Cas9

Accepted Manuscript Title: Suppression of Epstein-Barr virus DNA load in latently infected nasopharyngeal carcinoma cells by CRISPR/Cas9 Authors: Kit-San Yuen, Zhong-Min Wang, Nok-Hei Mickey Wong, Zhi-Qian Zhang, Tsz-Fung Cheng, Wai-Yin Lui, Chi-Ping Chan, Dong-Yan Jin PII: DOI: Reference:

S0168-1702(16)30855-3 http://dx.doi.org/doi:10.1016/j.virusres.2017.04.019 VIRUS 97127

To appear in:

Virus Research

Received date: Revised date: Accepted date:

30-12-2016 26-4-2017 26-4-2017

Please cite this article as: Yuen, Kit-San, Wang, Zhong-Min, Wong, NokHei Mickey, Zhang, Zhi-Qian, Cheng, Tsz-Fung, Lui, Wai-Yin, Chan, ChiPing, Jin, Dong-Yan, Suppression of Epstein-Barr virus DNA load in latently infected nasopharyngeal carcinoma cells by CRISPR/Cas9.Virus Research http://dx.doi.org/10.1016/j.virusres.2017.04.019 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Suppression of Epstein-Barr virus DNA load in latently infected nasopharyngeal carcinoma cells by CRISPR/Cas9 Kit-San Yuen, Zhong-Min Wang, Nok-Hei Mickey Wong, Zhi-Qian Zhang,

Tsz-Fung Cheng,

Wai-Yin Lui, Chi-Ping Chan, Dong-Yan Jin

School of Biomedical Sciences, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong

 Corresponding author.

E-mail address: [email protected] (D.-Y. Jin)

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Highlights:



CRISPR/Cas9 targeting of EBV essential genes is feasible



CRISPR/Cas9 targeting suppressed EBV DNA load



CRISPR/Cas9 suppression of EBV was progressive but incomplete



CRISPR/Cas9 targeting of EBV sensitized cells to chemotherapy

ABSTRACT

Epstein-Barr virus (EBV) infects more than 90 % of the world’s adult population. Once established, latent infection of nasopharyngeal epithelial cells with EBV is difficult to eradicate and might lead to the development of nasopharyngeal carcinoma (NPC) in a small subset of individuals. In this study we explored the anti-EBV potential of CRISPR/Cas9 targeting of EBV genome in infected NPC cells. We designed gRNAs to target different regions of the EBV genome and transfected them into C666-1 cells. The levels of EBV DNA in transfected cells were decreased by about 50%. The suppressive effect on EBV DNA load lasted for weeks but could not be further enhanced by re-transfection of gRNA. Suppression of EBV by CRISPR/Cas9 did not affect survival of C666-1 cells but sensitized them to chemotherapeutic killing by cisplatin and 5-fluorouracil. Our work provides the proof-of-principle for suppressing EBV DNA load with CRISPR/Cas9 and a potential new strategy to sensitize EBV-infected NPC cells to chemotherapy.

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Keywords: Epstein-Barr virus; CRISPR/Cas9; EBNA1; nasopharyngeal carcinoma

1.

Introduction Epstein-Barr virus (EBV) establishes an asymptomatic latent infection in most adults but

causes lymphoid and epithelial malignancies including nasopharyngeal carcinoma (NPC) in a small subset of people (Raab-Traub, 2012). During lytic replication >100 EBV proteins are expressed. In contrast, <15 EBV transcripts are produced from 5-100 copies of covalently closed circular episomal genome in latently infected cells. EBV infection is thought to confer growth and survival advantages to NPC cells (Lo et al., 2012; Lei et al., 2013; Tsao et al., 2015). In addition, serological tests suggest that EBV reactivation triggered through an unknown mechanism precedes and might therefore contribute to NPC development (Henle and Henle, 1976; Raab-Traub, 2002). Once latent infection is established, it is difficult to eliminate EBV genome completely. Although inhibitors of viral DNA polymerase and other compounds have been extensively tested as anti-EBV agents, none have been approved for clinical use (Lee et al., 2000; Gershburg and Pagano, 2005; Rafailidis et al., 2010; Whitehurst et al., 2013). Effective anti-EBV therapeutics are still in great need.

The incidence of NPC is remarkably higher in Southern China and some other areas of the world (Yu and Yuan, 2002). The etiology of NPC remains elusive but it is thought that latent infection of nasopharyngeal epithelial cells with EBV drives NPC development (Raab-Traub and Flynn, 1986). Current treatment options for NPC include chemotherapy and radiotherapy.

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They are effective but non-specific, causing severe side effects (Chan et al., 2005). In contrast, suppression or eradication of EBV in nasopharyngeal and NPC cells might have more specific prophylactic and therapeutic effects (Niedobitek, 2000). In this regard, RNA interference of EBV essential genes such as EBNA1 has been extensively tested for anti-EBV and antiproliferative potentials (Hong et al., 2006; Yin and Flemington, 2006; Ian et al., 2008). Moreover, small-molecule inhibitors of EBNA1 identified from high-throughput screening also showed promising effects in the suppression of EBV (Thompson et al., 2010). Finally, therapeutic use of EBV-specific T cells such as cytotoxic T lymphocytes and removal of latently infected cells through induction of lytic replication are also under intense investigation and clinical trials (Hutajulu et al., 2014; Manzo et al., 2015; Hui et al., 2012, 2016). However, methods that could eliminate EBV completely from latently infected NPC cells remain to be developed.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 nuclease (Cas9) system is a breakthrough genome-editing technique launched in 2013 (Cong et al., 2013; Gilbert et al., 2013). CRISPR/Cas9 editing of viral DNA is feasible. As such, the DNA genomes of EBV (Wang and Quake, 2014; Yuen et al., 2015; van Diemen et al., 2016) and multiple other viruses (Ebina et al., 2013; Bi et al., 2014; Hu et al., 2014; Dong et al., 2015; Kennedy et al., 2014, 2015; Lin et al., 2014; Seeger and Sohn, 2014; Liu et al., 2015; Zhen et al., 2015; van Diemen et al., 2016; Wang et al., 2016) were found to

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be edited effectively by CRISPR/Cas9. The high efficiency of editing makes CRISPR/Cas9 a powerful tool to combat retroviruses and DNA viruses. We have previously demonstrated the feasibility of CRISPR/Cas9 editing of EBV DNA and its utility in the construction of EBV mutants (Yuen et al., 2015). CRISPR/Cas9 has also been shown to suppress latent EBV infection in lymphoma cells. Interestingly, CRISPR/Cas9 targeting of EBNA1 in Raji or Namalwa cells reduced EBV DNA levels and arrested cell proliferation (Wang and Quake, 2014). Both are nonEBV-producer cell lines derived from Burkitt’s lymphoma. Whereas Raji carries a defective EBV genome, Namalwa contains two integrated copies of EBV DNA (Lawrence et al., 1998). Furthermore, CRISPR/Cas9 targeting of EBNA-1 and EBV origin of replication (OriP) in latently infected Burkitt’s lymphoma cell line Akata-BX1 led to the elimination of EBV genome (van Diemen et al., 2016). Akata-BX1 cells contain a recombinant EBV carrying a GFP marker in place of the thymidine kinase gene and the virus was reintroduced into EBV- Akata cells, which had lost its original EBV strain (Guerreiro-Cacais et al., 2007). In both studies, CRISPR/Cas9transfected cells were enriched by either cell sorting or drug selection to provide the proofof-concept for CRISPR/Cas9-mediated suppression or eradication of EBV. However, the antiEBV potential of CRISPR/Cas9 in EBV-infected NPC cells remains to be determined. It will also be of interest to see whether CRISPR/Cas9 editing could practically display anti-EBV activity in NPC cells without the need for target cell enrichment. Finally, if EBV is required for growth and survival of NPC cells (Choy et al., 2008; Lei et al., 2013; Tsao et al., 2015), the impact of

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the loss of EBV genome or significant reduction of EBV DNA load on NPC cell viability warrants further analysis.

In this study, we sought to evaluate the anti-EBV potential of CRISPR/Cas9 in NPC cells by targeting 3 different genomic elements, namely EBNA1, OriP and W repeats. The impacts of CRISPR/Cas9 editing on viral DNA load, infection titer, loss of episomes as well as viability and susceptibility to chemotherapy were documented. Our work provides the proof-of-concept for CRISPR/Cas9 suppression of EBV DNA accumulation in NPC cells and for sensitization of NPC cells to chemotherapy by suppressing EBV DNA load.

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2.

Materials and methods

2.1.

Plasmids gRNA-Cas9 co-expression plasmid PX459 (Ran et al., 2013) was a gift from Dr. Feng Zhang

(Massachusetts Institute of Technology, Cambridge, MA, USA). EBNA1-gRNA1, EBNA1-gRNA2, OriP-gRNA1, OriP-gRNA2, W-gRNA1 and W-gRNA2 were made by inserting the respective gRNA sequences into PX459. The gRNA sequences were as follows:

EBNA-1-gRNA1: 5-GCTTTGAGGTCCACTGCCGC-3

EBNA-1-gRNA2: 5-GTAGAAAGGACTACCGAGGA-3

oriP-gRNA1: 5-GTAAAAATGATTGGAATTGG-3

oriP-gRNA2: 5-GCAGAGAACCCCTTTGTGTT-3

W-gRNA1: 5-GTGTCCCCACGCGCGCATAA-3

W-gRNA2: 5-GGGCCCGGGCCCCCCGGTAT-3

2.2.

Cell culture and transfection EBV-infected HEK293M81 (kindly provided by Prof. Henri-Jacques Delecluse, DKFZ,

Heidelberg, Germany) and nasopharyngeal carcinoma cell line C666-1 were maintained in RPMI1640 medium (GIBCO, Life Technologies) supplemented with 10% fetal bovine serum

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(GIBCO, Life Technologies). HEK293M81 cells were transfected with Gene Juice (Novagen). C666-1 cells were transfected with TransIT-Keratinocyte Transfection Reagent (Mirus).

2.3.

PCR analysis CRISPR/Cas9-mediated EBV editing was performed as described (Yuen et al., 2015). Total

genomic DNA was purified using a Wizard Genomic DNA extraction kit (Promega). Four primers

(EBNA1-F:

5-ATGGTTCGGAGGATGGGGAATT-3;

GTTTTCCAACGCGAGAAGGT-3’;

OriP-F:

EBNA1-R:

5’-TCCCTCTGGGAGAAGGGTAT-3;

OriP-R:

55-

AACACTGTTTCGGGTTCCTG-3) were used in PCR amplification of EBNA1 or OriP gene. EBV DNA in culture medium was purified by using a MagMAX nucleic acid isolation kit (Life Technology).

2.4.

EBV infection of HEK293 cells HEK293M81 cells were seeded into 10 cm dishes and transfected with 4 mg of gp110 and

Zta expression plasmids to induce lytic replication as described (Yuen et al., 2015). Culture supernatant was collected 72 h post-transfection. HEK293 cells (1 × 105) in 6-well plates were infected with 200 µl of EBV-containing culture supernatant. After 1 day of infection, culture medium was replaced with 2 ml of fresh RPMI 1640 medium. Cells were incubated for an additional 2 days. Three days after infection, HEK293 cells were trypsinized and examined by using a FACSAria SORP cell sorter (BD Bioscience).

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2.5.

EBV DNA quantification Total genomic DNA was purified. DNA was diluted 1000-fold for quantitative PCR (qPCR)

analysis. The TaqMan® probes detecting the LMP1 region of EBV genome and GAPDH gene (probe Hs02758991_g1) were ordered from Applied Biosystems and the TaqMan® universal PCR Master Mix (Applied Biosystems) was used in the PCR reactions. qPCR was carried out in the StepOnePlusTM Real-Time PCR System (Applied Biosystems) using 40 cycles of amplification (95 oC for 10s and 60 oC for 1 min). Each sample was measured in triplicates. Primers for LMP1 qPCR were 5-ACCACGACAC ACTGATGAACAC-3 (forward) and 5CTAGAATCGT CGGTAGCTTG TTGA-3 (reverse). Amplicon size was 72 bp. Probe for LMP1 was 6FAM-ACTCCCTCCC GCACCC-MGB. The levels of EBV DNA relative to genomic GAPDH DNA were calculated from 2-ΔΔCT using the comparative CT method (Schmittgen and Livak, 2008). For absolute quantification, plasmid pGEM-LMP1 of 3221 bp in size was used for calibration. A standard curve of the natural logarithm of the copy number versus the CT value was generated with a serial dilution of pGEM-LMP1 from 20 to 200000 copies. Since the actual CT values obtained from the qPCR perfectly fit the linear regression line, the TaqMan® probe for LMP1 is highly sensitive for the detection of EBV DNA. The qPCR was performed in the linear range of the assay.

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2.6.

Proliferation assay Proliferation of C666-1 cells was measured by XTT (Gold Bio) reagent. The optimal cell

density for C666-1 in XTT assay was deduced by constructing a standard curve ranging from 103 to 105. Cells (5 × 104) were placed in 96 wells plate with 100 µl of growth medium one day before the addition of XTT reagent. Fifty microliters of XTT reagent (1 mg/ml) and 1 µl of electron coupling reagent PMS (1.25 mM of N-methyl dibenzopurazine methyl sulfate) were added to each well and incubated at 37 oC and an atmosphere of 6.5% CO2 for 4 h. The spectrophotometric absorbance of the samples at 450 nm was measured using a microplate reader. For drug treatment groups, cells were treated with 0.1 mM of cisplatin for 4 h or with 1 µM of 5-fluorouracil for 24 h. Viability of OriP-targeted cells was relative to that of cells infected with wild-type (WT) EBV. Viable cells (%) were derived from AbsWT/oriP / AbsWT × 100%.

3.

Results

3.1.

Feasibility of CRISPR/Cas9 editing of EBV genome in NPC cells C666-1 is the only NPC cell line constitutively carrying EBV (Cheung et al., 1999).

However, although the expression of some lytic transcripts in C666-1 cells can be induced by various agents (Hui et al., 2012, 2016), full lytic replication cycle cannot be completed and infectious virion cannot be produced efficiently from C666-1 cells (Tso et al., 2013), another cell line susceptible to lytic induction is also desired. HEK293M81 is a virus producer cell line

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for the M81 strain of EBV isolated from a Chinese patient with NPC (Tsai et al., 2013). HEK293 cells are considered to derive probably from kidney epithelial cells and HEK293M81 carries a recombinant EBV cloned in bacterial artificial chromosome. Compared to C666-1 cells, HEK293M81 cells are less or not representative of EBV+ NPC. However, because EBV viruses derived from NPC might have unique biological properties (Kwok et al., 2014), M81 is a desirable strain for NPC study. Whereas C666-1 served as an excellent model for latent infection of EBV in NPC cells, HEK293M81 allowed the assessment of titer and infectivity of M81. Thus, both C666-1 and HEK293M81 cell lines were used in our study. We have previously demonstrated the feasibility of CRISPR/Cas9 editing of EBV genome in HEK293 and C666-1 cells (Yuen et al., 2015). However, to achieve the goal of suppressing or curing EBV infection in NPC cells using CRISPR/Cas9, gRNA target sites should be carefully selected. In addition, the transfection efficiency of C666-1 cells has to be increased substantially.

Three EBV genomic regions, namely EBNA1, OriP and W repeats, were chosen as the gRNA target. Both EBNA1 and OriP are essential genes of EBV absolutely required for episome maintenance and replication. Although W repeats are non-essential, they were also chosen since the repeat region could provide multiple target sites for CRISPR/Cas9 editing, maximizing the suppressive effect. For each target region, we employed two gRNAs so that a major part of the region would be deleted (Fig. 1a). In addition, other forms of gene disruption such as frame-shift mutation could also be mediated by these gRNAs.

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Since C666-1 cells are difficult-to-transfect, we tested many different transfection reagents on this cell line and found that TransIT-Keratinocyte Transfection Reagent (Mirus) tailor-made for keratinocyte transfection gave the best transfection efficiency of about 80% (Fig. 1b). Less than 5% of C666-1 cells were transfected when any other transfection reagent was used. Thus, gRNA and Cas9 expression plasmids could be successfully introduced into C666-1 cells using the Mirus reagent.

Indeed, target regions in the EBV genome were effectively cleaved by CRISPR/Cas9 after expression of gRNAs and Cas9 in HEK293M81 and C666-1 cells for 2 days (Fig. 1c and d). The presence of the edited EBNA1 fragment of 901 bp and the edited OriP fragment of 313 bp as well as the diminution of the unedited DNA bands indicated the high efficiency of CRISPR/Cas9 editing. Consistent with the above result that C666-1 was efficiently transfected (Fig. 1b), CRISCPR/Cas9 cleavage was equally efficient in HEK293M81 and C666-1 cells (Fig. 1c and d). Thus, CRISPR/Cas9 editing of EBV genome in these cells was feasible. Since the sequence of the W repeats are highly redundant, the edited product would be indistinguishable from the unedited version, so PCR analysis of that region was not informative.

3.2.

CRISPR/Cas9 editing of EBV genome reduces virion production and infection titer As the first step to shed light on its biological consequence, we sought to assess how

CRISPR/Cas9 editing of EBV genome might affect virion production. The utility of serological

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markers of EBV lytic reactivation such as IgG and IgM antibodies against VCA and EA for early diagnosis of NPC supports the model that reactivation of EBV precedes NPC development (Henle and Henle, 1976; Raab-Traub, 2002). Based on the reasoning that suppressing lytic replication of EBV might have prophylactic and therapeutic effect, we compared the impact of CRISPR/Cas9 editing of the three genomic regions of EBV on virion production from HEK293M81 cells. gRNAs targeting EBNA1, OriP and W repeats were expressed in these cells before lytic induction. Virions were then collected from culture supernatant and EBV DNA levels were analyzed by qPCR. The yield of EBV DNA was significantly reduced when cells received EBNA1- or OriP-targeting gRNAs (Fig. 2a), indicating that disruption of these genes by CRISPR/Cas9 compromised EBV lytic replication.

Since EBV virions carrying a defective genome might not be revealed by PCR analysis of EBV DNA, the infection titer of EBV recovered from culture supernatant should be verified. To this end, we next performed infection experiment in HEK293 cells. Cells were infected with the same amount of EBV-containing supernatant. The percentages of freshly infected HEK293 cells were examined by flow cytometry. For the groups in which EBNA1 and OriP were targeted, the percentages of infected cells were reduced by 79% and 83%, respectively (Fig. 2b). Targeting of W repeats also resulted in a reduction of infection titer but to a lesser extent of about 40% (Fig. 2b). These results were generally consistent with the notion that CRISPR/Cas9 editing reduces virion production and infection titer.

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3.3.

CRISPR/Cas9 editing of EBV genome suppresses viral DNA load in C666-1 cells Another goal for CRISPR/Cas9 editing of EBV genome is to suppress or eradicate EBV in

latently infected NPC cells. The copy number of EBV genome in C666-1 has previously been determined to be as high as 50 (Lun et al., 2012; Yuen et al., 2015). This posed a significant technical challenge for suppression and ultimate eradication of EBV. Both reduction of EBV DNA load in gRNA-expressing cells and elimination of EBV from some of these cells resulted in the drop of EBV DNA levels. In light of this, we set out to analyze the steady-state levels of EBV DNA in gRNA-expressing C666-1 cells.

CRISPR/Cas9 editing of EBV essential genes has been shown to result in the loss of EBV genome from latently infected lymphoma cells after enrichment by drug selection (van Diemen et al., 2016). However, C666-1 cells are highly susceptible to puromycin. More than 98% of C666-1 cells die upon treatment with 3 µg/ml of puromycin for 1 day. They therefore cannot survive prolonged treatment with puromycin. In addition, antibiotic selection for gRNA-expressing cells is not possible when it enters the next phase of drug development. It remained to be seen whether CRISPR/Cas9 editing of EBV genome showed anti-EBV activity in C666-1 cells without drug selection. The magnitude and the time course of the suppressive effect should also be determined. Notably, qPCR analysis indicated a decline of EBV DNA load in C666-1 cells expressing gRNAs that target EBNA1, OriP or W repeats when total genomic DNA was collected 2 or 4 weeks after transfection (Fig. 3a). No antibiotics were added to

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enrich gRNA-expressing cells. When EBNA1 or OriP was targeted, the decline in EBV DNA load was visible 2 weeks after transfection and became more prominent 4 weeks after transfection (Fig. 3a). When the non-essential W repeats were cleaved, the change in EBV DNA levels was insignificant within the first 2 weeks but a 30% drop was seen after 2 additional weeks (Fig. 3a). Thus, CRISPR/Cas9 editing of either essential or non-essential EBV genes resulted in progressive decline of viral DNA load.

We noted that the EBV episomes were not completely eradicated from gRNA-expressing C666-1 cells. To investigate the cause, it would be of interest to determine whether the dose of gRNAs and Cas9 was insufficient or progressively decreased with time. With this in mind, two additional doses of gRNA-OriP-Cas9 plasmids were transfected into C666-1 cells. To our surprise, replenishing gRNA and Cas9 did not further decrease EBV DNA load (Fig. 3b). Thus, the dose of gRNA and Cas9 might not be a limiting factor in our experimental setting. Likewise, reintroduction of gRNA-W repeats had no additive effect on gRNA-OriP, whereas reintroduction of gRNA-EBNA1 exhibited a weak additive effect (Fig. 3c). This suggested that sequential introduction of different gRNAs might not be critical. Taken together, the suppression of EBV DNA accumulation by CRISPR/Cas9 editing appeared to be timedependent but gRNA dose-independent.

3.4.

CRISPR/Cas9 editing of EBV genome sensitized C666-1 cells to chemotherapy

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Because the above assays cannot distinguish suppression of EBV DNA load in many C6661 cells from complete elimination of EBV from a subset of C666-1 cells, we first performed in situ hybridization of EBER RNA to rule in or rule out the existence of EBV-free cells. We noted that all gRNA-OriP-expressing C666-1 cells harvested 4 weeks after transfection were EBER RNA-positive (data not shown). OriP-targeting gRNAs were chosen because they gave the best suppressive effect on EBV DNA load (Fig. 3a). Our results raised two possibilities: either EBVfree C666-1 cells were all dead or suppression of EBV DNA load by CRISPR/Cas9 editing was incomplete. To distinguish these two possibilities, we performed XTT assay to assess cell viability. Compared to mock-transfected cells, C666-1 cells transfected with gRNA-OriP-Cas9 plasmids for 4 weeks exhibited no change in viability or growth rate (Fig. 4). These results did not support that EBV-free C666-1 cells were dead or that suppressing EBV DNA load affected cell proliferation directly.

Because EBV might also induce chromosome instability (Shumilov et al., 2017), suppress DNA damage response (Gruhne et al., 2009; Whitehurst et al., 2012) and inhibit apoptosis (Choy et al., 2008; Banerjee et al., 2013), reducing EBV DNA load could affect the activation of cell cycle checkpoint and apoptosis through different mechanisms. On the other hand, most chemotherapeutic agents operate through DNA damage checkpoint and apoptosis. Thus, we next investigated whether and how CRISPR/Cas9 editing of EBV genome might influence chemotherapeutic killing of C666-1 cells. Interestingly, the viability of OriP-targeted C666-1

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cells were significantly reduced when treated cisplatin and 5-fluoruracil, two drugs commonly used in chemotherapy of NPC (Fig. 4). Thus, CRISPR/Cas9 editing of EBV genome sensitized C666-1 cells to chemotherapeutic killing.

4.

Discussion In this study, we demonstrated the feasibility and provided the proof-of-concept for the

use of CRISPR/Cas9 editing of EBV genome as an anti-EBV therapeutic in NPC cells. CRISPR/Cas9 editing of EBV genome led to suppression of viral DNA load and sensitization of NPC cells to chemotherapy. Our findings have implications in the design and development of anti-EBV and anti-NPC prophylactic and therapeutic agents. In particular, although our study was conducted in NPC cells, it will also be of interest to investigate whether suppression of EBV DNA load in nasopharyngeal epithelial cells in high-risk groups of people might reduce the risk for NPC development.

Among the three EBV genomic regions cleaved by CRISPR/Cas9 in this study, EBNA-1 and OriP were desirable targets in all assays. This was ascribed to their essentiality in EBV replication and maintenance (Rawlins et al., 1985; Yates et al., 1985). EBNA1 is the only viral protein expressed in all latency states seen in EBV-associated tumors. It is DNA-binding protein with multiple functions (Yates et al., 1985; Adams, 1987). OriP contains the dyad symmetry element and EBNA1 binding site (Rawlins et al., 1985; Reisman et al., 1985). It is not

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surprising that CRISPR/Cas9 targeting of these two regions might have deleterious effect on EBV. The weak additive effect of sequential targeting of OriP and EBNA1 was encouraging. Further optimization experiments might reveal a better combination of EBV-suppressing gRNAs. For W repeats, it was originally thought that multiple target sites might maximize the suppressive effect. However, only a mild negative effect of W repeat targeting was observed on EBV DNA load and infection titer. The number of W repeats varies among different EBV strains and it has been shown to be influential in the control of EBNA2/EBNA-LP coexpression (Tierney et al., 2011), but W repeats are non-essential. Plausibly, multiple site disruption in the repeat region might not be fatal to EBV episome. The mild suppressive effect observed could be attributed to the biological function of W repeats. Particularly, it remains to be determined whether W repeats play any role in viral maintenance in NPC. Taken together, our results indicated the anti-EBV potential of CRISPR/Cas9 targeting of either essential or nonessential viral genes.

In addition to the three EBV genomic regions targeted in our study, CRISPR/Cas9 editing also provides an opportunity to specifically target EBV genes that drive lytic replication. It will be of interest to see whether CRISPR/Cas9 targeting of lytic genes such as Ori-Lyt and Zta might effectively suppress EBV lytic cycle, during which thousands of copies of EBV genome are produced. This might be tested in induced HEK293M81 cells and immortalized nasopharyngeal epithelial NP460 cells carrying M81.

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Although CRISPR/Cas9 editing of EBV genome in NPC cells is feasible and results in a steady decline of EBV DNA load, the decline is slow and it takes 2-3 weeks before it is seen. This could be attributed at least in part to the high copy numbers of EBV genome in NPC cells. Because these cells harbor as high as 50 or 100 copies of EBV genome (Lun et al., 2012; Yuen et al., 2015), a suppressive effect could be masked and the unedited EBV genome might be sufficient to support growth, division and survival of the NPC cells. Therefore, stable and longterm expression of gRNAs and Cas9 in NPC cells is required for sustained suppression of EBV load.

EBV infection is required for NPC development and progression (Tsao et al., 2015). However, once NPC is well developed, the necessity of EBV for cell growth and survival of NPC cells remains to be clarified. Our results that suppression of EBV DNA load alone did not affect viability or proliferation of C666-1 cells implicated that EBV might not be absolutely required for survival or growth of NPC cells. Alternatively, insufficient suppression of EBV DNA load might be the reason for lack of effect on cell survival. This awaits further investigations. On the other hand, our demonstration that suppressing EBV DNA load by CRISPR/Cas9 sensitized C666-1 cells to chemotherapeutic agents suggested that EBV could confer a selection advantage to NPC cells by enabling them to survive stress such as cellular DNA damage induced by chemotherapeutic agents. This is generally consistent with previous findings on the inhibition of DNA damage response and DNA repair by EBV (Gruhne et al., 2009;

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Whitehurst et al., 2012). Moreover, we observed that prolonged expression of gRNAs and Cas9 in C666-1 cells led to progressive reduction of EBV DNA load, but all the survival cells were still EBV-positive. We have also made several other attempts to obtain EBV-free C666-1 cells but none was successful. The incomplete suppression of EBV DNA load could be explained by the high copy number of EBV genome in C666-1 cells, the lack of enrichment by drug selection, and the development of escape mutants that are no longer susceptible to CRISPR/Cas9 cleavage. Additionally, we cannot fully exclude the possibility that EBV-free cells are rapidly eliminated with no sign of apoptosis detectable in the XTT assay. Further investigations are required to clarify these issues. Particularly, it will be of interest to see whether prolonged CRISPR/Cas9 editing with multiple gRNAs might lead to complete eradication of EBV in some NPC cells.

Our results that CRISPR/Cas9 editing of EBV genome sensitized C666-1 cells to chemotherapy might provide a new strategy for combinatorial therapy of NPC. Co-delivery of chemotherapeutic drugs with CRISPR/Cas9 might reduce the dose of these drugs, thereby alleviating the side effects caused. As a first step towards further development of this idea of combinatorial therapy, the C666-1 xenograft model in mice could be used to perform a feasibility study. However, concerns about off-target effects and delivery vehicle have to be addressed before CRISPR/Cas9 technology can enter the next phase of medical applications. In our case, off-target cleavage of cellular DNA in non-NPC cells should be prevented. Since

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CRISPR/Cas9 only recognizes the target site with a 20 bp-long gRNA through simple base pairing, mis-binding of gRNA can result in off-target effects, leading to the introduction of double-strand breaks at non-desired positions (Semenova et al., 2011; Wiedenheft et al., 2011). Limiting the number of gRNAs used might be one strategy to reduce off-target effects. Moreover, a high-fidelity version of CRISPR/Cas9 nuclease Streptococcus pyogenes Cas9-HF1 (spCas9-HF1) was recently found (Kleinstiver et al., 2016). spCas9-HF1 is a quadruple substitution variant (N497A/R661A/Q695A/Q926A) of spCas9. The sequence variation allows spCas9-HF1 to retain its on-target activity but highly reduce the off-target activity. Genomewide analysis demonstrated that off-target effects were reduced to undetectable levels (Kleinstiver et al., 2016). This enzyme might prove useful in targeted editing of EBV in future.

Lentiviral and adeno-associated viral (AAV) vectors are the most common vehicles to deliver CRISPR/Cas9 in vivo (Senis et al., 2014; Shalem et al., 2014). In the context of NPC therapy, AAV vector might have some advantages. Particularly, epithelial cells in respiratory tract are efficiently transduced with AAV6 (Halbert et al., 2001). In addition, AAV vector can successfully deliver CRISPR/Cas9 in mouse model (Senis et al., 2014). Recombinant AAV has also been approved for clinical use in gene therapy of familial severe hypertriglyceridemia in Europe in 2012 (Gaudet et al., 2012). Thus, AAV might be the vector of choice to deliver CRISPR/Cas9 effectively to NPC cells.

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Acknowledgments This work was supported by the Hong Kong Research Grants Council (AoE/M-06/08 and C7011-15R), the Hong Kong Health and Medical Research Fund (HKM-15-M01) and SK Yee Medical Research Fund (2001).

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Figure Legends

Fig.1. CRISPR/Cas9 editing of EBV genome (a) Schematic diagram depicting the target sites for CRISPR/Cas9 editing in EBNA1 (upper), OriP (middle) and W repeat (lower) regions of the EBV genome. The target sites of gRNAs are highlighted with arrows. Also shown are binding sites of specific primers (EBNA1-F, EBNA1-R, OriP-F and OriP-R). (b) Transfection efficiency of C6661 cells. C666-1 cells were transfected with PX458 (GFP-CRISPR/Cas9 vector) using TransITKeratinocyte Transfection Reagent (Mirus) and the GFP signal was captured 24 h posttransfection. (c, d) Feasibility of CRISPR/Cas9 editing. gRNA-Cas9 co-expression plasmids were transfected into HEK293M81 (293M81) and C666-1 cells. Cells were harvested 48 h posttransfection and total genomic DNA was extracted using the Wizard® Genomic DNA extraction kit (Promega). EBNA1 and OriP specific primers were used in PCR analysis. The unedited EBNA1 (2272 bp), edited EBNA1 (901 bp), unedited OriP (2462 bp) and edited OriP (313 bp) DNA fragments were detected.

Fig.2. Suppression of EBV virion production and infection titer by CRISPR/Cas9 editing. (a) Virus recovery from culture medium. HEK293M81 cells were transfected with the gRNA-Cas9 co-expression plasmids 72 h before induction of lytic replication by transfection of Zta and gp110 expression plasmids. Cultured medium was collected 72 h post-induction. EBV virions in filtered and cell-free culture medium were concentrated by ultra-centrifugation (75,000 ×g for 2 h at 4 oC). EBV genomic DNA was purified using MagMAXTM nucleic acid isolation kit (Life

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Technology) and subjected to qPCR analysis. Results represent the means derived from three biological replicates and error bars indicate SD. The differences between the selected groups were statistically significant by Student’s t test (p = 0.0082 between mock and gRNA-EBNA1 as well as p = 0.014 between mock and gRNA-OriP). The actual copy numbers of EBV DNA in one milliliter of culture medium are 9.97 × 106, 5.12 × 106 and 5.60 × 106 for the mock, gRNAEBNA1 and gRNA-OriP groups, respectively, as determined by a standard curve created using a pGEM-LMP1 plasmid. (b) HEK293M81 cells were transfected with the gRNA-Cas9 coexpression plasmids 72 h before induction of lytic replication. Culture medium of the transfected HEK293M81 cells was collected. Fresh HEK293 cells were then infected with the same amount of culture medium containing the GFP+ recombinant virus. Percentage of GFP+ cells was analyzed by flow cytometry 72 h post-infection. The differences between the selected groups were statistically significant by Student’s t test (p = 0.0062 between mock and gRNA-EBNA1, p = 0.0072 between mock and gRNA-OriP, as well as p = 0.0088 between mock and gRNA-W repeats).

Fig.3. CRISPR/Cas9 suppression of EBV DNA load in C666-1 cells. (a) Progressive decrease of EBV DNA load. C666-1 cells were transfected with the gRNA-Cas9 co-expression plasmids and total genomic DNA was extracted 1, 2 and 4 weeks post-transfection. EBV DNA load was determined by Taqman qPCR. Data points in the curves represent the means derived from three biological replicates and error bars indicate S.D. (b) Impact of gRNA-OriP-Cas9 plasmid

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reintroduction on EBV DNA load. C666-1 cells were transfected with gRNA-OriP-Cas9 coexpression plasmids twice with an interval of 3 days. Cells were mock transfected with empty plasmids when gRNA-OriP-Cas9 plasmids were not given. Total genomic DNA was harvested 2 weeks after the first transfection. The difference between the mock and gRNA-OriP groups was statistically significant by Student’s t test (p = 0.024). (c) Impact of reintroduction of a second gRNA-Cas9 plasmid on EBV DNA load. C666-1 cells were transfected with gRNA-OriPCas9 and either gRNA-W repeats-Cas9 or gRNA-EBNA1-Cas9 sequentially with an interval of 3 days. The difference between the gRNA-OriP and gRNA-OriP + gRNA-EBNA1 groups was statistically significant by Student’s t test (p = 0.048).

Fig.4. CRISPR/Cas9 editing of EBV DNA sensitizes C666-1 cells to chemotherapeutic killing. C666-1 cells were either mock transfected or transfected with gRNA-OriP-Cas9 co-expression plasmids. Cell were incubated for 4 weeks to allow the suppression of EBV DNA load. Four weeks after transfection, cells were treated with 0.1 mM of cisplatin for 4 h or 1 µM of 5fluorouracil (5-FU) for 24 h. Cell viability was measured by XTT assay. The differences between mock-transfected and gRNA-OriP-Cas9-transfected groups treated with cisplatin (p = 0.018) or 5-fluorouracil (p = 0.0014) were statistically significant by Student’s t test.

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