Original Research Article

Targeted editing of avian herpesvirus vaccine vector using CRISPR/Cas9 nuclease

Prof. Venugopal Nair OBE,
Yongxiu Yao1,2, Andrew Bassett3 and Venugopal Nair1,2# 

1Viral Oncogenesis Group & 2UK-China Centre of Excellence on Avian Disease Research, The Pirbright Institute, Ash Road, Pirbright, Woking, Surrey, GU24 0NF, United Kingdom

3Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, CB10 1SA, United Kingdom

Herpesvirus of turkey (HVT) is an avian alphaherpesvirus used as live vaccine against Marek's disease for more than 40 years. Here, we describe the application of CRISPR/Cas9-based genome editing as a rapid and efficient method of introducing targeted mutations to the genome of a live herpesvirus vaccine vector using a transfection-infection system.

*Corresponding author:

Venugopal Nair
Tel: +44 (0)1483 231415
E-mail: venugopal.nair@pirbright.ac.uk 


Herpesvirus of turkey, vaccine, CRISPR/Cas9, targeted editing

Herpesvirus of turkey (HVT) or Meleagrid herpesvirus 1 is a naturally occurring, non-pathogenic alphaherpesvirus that was originally isolated from domestic turkeys in the late1960s (Kawamura et al., 1969; Witter et al., 1970). As a member of the genus Mardivirus (Fauqet, 2005), HVT is widely used as a live vaccine against Marek’s disease (MD) because of its genomic relatedness to Marek’s disease virus (MDV) (Afonso et al., 2001; Baigent et al., 2006). Despite the introduction of new generations of MD vaccine strains, such as SB-1 (Schat & Calnek, 1978) and CVI988 (Rispens et al., 1972) for better protection against increasing virulence of MDV strains, HVT-based vaccines are still being used widely (Gimeno et al., 2016), particularly in combination with other strains to exploit the synergistic protective effects (Witter & Lee, 1984). HVT is also used extensively as a vector platform for generating recombinant vaccines against a number of avian diseases. Recombinant HVTs expressing protective genes of avian pathogens such as avian influenza virus, Newcastle disease virus, infectious bursal disease virus, infectious laryngotracheitis virus, avian leukosis virus and Eimeria have been developed (Bublot, 2004; Cronenberg et al., 1999; Li et al., 2011; Reddy et al., 1996; Tsukamoto et al., 2002). Recombinant HVT vaccine candidates are generated usually by homologous recombination in virus-infected cells or through recombineering techniques on full-length genomes cloned in bacterial artificial chromosome (BAC) (Baigent et al., 2006). However, these methods are generally laborious often needing drug selection cassettes that need to be removed through additional steps such as the Cre-loxP or Flp-FRT site specific recombinase systems (Zhao, 2010).
Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nuclease-mediated genome editing has been used for manipulating genomes of several large DNA viruses, including herpes simplex virus type I, adenovirus, pseudorabies virus, vaccinia virus, Epstein-Barr virus and guinea pig cytomegalovirus (Bi et al., 2014; Bierle et al., 2016; Suenaga et al., 2014; Xu et al., 2015; Yuan et al., 2015; Yuen et al., 2015). Here we report the development of a simple transfection-infection methodology that utilizes CRISPR/Cas9-mediated genome editing to introduce targeted mutations of the HVT genome. By using drug selection to restrict viral replication in cells that express Cas9 and a virus-specific guide RNA (gRNA), we observed highly efficient generation and recovery of viral mutants. Transfection of a single virus-specific gRNA can engineer short deletion at the predicted Cas9 cleavage site. These results demonstrate that CRISPR/Cas9-mediated gene editing is an alternative to traditional recombination and BAC recombineering techniques for the genetic manipulation of HVT, and represent the first application of this technology in avian herpesvirus mutagenesis.
In order to determine whether the CRISPR-Cas9 system could be used to edit HVT genomes, first  attempt was made to edit the heterologous gene encoding green fluorescent protein (GFP) in the recombinant HVT (GFP HVT) to exclude the effects of viral gene inactivation on viral replication. In this construct, the GFP gene is cloned into the downstream of Thymidine kinase (TK) promoter in the US2 locus (Fig. 1) of the BAC clone of HVT (Baigent et al., 2006). One of the published gRNA sequences targeting the coding sequence of GFP gene (GFP-gRNA) (Shalem et al., 2014) was cloned into pX459-v2 vector (Addgene plasmid # 62988) that expresses the codon-optimized S. pyogenes Cas9 (Cas9) as a bicistronic mRNA with the puromycin-N-acetyltransferase gene and the gRNA sequence driven by the U6 promoter, by introducing synthesized oligo-DNA primers corresponding to the target sequence into BbsI restriction sites. The primer sequences used for guide RNA cloning are listed in Table 1. The pX459-v2 vector without any gRNA sequences was used as control to investigate the effect of Cas9 expression on replication in the absence of targeted Cas9 cleavage of the HVT genome. 
2.0μg of either GFP-gRNA or control construct was transfected into DF-1 cells using Lipofectamine® 2000 Transfection Reagent (Invitrogen). To maximize the chance of viral genome being edited, the transfected DF-1 cells were selected with puromycin (1µg/ml) at 24 hours post transfection (hpt). After 72 hours of drug treatment (96 hpt), the cells were infected with GFP HVT virus at an MOI of 0.01. DNA was extracted by incubation the infected cells in 1X squishing buffer (1 mM EDTA, 25 mM NaCl, 200ug/ml Proteinase K and 10 mM Tris-HCl, pH 8) at 65ºC for 30 minutes followed by inactivation of the proteinase K at 95C for 2 minutes when extensive cytopathic effects were visible. GFP expression continued to be high in the control transfected cells. High resolution melting analysis (HRMA) was carried out on DNA samples to assay for evidence of Cas9 cleavage and repair by non-homologous end joining (NHEJ) pathways. HRMA is a post-PCR analysis method used to identify variation in nucleic acid sequences based on detecting small differences in melting (dissociation) temperatures. Indel mutations result in a change in the melting temperature of PCR products spanning the target site which can be detected by analysis of the loss of fluorescent signal with increasing temperature.  The primers used in HRMA were designed to generate amplicons of ~200 bp traversing across the CRISPR target site using primerBLAST. The complete list of HRMA primers used is presented in Table 2. HRM results were generated using MeltDoctor HRM Master Mix on a 7500 Fast Real-Time PCR Instrument and analyzed using Applied Biosystems™ HRM Software v3.0 according to the manufacture’s instruction. The HRM run includes 40 cycles of  amplification (cycling conditions: Denature 95 °C 15 seconds; Anneal/extend 60 °C 1 minute) and melting of the DNA (Denature 95 °C 10 seconds, Anneal 60 °C 1 minute, High resolution melting 95 °C 15 seconds, Anneal 60 °C 15 seconds) to generate HRM fluorescence data. As shown by the melt curves, the genotypes were clearly distinguishable in the aligned HRM profile between wild type sequence (GFP-WT) and mutant (GFP-Mu) sequences (Fig. 2a).  The efficiency of GFP editing was extremely high as the GFP positive cells disappeared at the second passage (Fig. 2b). Following HRM, the genotype of each sample was identified by sequencing. As shown in the sequence chromatogram, a single nucleotide T was deleted from the predominant allele compared to the corresponding region of the wild type sequence (Fig. 2c top panel) that led to the premature stop codon inactivating the GFP expression (data not shown). These were in agreement with the results obtained using Tracking of Indels by Decomposition (TIDE) (http://tide-calculator.nki.nl/) programme (Brinkman et al., 2014), a web tool for the rapid assessment of CRISPR-Cas9-based genome editing of a target locus (Fig. 2c bottom panel). The overall editing efficiency quantified by TIDE was 98.9% and the predominant allele (70%) contained a single nucleotide deletion. Low frequency mutations of 3- and 4-nucleotide deletions were also observed. Thus CRISPR/Cas9 nuclease was very efficient in cleavage and repair of GFP encoded within the HVT genome at the predicted cleavage site. However, the clonal effects where the single nucleotide mutated virus may serve as a template for homology-directed repair of the other double strand breaks (DSBs) cannot be ruled out. 
Having demonstrated that the heterologous GFP gene cloned into the HVT genome could be efficiently edited by CRISPR/Cas9 system, we examined the application on to the virus-encoded genes. Glycoprotein B (gB) is an essential gene for growth of all herpesviruses (Pereira, 1994), including HVT. Two gRNAs, gB-gRNA-8 and gB-gRNA-18 targeting the coding sequence of the gB gene were designed (Fig. 1) using CRISPR guide RNA designing software (http://crispr.mit.edu/) and cloned into the CRISPR/Cas9 vector pX459-v2 and the resulting vector DNA was transfected into DF-1 cells. Again, the transfected cells were selected with puromycin for 3 days before infection with GFP HVT virus. The indel mutations were detected from both guide RNA transfections by HRMA (Fig. 2d). Compared to the control infected cells that showed extensive cytopathic changes of virus infection by GFP positive cells, the numbers of GFP-positive cells from the gB-gRNA-8 transfected cells decreased gradually and only single GFP-positive cells remained at second passage, and disappeared totally at the third passage (data not shown). Nevertheless, virus replication was not affected in the cells transfected with the gB-gRNA-18 as was evident from the appearance of GFP-positive virus plaques (Fig. 2e). We carried out sequencing of the PCR products surrounding the target site of both gB-gRNAs using the same primer used in HRMA. Poly Peak Parser which is able to separate chromatogram data containing ambiguous base calls into wild-type and mutant allele sequences, was used to identify specific indels. Consistent with the HRMA results, mutations in the gB gene were detected with both guide RNA transfections but not with the control (Fig. 2f). DNA sequence from cells with decreased virus and GFP positive cells production (gB-Mu-8 mutated virus) showed one nucleotide deletion which leads to frameshift and premature stop codon. On the other hand, sequence from cells infected with gB-Mu-18 mutated virus showed in-frame mutations (one amino acid deletion) in gB. Consistently, TIDE analysis showed that the single-nucleotide deleted population (34.5% of sequences) was the predominant mutant apart from the wild type (40.7% of sequences) in the gB-gRNA-8–transfected cells. It confirmed that the one nucleotide deletion which led to frameshift that introduced a premature stop codon in the essential gB allele could have accounted for the decreased virus production and GFP-positive cells. On the other hand, sequence from gB-gRNA-18 transfected cells that showed continuous virus growth carried an in-frame mutation with one amino acid deletion in gB as the predominant allele (60.2% of sequences) by TIDE analyses (Fig. 2f). The reasons for the clonal nature of the appearance of this single population are not fully clear. However, the possibility that this mutation generated early was used as template for subsequent repair leading to this predominant clonal line cannot be discounted. Nevertheless, these results further confirmed the importance of gB for HVT growth, and that the deletion of the single amino acid in gB did not affect its function. 
We also examined editing of two other HVT genes, glycoprotein E (gE) and I (gI). These two glycoproteins that form a disulphide-linked heterodimer are nonessential for the growth of a number of herpesviruses such as HSV-1, PRV, bovine herpesvirus 1 (BHV-1), and feline herpesvirus (Balan et al., 1994; Mijnes et al., 1996; Yoshitake et al., 1997; Zuckermann et al., 1988), while they are essential for MDV-1 in cell culture (Schumacher et al., 2001). Using mutagenesis tools on the BAC clone, we have shown that both gE and gI are nonessential for HVT growth (unpublished), and wanted to confirm this further using CRISPR/Cas9-mediated genome editing tools. The gRNAs targeting the coding sequence of gI (gI-gRNA-4) and gE (gE-gRNA-5) were designed (Fig. 1). DF-1 cells transfected with the vector DNA carrying the CRISPR/Cas9 cassette were selected with puromycin and infected with GFP HVT virus. Mutations were detected from both transfection/infections by HRMA (Fig. 2g) and all the mutant viruses grew well despite the mutations introduced (Fig. 2h). When the nucleotide sequence chromatogram data of the PCR products from both transfection/infections were analysed by TIDE, both of them showed high editing efficiencies of 91.2% and 95.4% respectively (Fig. 2i). For gI-gRNA-4 transfection, 28.1% of sequences with 1-nucleotide deletions and 21.4% of sequences with 3-nucleotide deletions were the predominant populations although other types with 2- (4.9%), 4- (10%), 5- (11.8%), and 6- (13.1%) nucleotide deletions existed at low frequencies. For gE-gRNA-5 transfection, 42.8% of sequences with 6-nucleotide deletions and 45.4% of sequences with 3-nucleotide deletions were the predominant populations, and only 4.2% of sequences with one-nucleotide deletion were present. For more accurate assessment of the efficiency of Cas9 cleavage and repair and to characterize the actual sequence of the variants, gI-Mu-4 and gE-Mu-5 mutated virus population were further analysed by plaque purification. Eleven potential gI-edited plaques and twenty-three gE-edited plaques were picked and the regions corresponding to the HRMA were amplified by PCR and sequence determined.  All of the plaques analysed showed the mutations demonstrating the efficiency of the CRISPR/Cas9-mediated editing of herpesvirus genomes. A single C to A silent mutation was present in all 11 gI-edited plaques (Fig. 2i. top). For the 23 gE-edited plaques analysed, sequence of 3 nucleotides AAC was missing in 13 clones which is consistent with the TIDE analysed result showing 45.4% of the most predominant sequences with 3-nucleotide deletions, with another 8 clones showing an additional T to C substitution, suggesting that these are the dominant mutations. Two other types of mutations including one single nucleotide substitution and 6 nucleotide deletion were found in one plaque only (Fig. 2i bottom). Regardless of the mixed nature of the mutations analysed by TIDE especially for the gI-Mu-4 edited virus, all of the isolated viruses had silent or in-frame mutations, perhaps suggesting the existence of selection pressure to maintain these ORFs in spite of the non-essential nature of these genes as shown by BAC mutagenesis strategies (unpublished). Thus, our experiments demonstrated the efficiency of CRISPR/Cas9-cleavage and repair of the HVT genome to generate gene-disrupting indel mutations.
Taken together, we report the use of CRISPR/Cas9-mediated gene editing to introduce targeted mutations into the genome of the live attenuated HVT vaccine vector. CRISPR/Cas9 gene editing has recently been employed to manipulate the genomes of a number of DNA viruses, and this study represents the first application of the technology to an avian herpesvirus vaccine strain. Using a simple transfection/infection methodology, we demonstrated that CRISPR/Cas9-based genome editing can efficiently be used to generate HVT mutants. Detection of several single GFP-positive cells indicating a defect in the cell-to-cell spread of viruses, accompanied by significant frameshift mutations leading pre-mature stop codon confirmed the essential nature of gB gene in HVT, as in other herpesviruses (Pereira, 1994). This was further evident from the predominant population of single amino acid deleted in-frame mutant seen with the gB-gRNA-18 editing, that is thought not to affect gB function. All isolated gE and gI-edited viruses had only silent or in-frame mutations. Although we have shown that gE and gI are nonessential for HVT replication (unpublished), we would speculate that there is still potential selective advantage for these viruses with fully intact genes, as seen in Pseudorabies virus (Peng et al., 2016). 
In this study, we have shown that HVT genome can be easily edited using the CRISPR/Cas9 nuclease. Although we only generated indel mutations in this study, the CRISPR/Cas9 system also allows rapid generation of knock-in HVT mutants as demonstrated with other viruses (Bi et al., 2014; Xu et al., 2015). As HVT is widely used as a vaccine vector for expression of heterologous antigens, engineering HVT genome using the CRISPR/Cas9 nuclease for the development of new, highly immunogenic, multivalent vectored vaccines can be furthered explored.
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Figure-1Fig. 1 Schematic diagram showing the positions of the gRNAs in the HVT genome
The internal and terminal repeat long (TRL/IRL) and short (TRS/IRS) regions flanking the unique long (UL) and short (US) regions of the genome are shown. Genomic positions and orientations of GFP and the gB, gE and gI genes are shown. The predicted cleavage sites are indicated () at the top of the gene for the forward target sequences.
Figure-1 Figure-1 Figure-1 Fig. 2 Disruption of GFP, gB, gI and gE genes of GFP HVT by CRISPR/Cas9 nuclease in DF-1 cells
HRMA (a, d and g) was performed on genomic DNA samples from infected DF-1cells (b, e and h) followed by DNA sequencing (c, f and i); (a) High Resolution Melt Curves of GFP wild type (black, GFP-WT) and GFP mutant (red, GFP-Mu); (b) Infected cells showing no GFP positive cells present in GFP-Mu virus infected cells whereas many GFP positive cells present in the GFP-WT infection, although both are heavily infected; (c) Top panel: Nucleotide sequence chromatogram showing a single nucleotide T present in wild type GFP sequence (top row) but not mutant GFP sequence (bottom row) indicated by blue arrow. Target sequence is underlined and cleavage site is indicated by a black arrow. Bottom panel: Indel spectrum of GFP mutant virus in DF-1 cells determined by TIDE analysis; (d) High Resolution Melt Curves of gB wild type (black, gB-WT), gB mutant 8 (red, gB-Mu-8) and gB mutant 18 (blue, gB-Mu-18); (e) Infected cells showing single GFP positive cells present in gB-Mu-8 infected cells and both gB-WT and gB-Mu-18 are heavily infected; (f) The nucleic acid sequences of the targeting sites on HVT gB gene and the mutated gene sequences induced by gB-targeting Cas9/gRNA and indel spectrum determined by TIDE. Nucleic acid numbers for the gB gene are indicated in the figure. Target sequence is underlined, PAM sequence is in blue and cleavage site is indicated by an arrow. Deleted nucleotides are in red font. (g) High Resolution Melt Curves of gI wild type (black, gI-WT) and gI mutant 2 (red, gI-Mu-2) also gE wild type (black, gE-WT) and gE mutant 5 (red, gE-Mu-5); (h) DF-1 cells infected by gI-Mu-4 (top), WT (middle) and gE-Mu-5 (bottom); (i) The nucleic acid sequences of the targeting sites on HVT gI and gE genes and the mutated gene sequences of isolated mutant viruses induced by gI/gE-targeting Cas9/gRNA and indel spectrum determined by TIDE. Nucleic acid numbers for the gI/gE gene are indicated in the figure. Target sequence is underlined, PAM sequence is in blue and cleavage site is indicated by an arrow. Deleted nucleotides are in red font with frequencies of the mutants shown in brackets. 

Published: 15 November 2016

Reviewed By : Dr. Wei Gang Hu.Dr. Ken Draper.Dr. Carlos Augusto Pereira.

Copyright: Copyright: © 2016 Venugopal Nair. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.