key: cord-0882868-fkj0fm5v
authors: Peng, Qi; Fang, Liurong; Ding, Zhen; Wang, Dang; Peng, Guiqing; Xiao, Shaobo
title: Rapid manipulation of the porcine epidemic diarrhea virus genome by CRISPR/Cas9 technology
date: 2019-11-08
journal: J Virol Methods
DOI: 10.1016/j.jviromet.2019.113772
sha: 661dbf3aaf3b32f2f8514e5c51d6bc4999667643
doc_id: 882868
cord_uid: fkj0fm5v

Porcine epidemic diarrhea virus (PEDV) is a highly pathogenic enteric coronavirus causing lethal watery diarrhea in suckling piglets. Reverse genetics is a valuable tool to study the functions of viral genes and to generate vaccine candidates. In this study, a full-length infectious cDNA clone of the highly virulent PEDV strain AJ1102 was assembled in a bacterial artificial chromosome (BAC). The rescued virus (rAJ1102) exhibited similar proliferation characteristics in vitro to the wildtype AJ1102. Using CRISPR/Cas9 technology, a recombinant virus rAJ1102-ΔORF3-EGFP in which the ORF3 gene was replaced with an EGFP gene, was successfully generated, and its proliferation characteristics were compared with the parental rAJ1102. Importantly, it just took one week to construct the recombinant PEDV rAJ1102-ΔORF3-EGFP using this method, providing a more efficient platform for PEDV genome manipulation, which could also be applied to other RNA viruses.

Porcine epidemic diarrhea (PED) is a highly contagious acute diarrheal disease characterized by watery diarrhea, vomiting and dehydration, with an 80%-100% mortality rate in neonatal piglets (Jung and Saif, 2015) . PED was first recognized in the United Kingdom and Belgium in the 1970s (Wood, 1977) , and sporadically or endemically appeared in Europe and Asia before 2010 (Van Reeth and Pensaert, 1994) . In late 2010, a large-scale outbreak of PED, which was caused by a highly virulent PED virus (PEDV) variant (GII genogroup), occurred in China (Li et al., 2012; Sun et al., 2016) . In April 2013, the Chinese-like PEDV variant also emerged in the United States and rapidly spread across the country (Stevenson et al., 2013; Vlasova et al., 2014) . Currently, PEDV has been reported in many countries and causes high economic losses (Jung and Saif, 2015; Lee, 2015; Song et al., 2015; Wang et al., 2019; Zhang and Yoo, 2016) .

PEDV is an enveloped, single-stranded positive-sense RNA virus, belonging to the genus Alphacoronavirus within the Coronaviridae family in the order Nidovirales (Song and Park, 2012) . The complete genome of PEDV is approximately 28 kb, encoding at least seven open reading frames (ORFs). The two large ORFs, 1a and 1b, occupy almost two-thirds of the genome, encoding two large replicase proteins, pp1a and pp1b, which are post-translationally cleaved into 16 nonstructural proteins (nsps), nsp1-nsp16. The remaining ORFs, ORF2-6, encode spike (S) protein, envelop (E) protein, membrane (M) protein, nucleocapsid (N) protein, and one accessory protein, ORF3 (Duarte et al., 1993) .

Reverse genetics systems are valuable tools to study the functions of viral genes and to generate recombinant viruses with defined genetic changes as vaccine candidates. In 2013, Li et al. first reported a reverse genetics system for the Korean classical PEDV vaccine strain DR13 based on a targeted RNA recombination method (Li et al., 2013) . Following this, Jengarn et al. engineered an infectious cDNA clone of the Thailand classical PEDV strain AVCT12 into a bacterial artificial chromosome (BAC) using eight contiguous cDNA fragments (Jengarn et al., 2015) . In 2016, Beall et al. constructed infectious cDNA clones of a highly pathogenic US PEDV strain PC22A using in vitro ligation of contiguous cDNA fragments, and performed in vitro transcription to generate infectious viral RNA (Beall et al., 2016) . Using a similar strategy, Fan et al. developed an infectious cDNA clone for a Chinese PEDV variant strain, AH2012/12 (Fan et al., 2017) . Li et al. developed a reverse genetics system for two Chinese PEDV strains with differing virulence by ligation of cDNA fragments into BACs one by one . Using established reverse genetics systems, the functions of https://doi.org/10.1016/j.jviromet.2019.113772 Received 12 March 2019; Received in revised form 6 November 2019; Accepted 7 November 2019 some PEDV proteins, such as S protein and ORF3, in modulating PEDV pathogenicity have been examined (Beall et al., 2016; Hou et al., 2017 Hou et al., , 2019 Kaewborisuth et al., 2018; Wang et al., 2018) . Several recombinant PEDV vaccine candidates have also been generated using reverse genetics systems (Hou et al., 2019; Kao et al., 2018; Wang et al., 2018) . Although infectious clone systems for PEDV using various strategies (Teeravechyan et al., 2016) have become established, approaches to generate a new mutant PEDVs with defined genetic changes using infectious clones remains a tedious process, usually requiring constructing and ligating a set of contiguous cDNA fragments. A simple and rapid method for manipulation of the full-length infectious clone is desirable.

In this study, a full-length infectious clone of PEDV strain AJ1102 was generated and a simple method to construct recombinant PEDV was developed based on the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology, providing a more efficient platform for PEDV genome manipulation.

Vero cells (ATCC CCL-81) were maintained in Dulbecco's Modified Eagle Medium (DMEM) (Gibco) containing 10% fetal bovine serum (FBS) in a 37°C, 5% CO 2 humidified atmosphere. PEDV strain AJ1102, a highly virulent PEDV variant isolated from a neonatal piglet with acute diarrhea in China in 2011 (Bi et al., 2012) , was propagated in Vero cells supplemented with trypsin (10 μg/mL). The mAb against PEDV N protein was produced at Huazhong Agricultural University as described previously (Ding et al., 2014) .

A low-copy number BAC vector (pBeloBAC11) was used to construct the infectious cDNA clone of AJ1102 (F12), the 12th passage of AJ1102 strain. The pBeloBAC11 was modified to incorporate the CMV promoter, PEDV 5′ UTR, N-terminal of ORF1a (the first 800 nucleotides; nts), restriction enzyme site of PacI, C-terminal end of the N gene (nt 26,888-27,701) , PEDV 3′ UTR, a 28-residue poly(A) tail, hepatitis delta virus (HDV) ribozyme self-cleavage site and bovine growth hormone (BGH) termination sequences (Fig. 1A) , generating an intermediate BAC plasmid, pBAC-M-PEDV. Total viral RNA was extracted from Vero cells infected with PEDV strain AJ1102 (F12) by using a TaKaRa MiniBEST Viral RNA/DNA Extraction Kit (TaKaRa). The cDNA was synthesized from genomic RNA using SuperScript IV Reverse Transcriptase (Invitrogen) in accordance with the manufacturer's instructions. Five overlapping DNA fragments (A: nt 1, 050-7,173; B: nt 7,151-13,036; C: nt 12,975-17,617; D: nt 17,598-22,217; E: 22,199-26,929) were amplified with SuperFi™ Green PCR Master Mix (Invitrogen) in accordance with the manufacturer's instructions, and then assembled into the modified BAC vector pBAC-M-PEDV. First, fragments A and B were ligated into pBAC-M-PEDV by homologous recombination with an Infusion Clone Kit (TaKaRa), resulting in pBAC-AB. Using the same method, fragments C, D, and E were then ligated into pBAC-M-PEDV, resulting in the plasmid pBAC-CDE. Finally, plasmids pBAC-AB and pBAC-CDE were double digested with PacI and SacII, then ligated together with T4 DNA ligase (ThermoFisher Scientific) to create the construct pBAC-AJ1102. The primers used to construct the full-length cDNA clone are listed in Table 1 .

Confluent Vero cells in six-well culture plates were transfected with BAC plasmids harboring the complete or edited genome (6 μg/well) using Lipofectamine® 3000 (Invitrogen). At 6 h post-transfection, the cells were rinsed twice with DMEM and supplemented with 2 mL of DMEM containing 10 μg/mL of trypsin (Sigma), then placed in a 37°C, 5% CO 2 incubator to facilitate the recovery of infectious virus. Cells were observed daily for the appearance of CPE.

The sgRNA templates were amplified with forward primers sgRNA-ΔORF3a/b and reverse primer scaffold oligo using SuperFi™ Green PCR Master Mix (Invitrogen) at 98°C for 3 min and 34 cycles of 98°C 30 s, 55°C 30 s, 72°C 30 s, followed by a final extension at 72°C for 5 min. The amplification products were then purified using a DNA extraction kit (Omega Bio-tek) and transcribed at 37°C overnight with a T7 transcription kit (NEB) according the manufacturer's instructions. The sgRNAs were phenol chloroform extracted and eluted in RNase free water.

The specific cleavage reaction was conducted in a 50 μL-mixture containing 5 μg pBAC-AJ1102, 5 μL of Cas9 nuclease (NEB), 20 μg sgRNAs (10 μg for each sgRNA) and 5 μL of 10 × NEB Cuterbuffer 3.1 at 37°C for 2.5 h. The cleaved pBAC-AJ1102 was purified with a DNA Cycle Pure Kit (Omega Bio-tek) and verified by electrophoresis in 0.8% agarose gel. The recombinant pBAC-AJ1102-ΔORF3-EGFP was constructed by homologous recombination using an Infusion Clone Kit (Clontech) in a mixture containing the cleaved pBAC-AJ1102 and a DNA fragment with an EGFP gene, E gene, partial sequences of S gene and M gene, and two 20 bp homologous arms.

Vero cells seeded in 12-well plates were infected with wtAJ1102 and rAJ1102 for 24 h. The cells were fixed with 4% paraformaldehyde for 15 min and then permeabilized with cold methanol for 10 min at room temperature, followed by washing three times with PBS, and blocked with 5% bovine serum albumin for 1 h. Afterwards, the cells were washed thrice with PBS and then incubated with mAb against PEDV N protein at room temperature for 1 h. After three washes, the cells were stained with fluorescence isothiocyanate (FITC)-conjugated goat anti-mouse IgG. Cell nuclei were stained with 0.01% 4′,6-diamidino-2-phenylindole (DAPI) for 15 min at room temperature. After three washes with PBS, fluorescent images were visualized with a fluorescence microscope (Nikon).

Vero cells seeded in six-well plates were infected or mock infected with viruses for 24 h and harvested with lysis buffer (4% SDS, 3% DTT, 0.065 mM Tris-HCl, [pH 6.8], 30% glycerine) supplemented with protease inhibitor (PMSF, Beyotime). The cell lysates were boiled at 100°C for 10 min before separation by SDS-PAGE and electroblotting onto polyvinylidene difluoride membranes (Millipore). The membranes were first blocked with 10% skimmed milk and then inoculated with the corresponding primary antibodies, followed by horseradish peroxidaseconjugated secondary antibody, and visualized using chemiluminescent substrate (Bio-Rad).

Monolayers of Vero cells in 12-well plates were incubated with 500 μL of 10-fold serially diluted parental or rescued PEDV for 1 h at 37°C with periodic gentle rocking. Following this, the cells were washed thrice with DMEM to remove unabsorbed viruses, then overlaid with 1 mL of DMEM containing 1.5% methylcellulose and 10 μg/mL 

Primer sequences used in this study. Peng, et al. Journal of Virological Methods 276 (2020) 113772 trypsin. After incubation at 37°C for 48 h, the cells were then fixed with 4% paraformaldehyde and stained with 0.1% crystal violet.

Vero cells in 12-well plates were inoculated with parental or recombinant PEDV at a multiplicity of infection (MOI) of 0.1 in triplicate wells. One hundred microliters of virus mixture was added to each well. After 1 h inoculation at 37°C for virus attachment, the inocula were aspirated and cells were washed once with DMEM, then 1 mL of DMEM medium containing 10 μg/mL trypsin was pipetted into each well. Cell supernatants and infected cells were collected at various time-points for virus titration by TCID 50 assay.

To facilitate assembly of the complete cDNA, an intermediate BAC plasmid, termed pBAC-M-PEDV, was constructed as the backbone to harbor the complete genome of PEDV strain AJ1102 (F12). Then, five overlapping DNA fragments (A-E) were amplified and assembled into pBAC-M-PEDV (Fig. 1A) . The resulting plasmid, pBAC-AJ1102, was digested with KpnI for restriction fragment length polymorphism analysis (RLFP) (Fig. 1B) and re-sequenced. The results showed the fulllength cDNA clone of PEDV AJ1102 (F12) was successfully constructed, and there was no deletion or insertion compared with the original PEDV strain AJ1102 (F12) sequence (GenBank accession number MK584552) (data not shown).

After transfection of pBAC-AJ1102 into Vero cells, evident CPE, characterized by cell fusion and syncytium formation was observed at 24 h post-transfection. The rescued rAJ1102 was confirmed by indirect immunofluorescence assay (IFA) using mouse anti-PEDV N protein monoclonal antibody (mAb) (Fig. 2A) . The growth curves of the rescued rAJ1102 and the parental PEDV AJ1102 (F12) were further compared. As shown in Fig. 2B , rAJ1102 demonstrated similar proliferation properties to the parental PEDV strain, the titers of which peaked at 36 h post-infection (hpi). Plaque assay was also conducted and the results showed that there is no significant difference in plaque morphology between rAJ1102-infected cells and cells infected with the parental PEDV AJ1102 (F12) (Fig. 2C ).

To prove the idea that CRISPR/Cas9 technology is a simple tool that can be used to edit the genomic cDNA of PEDV in BAC plasmids, the ORF3 gene was chosen as a target because previous studies have demonstrated that the ORF3 gene is nonessential for PEDV replication and could be replaced with other foreign genes (Beall et al., 2016 ). An enhanced green fluorescent protein (EGFP) gene was designed to replace the ORF3 gene. To this end, two sgRNAs (sgRNA-ΔORF3a and sgRNA-ΔORF3b) targeting the upstream and downstream sequences of the ORF3 gene, respectively, were synthesized. Each sgRNA contained a cleavage site which was located at three nucleotides upstream of the protospacer adjacent motif (PAM) (Fig. 3A) . The sgRNAs were generated by T7 in vitro transcription of templates amplified with primers of sgRNA-ORF3a/b and scaffold oligo (Table 1) by overlapping PCR (Fig. 3B) . To cleave the BAC plasmid pBAC-AJ1102, a mixture containing S. pyogenes Cas9 protein (1 μM), sgRNA-ΔORF3a and sgRNA-ΔORF3b (300 nM), pBAC-AJ1102 (5 μg) and the Cas9 nuclease buffer (1×) was incubated at 37°C for 2.5 h. As shown in Fig. 3C , pBAC-AJ1102 was cleaved at the designed sites, producing a linearized BAC (34.1 kb) and a 2.2 kb fragment containing ORF3 gene, E gene and partial sequences of S gene and M gene. At the same time, a 2.3 kb fragment containing the cDNA of EGFP, E gene, partial sequences of S gene and M gene, and a 20 bp homologous arm at both terminals (homologous arm 5'-AGAGGCTATTAGTCAAACTT-3' at 5' terminal, homologous arm 5'-AACTGGGTCTTTTTTGCTTT-3' at 3' terminal), was generated by fusion PCR with primers listed in Table 1 . Then the linearized BAC fragment was purified and ligated with the 2.3 kb fragment by homologous recombination (Fig. 3A) . After transformation of DH10B competent cells, the recombinant BAC plasmid, pBAC-AJ1102-ΔORF3-EGFP, was obtained. Sequence analysis confirmed that the ORF3 gene was successfully replaced by the EGFP gene (Fig. 3D ). To evaluate the success rate and reproducibility of this method, three independent experiments were performed. For each experiment, ca. 20 clones were picked. The percentage of positive clones in three independent experiments was 47% (8/17), 38% (8/21), and 47.8% (11/ 23), respectively, and the mean positive rate was 44%, demonstrating that this method is an efficient technology for manipulation of the fulllength infectious clone of PEDV genome.

The plasmid, pBAC-AJ1102-ΔORF3-EGFP, was transfected into Vero cells and evident CPE characterized by syncytium formation could be observed at 24 h after transfection (Fig. 4A) . Furthermore, EGFP was expressed in cells transfected with pBAC-AJ1102-ΔORF3-EGFP, while not in mock-transfected cells (Fig. 4A) . The rescued virus, termed rAJ1102-ΔORF3-EGFP, was passaged in Vero cells and the 3 rd passage virus was inoculated onto cell monolayers. Nearly all cells were infected and fluorescence could be observed (Fig. 4B) . To further identify the recombinant virus rAJ1102-ΔORF3-EGFP, Vero cells were infected with the parental PEDV AJ1102, the rescued virus rAJ1102 or rAJ1102-ΔORF3-EGFP. At 24 h after infection, cells were collected for Western blot with antibodies against PEDV N protein and EGFP. As shown in Fig. 4C , all three viruses expressed N protein, while only rAJ1102-ΔORF3-EGFP-infected cells expressed EGFP, which further confirmed that EGFP was successfully inserted into the PEDV genome.

To further characterize the proliferation properties of rAJ1102-ΔORF3-EGFP, the growth curve of rAJ1102-ΔORF3-EGFP was determined in Vero cells. As shown in Fig. 4D , rAJ1102-ΔORF3-EGFP reached a peak titer (TCID 50 = 10 -6.7 /mL) at 36 hpi. Compared with the growth curve of rAJ1102 (Fig. 4D) , rAJ1102-ΔORF3-EGFP attained similar titers in Vero cells, consistent with a previous study that demonstrated that ORF3 is not essential for PEDV replication in vitro (Jengarn et al., 2015) .

Coronaviruses are single-stranded positive-sense RNA viruses, and contain the largest genome among the RNA viruses, making them difficult to be manipulated directly. In this study, an infectious clone of PEDV strain AJ1102 was constructed based on the BAC system, and a strategy to edit the genome in an in vitro plasmid system was developed by using CRISPR/Cas9 technology. The results clearly showed this is a simple and rapid method to manipulate the full-length cDNA clone of PEDV, providing a more efficient platform for basic studies and vaccine development for PEDV.

CRISPR/Cas9 technology has been applied to engineer the genomes of many organisms or large DNA plasmids with high efficiency and specificity (Jiang et al., 2015; Mali et al., 2013; Moradpour and Abdulah, 2019; Wang et al., 2015) . For example, Wang et al. used the Cas9/sgRNA complex to cut a 22 kb plasmid, and then a 783 bp DNA fragment was inserted into the linearized plasmid seamlessly through Gibson assembly to select 4 clones which were all positive (Wang et al., 2015) . Jiang et al. used RNA-guided Cas9 to cut bacterial chromosomes Q. Peng, et al. Journal of Virological Methods 276 (2020) 113772 and the cleaved target genome segment was ligated to a PCR amplified vector backbone through Gibson assembly (Jiang et al., 2015) . The cloning efficiency ranged from 21.5%-65.8% (21.5% for 100 kb and 65.8% for 50 kb fragment) (Jiang et al., 2015) . In the present study, CRISPR/Cas9 nuclease cleavage was combined with the in-fusion clone method to edit the viral genome in vitro using the infectious clone of PEDV as a model. Three independent experiments showed that the recombinant efficiency is 38%-47.8%. To the best of our knowledge, this is the first time this approach has been used to manipulate an infectious clone of RNA virus by CRISPR/Cas9 technology. Relative to previous strategies used to manipulate infectious clones of large RNA viruses by constructing and ligating a set of contiguous cDNA fragments, this method has demonstrable advantages in improving the quality and efficiency of constructing recombinant RNA viruses. First, two sgRNAs targeting the genes or regions of interest can be easily designed and generated using a T7 in vitro transcription kit. Second, cleavage of the target genes or regions can be completed within 3 h in a simple nuclease buffer system containing BAC plasmid, Cas9 nuclease, and sgRNAs. Third, the cleaved BAC plasmids can be easily ligated with a DNA fragment with a 20 bp homologous arm using an infusion clone kit to obtain the desired BAC plasmid containing the mutation, insertion or deletion of interest. In our experience, the whole process from sgRNA generation, BAC plasmid cleavage, homologous recombination, transformation, BAC plasmid extraction, transfection and final recovery of recombinant virus, could be completed within one week. Most importantly, a DNA template library with different mutations can be prepared to construct numerous recombinant PEDVs simultaneously. In other words, a high throughput method to construct recombinant viruses can be developed. This will dramatically reduce the time and cost for manipulation of the PEDV genome. Q. Peng, et al. Journal of Virological Methods 276 (2020) 113772 In addition to the obtained rAJ1102-ΔORF3-EGFP, the CRISPR/ Cas9 technique was used to construct another recombinant PEDV, in which a continuous 12-nt motif was successfully inserted into the Cterminal of the S gene of PEDV strain AJ1102 (F12) (data not shown), further demonstrating that CRISPR/Cas9 is a more efficient technology for manipulation of the PEDV genome. Furthermore, this strategy was also used to edit the full-length infectious clones of other RNA viruses with larger genomes, including transmissible gastroenteritis virus (TGEV), another swine enteric coronavirus, and porcine reproductive and respiratory syndrome virus (PRRSV), a member of the family Arteriviridae (unpublished data). Collectively, this study has established a more efficient platform for PEDV genome manipulation, and the method described in this study is also applicable to other RNA viruses.

Accessory proteins are genus-specific for coronaviruses and only a single accessory protein gene, the ORF3 gene, has been identified in the PEDV genome. PEDV ORF3 encodes an ion channel protein, but it is not essential for PEDV replication (Beall et al., 2016; Wang et al., 2012; Wongthida et al., 2017) . Thus, the ORF3 gene was chosen as a model to be replaced with an EGFP gene using the method described in this study. Consistent with previous studies, the rescued rAJ1102-ΔORF3-EGFP possessed similar proliferation properties in vitro compared with the wildtype AJ1102 virus. The insertion of reporter genes such as EGFP will be very useful for the study of various molecular and virological aspects of PEDV infection in vitro and in vivo. For example, rAJ1102-ΔORF3-EGFP can be exploited to establish convenient virus neutralization assays that provide answers within hours rather than days, and this reporter PEDV can also be used to investigate the transportation dynamics of PEDV in intestinal tract of piglets in vivo.

In summary, the CRISPR/Cas9 technology was successfully used to generate a recombinant reporter PEDV that demonstrates in vitro replication properties similar to the parental strain. Application of this technique can generate such recombinant viruses within a week, thus establishing a rapid and efficient platform for manipulation of not only the PEDV genome, but also of other RNA viruses.

Not applicable.

The authors declared no potential conflicts of interest.

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Complete genome sequence of porcine epidemic diarrhea virus strain AJ1102 isolated from a suckling piglet with acute diarrhea in China

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Cytopathic effect and fluorescence were visualized at 24 h post-infection (hpi). (C) Vero cells were infected with rAJ1102-ΔORF3-EGFP, rAJ1102 or parental AJ1102 (F12) (MOI = 0.1)

porcine epidemic diarrhea virus, China

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We thank Dr. Gang Wang for technical assistance. This work was supported by the National Key R&D Plan of China (2016YFD0500103), the National Natural Science Foundation of China (31730095; 31672569), and the Major S&T Project of Hubei Province (2017ABA138).