an episome that is maintained at constant copy number and (Adams and Lindahl, 1975). EBV has two subtypes, EBV-1 and EBV-2, which differ at the EBNA locus and infects lymphocytes and also epithelial cells, which are the primary site of initial replication (Lemon et al., 1977). EBV can enter a latent state in B cells, where the circular episomal EBV genome expresses only a few of the proteins that it encodes. The proteins expressed during latency may be involved in the events which lead to cellular transformation, especially EBV nuclear antigen 1 (EBNA-1, Rowe et al., 1992), which is a sequence-specific DNA-binding phosphoprotein that is involved in the maintenance of the latent EBV episome, viral DNA replication and cellular transformation (Rawlins et al., 1985). Another viral protein, EBNA-2 regulates expression of the other latency-associated EBV genes, LMP-1 and LMP-2 (Abbot et al., 1990). LMP-1 activates a number of pathways involved in cell transformation, induces invasion and metastasis factors and acts together with other EBV latency-associated proteins and RNAs (EBV-encoded small RNA-1 and -2 (EBER1 and EBER2), and the BamHI rightward transcripts (BARTs)) via multiple molecular mechanisms to effect cellular transformation by EBV in Burkitt’s lymphoma and nasopharyngeal carcinoma (Marquitz and Raab-Traub, 2102; Wakisaka and Pagano, 2003).

EBV has also been investigated using the CRISPR/Cas9 system (Wang and Quake, 2014). Cells derived from a patient with Burkitt’s lymphoma with latent EBV infection (Raji cells) showed a marked reduction in proliferation and decline in viral load as well as restoration of the apoptosis pathway in the cells after treatment with a CRISPR/Cas9 vector targeted to the viral genome using gRNAs specific for EBNA1, EBNA3C and LMP1 and others. SURVEYOR assays confirmed efficient editing of individual sites within the EBV genome. Yuen et al (2015) reported CRISPR/Cas9-mediated editing of EBV in human cells using two gRNAs to make a targeted deletion of 558 bp in the promoter region for BART in several human epithelial cell lines latently infected with EBV including nasopharyngeal carcinoma C666-1 cells. This resulted in the loss of BART miRNA expression and activity indicating the feasibility of CRISPR/Cas9-mediated editing of the EBV genome. No off-target cleavage was found by deep sequencing.

HIV-1

After 30 years since its discovery, HIV-1/AIDS remains a major public health problem worldwide. The development of highly effective Combination Antiretroviral Therapy that powerfully inhibits viral replication in cells that support HIV-1 infection and robustly reduces plasma viremia, has meant that HIV-1 infection is now survivable rather than always fatal. Despite this however, AIDS remains incurable since HIV-1 can undergo permanent integration into the host genome, thus providing a persistent reservoir of virus and the risk of viral reactivation even after effective antiretroviral therapy. As well as DNA viruses, retroviruses are amenable to CRISPR/Cas9 manipulation because they have a DNA intermediate in their life cycle. The CRISPR/Cas9 system has been used to target the HIV-1 LTR integrated in the genome of latently infected cells in culture (Ebina et al., 2013; Hu et al., 2014a). In this way, it was possible to inactivate viral gene expression and replication in latently infected microglial, promonocytic, and T cells (Hu et al., 2014a). This is a potential therapeutic advance in overcoming the stumbling block in eliminating all virus from HIV-1-infected individuals, i.e., permanently integrated DNA proviral copies of HIV-1 in the host genome, the reservoir of virus that is unreachable by current antiviral therapies. With CRISPR/Cas9, it was possible to identify specific targets in the HIV-1 proviral genome that completely excised integrated provirus (Hu et al., 2014a). Moreover, the presence of HIV-1-specific gRNAs and Cas9 in cells prevents HV-1 infection, i.e., CRISPR/Cas9 has both therapeutic and prophylactic potential (Hu et al., 2014a). Cas9/gRNAs caused neither genotoxicity nor off-target editing to the genome of host cells as revealed by whole-genome sequencing analysis and SURVEYOR assays. Recently, Zhu et al. (2015) tested 10 sites in HIV-1 DNA for CRISPR/Cas9 in JLat10.6 cells that are latently infected by HIV-1. Sequencing results showed that each target site in HIV-1 DNA was efficiently mutated by CRISPR/Cas9 with the target site in the second exon of Rev exhibiting the highest degree of mutation, with the result that HIV-1 gene expression and virus production were reduced 20-fold. In another recent study, Liao et al. (2015) also demonstrated that CRISPR/Cas9 system disrupted latently integrated HIV-1 genome and provided long-term adaptive defense against new viral infection, expression and replication in human cells. They showed that engineered human-induced pluripotent stem cells stably expressing HIV-targeted CRISPR/Cas9 could be efficiently differentiated into HIV reservoir cell types and maintain their resistance to HIV-1 challenge.

Another approach to employ CRISPR/Cas9 against HIV-1 would be to target cellular genes encoding proteins required for HIV-1 infection such as CCR5, a coreceptor for HIV-1 entry (Manjunath et al., 2013; Stone et al., 2013). Wang et al. (2014) showed that transduction of lentiviral vectors expressing Cas9 and CCR5-specific gRNAs into HIV-1 susceptible human CD4+ cells gave a high frequency of CCR5 gene disruption and became not only resistant to R5-tropic HIV-1, including transmitted/founder (T/F) HIV-1 isolates but also had a selective advantage over cells with undisrupted CCR5 during R5-tropic HIV-1 infection. No mutation was detected at potential off-target sites that were highly homologous, particularly CCR2.

Thus the CRISPR/Cas9 system has the potential to be developed to provide a specific and efficacious approach against HIV-1/AIDS that can be employed both prophylactically and therapeutically. The feasibility of HIV-1 eradication by CRISPR/Cas9 in animal models and in a clinical setting with individuals with HIV-1/AIDS remains under investigation.

CONCLUSION

The CRISPR/Cas9 system is a new and powerful tool for precise, simple and easy to use genetic engineering of different DNA targets. Although it has only been available to be used in eukaryotic cells for only about two years, it has already found many potential applications to human diseases including genetic disorders, cancer and viruses. So far, CRISPR/Cas9 has been applied to four human viruses as discussed above and summarized in Table 1.

TABLE 1.

Application of CRISPR/CAS9 to specific human viruses

Virus	Target	Cells	Results	Reference
HPV18	E6 or E7	HeLa	Induction of p53 or Rb Cell cycle arrest	Kennedy et al., 2014
HPV16	E6 or E7	SiHa	Induction of p53 or Rb Cell cycle arrest	Kennedy et al., 2014
HPV16	E7	SiHa and CaSki	Apoptosis and growth inhibition	Hu et al., 2014b
HPV16	E6, E7 or E6/E7 promoter	SiHa	p53 and p21 induction, Inhibition of cell proliferation and tumorigenesis	Zhen et al., 2014
HBV	P1 (1,292–1,314) & XCp (1,742–1,764)	Huh-7	Reduction in HBsAg level in medium	Lin et al., 2014
	P1 (1,292–1,314) & XCp (1,742–1,764)	Mouse liver in vivo	Reduction in HBsAg level in serum	Lin et al., 2014
	ENII-CP/X & Pre-C	HepG2 with HBVR	8-fold inhibition of HBV infection	Seeger and Sohn, 2014
	HBsAg, core, and/orRT.	HepAD38	Reduction in viral DNA and cccDNA levels	Kennedy et al., 2015
	HBsAg, HBx	HepG2.2.15	Reduction in HBsAg level In medium	Zhen et al., 2015
		Mouse liver	Reduction in HBsAg level In serum
EBV	EBNA1, EBNA3C LMP1 and others	Raji	Reduction in proliferation and viral load, apoptosis	Wang and Quake, 2014
	BART promoter	C661-1 cells and others	Loss of miRNA expression	Yuen et al., 2015
HIV-1	LTR	T cells	Loss of HIV-1 gene expression	Ebina et al., 2013
	LTR	Microglia, promonocytes, T cells	Excision of provirus Prevention of HIV-1 infection	Hu et al., 2014a
	CCR5	CD4+ cells	Resistance to R5-tropic HIV-1	Wang et al., 2014

There are many other human disease-associated viruses that are likely susceptible to this approach. Examples include herpesviruses such as Kaposi's sarcoma-associated herpesvirus (KSHV, HHV8), which causes Kaposi’s sarcoma (Chang et al., 1994) and polyomaviruses such as Merkel cell carcinoma virus (MCV), which causes Merkel cell carcinoma (Feng et al., 2007), polyomavirus JC (JCV), which causes progressive multifocal leukoencephalopathy (Padgett et al., 1971) and polyomavirus BK (BKV), which causes polyomavirus-associated nephropathy (PVAN, Gardner et al., 1971).

While there are many examples of the efficacious use of CRISPR/Cas9 in cell culture as described in this review, application to the clinic will require a method of safe and efficient delivery. For basic research studies in cell culture, plasmid transfection or viral transduction with lentiviral or adenoviral vectors have been employed. Lentivirus transduction in particular has been used in many labs because of the high efficiency of delivery and stable gene expression. However, the safety of lentiviruses is compromised by its unfavorable insertion-mediated mutagenesis effect, whereby the silencing or activation of unexpected genes may occur. The generation of integrase-deficient lentivirus vectors (IDLV) has recently provided a means to reduce the risk of virus-mediated insertions and improve the safety of gene delivery (Liu et al., 2014). Immunogenicity of the viral vector is another problem both for lentiviruses (Rothe et al., 2013) and adenoviruses (Campos and Barry, 2007). Another possibility is to use adeno-associated virus (AAV), which is only mildly immunogenic, to deliver Cas9 (Howes and Schofield, 2015; Platt et al., 2014; Senis et al., 2014; Swiech et al., 2015). AAV is a small virus but the widely used Cas9 gene from Streptococcus pyogenes (spCas9) is 4.1 Kbp long so the constructs used in these studies necessarily had to use minimal promoter and polyadenylation signals. To improve delivery, it is possible to split spCas9 into functional N-terminal and C-terminal fragments and deliver and express them as separate polypeptides, which can be recruited by the gRNA into a ternary complex that recapitulates the activity of full-length spCas9 and catalyzes site-specific DNA cleavage (Wright et al., 2015; Zetsche et al., 2015). In addition, shorter size of functional Cas9 from Staphylococcus aureus (saCas9) has been identified and exhibits similar cleaving efficiency to spCas9. Thus the field of the CRISPR/Cas9 system is rapidly evolving and holds much promise for the future.