Immunology and Infection
Published: January 7th, 2019
Herpesvirus of turkeys (HVT) is widely used as a vector platform for the generation of recombinant vaccines against a number of avian diseases. This article describes a simple and rapid approach for the generation of recombinant HVT-vectored vaccines using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.
Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian influenza (AI), Newcastle disease (ND), and infectious bursal disease (IBD), using bacterial artificial chromosome (BAC) mutagenesis or conventional recombination methods. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system has been successfully used in many settings for gene editing, including the manipulation of several large DNA virus genomes. We have developed a rapid and efficient CRISPR/Cas9-mediated genome editing pipeline to generate recombinant HVT. To maximize the potential use of this method, we present here detailed information about the methodology of generating recombinant HVT expressing the VP2 protein of IBDV. The VP2 expression cassette is inserted into the HVT genome via an NHEJ (nonhomologous end-joining)-dependent repair pathway. A green fluorescence protein (GFP) expression cassette is first attached to the insert for easy visualization and then removed via the Cre-LoxP system. This approach offers an efficient way to introduce other viral antigens into the HVT genome for the rapid development of recombinant vaccines.
Marek's disease (MD) is a lymphoproliferative disease of chickens induced by serotype 1 (Gallid Herpesvirus 2 [GAHV-2]) of the genus Mardivirus in the subfamily of Alphaherpesvirinae. Mardivirus also includes two nonpathogenic serotypes: serotype 2 (GaHV-3) and serotype 3 (MeHV-1, historically known as HVT) which are used as vaccines against MD. Live HVT vaccine (FC-126 strain) is the first generation of MD vaccine used in the early 1970s and is still being used widely in bivalent and polyvalent vaccine formulations to provide an enhanced protection against MD. HVT is also widely used as a vaccine vector to induce the protection against a number of avian diseases due to its versatility and safety for both in ovo and subcutaneous hatchery administration and capability to provide a lifelong immunity. The strategy to generate recombinant HVT vaccines is based on either conventional homologous recombination in virus-infected cells, overlapping cosmid DNAs, or BAC mutagenesis2. However, these methods are generally time-consuming and labor-intensive, requiring the construction of transfer vectors, the maintenance of the viral genome in Escherichia coli, plaque purifications, and the removal of the BAC sequence and selection marker from the edited viruses3,4.
CRISPR/associated (Cas9) is the most popular gene editing tool in recent years due to its versatility and specificity. The CRISPR/Cas9 system has been successfully used in the efficient generation of genetically modified cells and animal models5,6,7,8,9,10, as well as in the manipulation of several large DNA virus genomes11,12,13,14,15,16,17,18,19,20. After reporting a simple and efficient method using the CRISPR/Cas9 system to edit the HVT genome21, we developed a pipeline for the rapid and efficient generation of recombinant HVT22.
In order to extend the potential application of this method, we describe the detailed methodology for the generation of recombinant HVT vaccine expressing the VP2 gene of IBDV at the UL45/46 locus in this report. The approach combines NHEJ-CRISPR/Cas9 to insert the VP2 gene tagged with GFP reporter gene and a Cre-LoxP system to remove the GFP expression cassette later. Compared to traditional recombination and BAC recombineering techniques, we demonstrate that NHEJ-CRISPR/Cas9 together with a Cre-Lox system is a rapid and efficient approach to generate recombinant HVT vaccine.
1. Preparation of Cas9/gRNA Expression and Donor Constructs
2. CRISPR/Cas9-mediated Knock-in: Transfection and Infection
3. Harvesting and Purification of the HVT Recombinant Virus
4. Excision of the Fluorescent Reporter Gene via the Cre-lox System
5. Plaque Purification
6. Verification of the Recombinant HVT
7. Stability of the Recombinant Virus
The strategy used for the generation of the recombinant HVT vaccine is outlined in Figure 1, which includes how the donor plasmid is constructed (Figure 1A) and procedures to generate the recombinant HVT (Figure 1B). Five to thirty GFP-positive plaques surrounded by wild-type plaques can be observed in gene knocking-in wells under the fluorescence microscope 3 d posttransfection and -infection. The purified virus obtained after single-cell sorting (Figure 2A) was analyzed by 3' junction PCR, which shows a PCR product of the expected size (Figure 2A, bottom panel). After the excision of the GFP reporter by Cre recombinase, over 50% of the plaques lost their GFP expression. The purified plaque after the GFP excision (Figure 2B) by single-cell sorting was further confirmed by 5' junction PCR, which shows the right-sized PCR product (Figure 2B, bottom panel). Figure 3 shows the sequencing results of both junction PCR products with different colored elements. In Figure 4, the protein expression was confirmed by IFA with VP2-specific monoclonal antibody and anti-HVT chicken serum. As expected, cells infected with the parental HVT can only be stained by anti-HVT serum (green), while recombinant HVT-infected cells clearly showed the expression of VP2 gene (red).
Figure 1: Strategy for the generation of a recombinant HVT-vectored vaccine. (A) This panel shows a schematic representation of the cloning strategy for donor plasmid construction. The key elements include two Cas9 target sites (sgA) for releasing insert, a reporter GFP cassette flanked with LoxP sequences for the excision of GFP, and the VP2 expression cassette. (B) This panel shows an overview of a two-step gene knock-in strategy. The insert fragment of the GFP and the VP2 expression cassettes is released by Cas9/sgA cleavage and inserted into the HVT genome at UL45/46 loci via NHEJ-CRISPR/Cas9. The GFP-positive recombinant virus is then sorted and purified by single-cell fluorescence-activated cell sorting (FACS). Subsequently, the GFP reporter gene is excised by Cre recombinase and the recombinant virus is purified and characterized. Please click here to view a larger version of this figure.
Figure 2: Verification of the recombinant HVT. (A) This panel shows a GFP-positive plaque (HVT-GFP-VP2) visualized under the fluorescence microscope (top panel) and the PCR verification of HVT-GFP-VP2 with primers VP2-F & UL46-R1 for the 3' junction.(B) This panel shows a plaque (HVT-VP2) visualized after the GFP excision of HVT-GFP-VP2, using Cre recombinase and PCR verification of HVT-VP2 with primers UL45-F1 and VP2-R1 for the 5' junction. Please click here to view a larger version of this figure.
Figure 3: Sequence analysis of the recombinant HVT virus. (A) The sequences of the key elements in different colors in this panel are the HVT intergenic region between UL45/46 with the gRNA target sequence underlined and an arrow showing the Cas9 cleavage site, the VP2 expression cassette with the end sequences in italic lowercase, the sg-A target sequence in red with the arrow showing the Cas9 cleavage site, the LoxP site sequence in green, and two SfiI sites sequences in blue. (B) This panel shows the sequencing results of the 5' and 3' junctions and a schematic presentation of HVT-VP2 with key elements with corresponding colors presented in sequences. Please click here to view a larger version of this figure.
Figure 4: Characterization of the recombinant HVT-VP2. This panel shows the confirmation of the successful expression of VP2 in infected CEFs by indirect immunofluorescence assay (IFA) with anti-VP2 monoclonal antibody HH7 (red). HVT infection is confirmed by IFA with HVT-infected chicken serum (green). The scale bar = 20 µm. Please click here to view a larger version of this figure.
The CRISPR/Cas9 system has become a valuable tool in gene editing. The traditional technologies for recombinant HVT vector development, such as homologous recombination13 and BAC mutagenesis technology25, usually involve several rounds of vector cloning and selection, as well as large-scale screening, which may take several months. The protocol described here using an NHEJ-CRISPR/Cas9-based strategy combined with the Cre-Lox system and single-cell sorting is more a convenient, efficient, and faster approach in recombinant vaccine generation. Using this pipeline, the recombinant virus can be obtained within only 1 - 2 weeks24, and plaque purification steps can also be reduced to a single-round separation using fluorescence-activated cell sorting17. The whole process, from gRNA design and donor construction to obtaining the purified recombinant HVT virus, can be achieved within 1 month. The critical steps for successful recombinant HVT generation include the high-efficiency gRNA selection for targeting the viral genome to ensure efficient cleavage for the foreign gene insertion, the high transfection efficiency to maximize the chance for Cas9/gRNAs and the virus to meet in the same cell for editing, and the 12 hour interval between the transfection of the donor and gRNA plasmids and the viral infection to allow Cas9 and gRNA to be expressed at a reasonable level before the virus gets into the cells.
The limitation for the HVT recombinant generation is the complexity of the identification of GFP-positive clones by junction PCR. The GFP-VP2 cassettes could be inserted in either orientation. The junction PCR described here is only for the identification of the insert in the sense orientation. In case of the insert in antisense orientation, PCR using the primer pairs described would not work, and the internal primers could be swapped for this purpose. Another potential problem is that the donor construct can only be used for one gene insertion in the same virus due to the existence of the remaining LoxP sequence after the GFP removal by Cre treatment. A new donor construct with a variant LoxP sequence could be used instead for a multiple insertion purpose.
NHEJ and HDR (homology-directed repair) are the two pathways to repair the double-stranded breaks (DSBs) created by Cas926,27. NHEJ is more efficient as it occurs throughout the cell cycle28, whereas HDR is less efficient and only occurs during S and G2 phases6,29,30. We exploited the more efficient NHEJ repair pathway here to introduce the foreign genes into the targeted locations. Although the NHEJ repair may introduce indels by joining noncompatible or damaged DNA ends through a homology-independent mechanistically flexible process31,32 between the cleaved donor sequence and genomic DNA, the indels can only occur at the cleavage sites of sgA, and the foreign gene-expression cassette is not affected. Another advantage of this approach is that NHEJ is free from the restriction of homology arm construction, making the cloning step very straightforward. This prompts a great potential for the application of NHEJ for foreign gene insertion. The introduction of a universal gRNA target site at both ends of the foreign gene cassette makes the process more rapid as the donor template could be constructed straightaway with no need for the specific gRNA selection. The backbone of the donor plasmid containing sgA target sites, LoxP sites, and PacI and SfiI sites can also be shared widely between different reporter genes, foreign gene-expression cassettes, and different virus vectors, giving this new approach the advantage of customization.
The HVT-harboring VP2 insert was used to describe the protocol in this manuscript; however, the same approach can be used to insert more viral genes at different genomic locations of the HVT genome using the gRNA targeting the desired corresponding sequence for the development of multivalent recombinant HVT vectored vaccines. Other MDV vaccine strains, such as SB-1 and CVI988, other avian herpesviruses, including infectious laryngotracheitis virus and duck enteritis virus, and also other avian DNA viruses, such as pox viruses and adenoviruses, can also be engineered using the same approach for multivalent recombinant vaccine development. The development of new multivalent vectored vaccines using the CRISPR/Cas9 system platform described here will be highly beneficial for the poultry industry to protect against multiple poultry diseases.
The authors have nothing to disclose.
The authors thank Pippa Hawes for helping with the confocal imaging. This project was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) grants BBS/E/I/00007034 and BB/L014262/1.
|M199 medium, Earle's Salts
|cell culture medium
|Fetal Bovine Serum
|cell culture medium
|Penicillin and Streptomycin
|Tryptose Phosphate Broth
|cell culture medium
|HVT Fc126 strain
|Avian Disease and Oncology Laboratory
|wildtype HVT virus
|Cas9 and gRNA expression vector
|pGEM-T Easy Vector Systems
|donor vector cloning
|Subcloning Efficiency DH5α Competent Cells
|donor vector cloning
|donor vector cloning
|T4 DNA Ligase
|QIAprep Spin Miniprep Kit
|Cell Culture 6-well Plate
|GoTaq Master Mixes
|junction PCR amplification
|Platinum Pfx DNA Polymerase
|PCR amplification of full insert
|Goat anti-Chicken IgY (H+L) Antibody, Alexa Fluor 488
|Goat anti-Mouse IgG Antibody, Alexa Fluor 568
|Confocal laser scanning microscope
|Leica TCS SP5 LASAF
|FACS cell sorter
|single cell sorting
|Paraformaldehyde (4% in PBS)
|Santa cruz biotechnology
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