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.

1. Preparation of Cas9/gRNA Expression and Donor Constructs
<ol>
	<li>Construction of Cas9/gRNA expression plasmids
	<ol>
		<li>Design a gRNA sequence targeting intergenic region between UL45 and UL46 genes of HVT as described previously22. Align the guide-RNA target sequence against the HVT genome to rule out any potential off-target sequences in the HVT genome. Synthesize and clone the gRNA sequence targeting UL45/46 region and sg-A sequence from published data23 into pX459-V2 as described previously22. Verify the cloned gRNA sequence by Sanger sequencing using the U6-Fwd primer24.</li>
	</ol>
	</li>
	<li>Construction of donor plasmid
	<ol>
		<li>To generate a donor plasmid containing the VP2 expression cassette tagged with a removable GFP reporter cassette, design oligos Donor-F and Donor-R containing the following elements (Figure 1A and Figure 3A): an sg-A target sequence at both ends, a PacI site flanked with two LoxP sequences for the GFP reporter cassette cloning and excision, and two SfiI sites for the cloning of the VP2 expression cassette.</li>
		<li>Clone the sequence into a pGEM-T-easy vector and, then, clone the GFP and VP2 gene cassettes into the resulting vector to generate the donor plasmid designated as pGEM-sgA-GFP-VP222. 
		NOTE: Any cloning vector can be used to construct donor plasmid.</li>
	</ol>
	</li>
	<li>Plasmid DNA preparation
	<ol>
		<li>Prepare both donor plasmid and Cas9/gRNA expression plasmid DNAs using a commercial DNA extraction kit according to the manufacturer&#39;s instructions.</li>
	</ol>
	</li>
</ol>
2. CRISPR/Cas9-mediated Knock-in: Transfection and Infection
<ol>
	<li>The day before transfection, prepare chick embryo fibroblasts (CEFs) for the transfection/infection, using 10-day old embryos in M199 medium supplemented with 5% fetal bovine serum (FBS), 10% tryptose phosphate broth, 100 U/mL penicillin-streptomycin, and 0.25 &#181;g/mL fungizone. Seed 1.3 x 106 cells per well into each well of a 6-well plate in 2.5 mL of medium. 
	NOTE: CEF cells can be kept for 3 d at 4 &#176;C.</li>
	<li>Transfect CEF cells (prepared in step 2.1) with 0.5 &#181;g of UL45/46-gRNA, 0.5 &#181;g of sg-A, and 1 &#181;g of pGEM-sgA-GFP-VP2 using an appropriate transfection reagent according to the manufacturer&#39;s instructions. Incubate the cells for 12 h in an incubator (at 38.5 &#176;C with 5% CO2).</li>
	<li>12 h posttransfection of Cas9/gRNA and donor plasmids, dilute the HVT virus stock with M199 culture medium to 1 x 105 pfu/mL. Add 130 &#181;L of the diluted virus into each well of the transfected cells and set one well of untransfected cells as a negative control with the same quantity of virus. Incubate the transfected/infected cells for 3 d at 38.5 &#176;C with 5% CO2. Carry out all procedures using the Joint Code of Practice (JCoPR) approved by the funders.</li>
</ol>
3. Harvesting and Purification of the HVT Recombinant Virus
<ol>
	<li>Fluorescence-activated cell sorting
	<ol>
		<li>Prepare two 96-well plates preseeded with 2 x 104 CEF cells per well the day before sorting.</li>
		<li>Trypsinize transfected/infected CEFs 3 d postinfection. Aspirate the medium from each well and rinse the cell sheet with phosphate-buffered saline (PBS). Add 1 mL of 0.05% trypsin-EDTA (0.48 mM) to trypsinize the cells in the 38.5 &#176;C incubator for approximately 5 min.</li>
		<li>Resuspend and transfer the cells into a 1.5 mL microcentrifuge tube with 50 &#181;L of FBS. Centrifuge at 200 x g for 5 min.</li>
		<li>Resuspend the cells in 1 mL of PBS with 1% FBS. Count the cell numbers using a hemocytometer and adjust the number of cells to 1 x 106 cells/mL. 
		NOTE: Cells can be kept on ice for 1 - 2 h.</li>
		<li>Transfer the cells to a polystyrene sorting tube through its strainer cap. Sort the single cells expressing GFP into 96-well plates seeded with CEFs using the cell sorter according to the manufacturer&#39;s instruction. Incubate the sorted cells for 5 d at 38.5 &#176;C with 5% CO2. 
		NOTE: One passage of the recombinant virus may be needed before sorting if there are too many single GFP-expression cells and few GFP-positive plaques in the original well.</li>
	</ol>
	</li>
	<li>Passaging of recombinant viruses
	<ol>
		<li>Prepare 6-well plates seeded with 1.3 x 106 CEF cells per well the day before the passage. 5 d postsorting, check the 96-well plates under a fluorescence microscope. Mark the wells containing a single GFP-positive plaque. 
		NOTE: See Figure 2A for a representative GFP-positive plaque.</li>
		<li>Trypsinize each GFP-positive well with 50 &#181;L of trypsin-EDTA for 3 min, add 50 &#181;L of culture medium, resuspend and transfer the cells into one well of a 6-well plate with CEFs. This will be the first generation of recombinant HVT.</li>
		<li>Harvest the first generation of recombinant viruses 3 d later, freeze down one vial of each in 1 mL of freezing medium containing 10% fetal calf serum (FCS), 10% dimethyl sulfoxide (DMSO), and 80% culture medium, and store the viruses in liquid nitrogen. 
		NOTE: The harvest time varies from 2 - 4 d depending on the amount and proliferation capacity of the virus.</li>
		<li>Collect 1 x 105 cells of each first generation of viruses, centrifuge them, and discard the supernatant. Store the cells at -20 &#176;C for DNA extraction.</li>
	</ol>
	</li>
	<li>Detection of genomic insertion by polymerase chain reaction
	<ol>
		<li>For the DNA extraction, defrost and resuspend the cell pellet from step 3.2.4 with 50 &#181;L of squishing buffer (10 mM Tris-HCl [pH 8], 1mM EDTA, 25 mM NaCl, and 200 &#181;g/mL Proteinase K) and lyse the samples at 65 &#176;C for 30 min and, then, at 95 &#176;C for 2 min to inactivate the Proteinase K.</li>
		<li>Perform a polymerase chain reaction (PCR) with 3&#39; junction primers (Table 1) using 1 &#181;L of DNA sample.For each sample, prepare the following 20 &#181;L reaction mix on ice: 2x PCR Master Mix (10 &#181;L), 10 &#181;M upstream primer (0.5 &#181;L), 10 &#181;M downstream primer (0.5 &#181;L), DNA template (1 &#181;L), and nuclease-free water (8 &#181;L). The amplification program is: 95 &#176;C for 2 min; 95 &#176;C for 30 s, 55 &#176;C for 30 s, and 72 &#176;C for 40 s for 35 cycles; 72 &#176;C for 7 min. Load 2 &#181;L of the amplification products to one well of 1% agarose gel for gel electrophoresis. 
		NOTE: See Figure 2A for a representative result of the 3&#39; junction PCR.</li>
	</ol>
	</li>
</ol>
4. Excision of the Fluorescent Reporter Gene via the Cre-lox System
<ol>
	<li>To remove the GFP gene from the recombinant virus, transfect 2 &#181;g of Cre recombinase expression plasmid into the 6-well plate preseeded with CEF cells, using a transfection reagent following the manufacturer&#39;s instruction.</li>
	<li>12 h posttransfection, defrost one vial of recombinant virus from liquid nitrogen, resuspend gently, seed 50 &#181;L into each well of transfected cells, and set one well as the negative control with the same amount of virus. Incubate for 3 d at at 38.5 &#176;C with 5% CO2.</li>
</ol>
5. Plaque Purification
<ol>
	<li>Fluorescence-activated cell sorting
	<ol>
		<li>Seed two 96-well plates with 2 x 104 CEF cells per well the day before sorting.</li>
		<li>Follow the procedures described in step 3.1 72 h postinfection (from step 4) to prepare the infected cells for sorting. Sort the single nonfluorescence cells into 96-well plates seeded with CEFs.</li>
	</ol>
	</li>
	<li>Passaging of the recombinant viruses and PCR confirmation
	<ol>
		<li>Choose 5 - 10 single nonfluorescence plaques 5 d postsorting, trypsinize them with 50 &#181;L of trypsin-EDTA at 38.5 &#176;C for 3 min, and add 50 &#181;L of culture medium to resuspend the cells. 
		NOTE: See Figure 2B for a representative GFP-negative plaque.</li>
		<li>Pass half of the cells into each well of a 6-well plate preseeded with CEFs as the second generation.</li>
		<li>Centrifuge the remaining cells of each clone at 200 x g for 5 min, discard the supernatant, and resuspend the cells with 50 &#181;L of squishing buffer for DNA extraction.</li>
		<li>Perform PCR with 5&#39; junction primers (Table 1) using 1 &#181;L of DNA template with the same PCR reaction condition as described in step 3.3.2. 
		NOTE: See Figure 2B for a representative result of 5&#39; junction PCR.</li>
		<li>Based on the PCR results, choose three to five positive clones of recombinant HVT for further passages and verification.</li>
	</ol>
	</li>
</ol>
6. Verification of the Recombinant HVT
<ol>
	<li>Indirect immunofluorescence assay
	<ol>
		<li>Infect CEFs with the second generation of recombinant HVT obtained from step 5.2 in the 24-well plate seeded with 2.5 x 105 CEFs the day before infection. Remove the cell culture medium 48 h postinfection and add 500 &#181;L of 4% paraformaldehyde in PBS to fix the cells. Incubate the plate at room temperature for 30 min and, then, remove the fixative and wash the cell layer 3x with PBS. 
		NOTE: The cells may be stored at 4 &#176;C at this step for several weeks.</li>
		<li>Remove the PBS and add 500 &#181;L of 0.1% Triton X-100 to permeabilize the cells for 15 min. Wash the cell layer 3x with PBS.</li>
		<li>Block nonspecific binding by adding blocking buffer (5% bovine serum in PBS) for 1 h.</li>
		<li>Dilute the primary antibody anti-VP2 monoclonal antibody HH7- or HVT-infected chicken serum at 1:200 in blocking buffer, add 200 &#181;L per well, and incubate at room temperature for 1 h. Wash the cell layer 3x with PBS.</li>
		<li>Dilute the secondary antibody goat anti-mouse IgG Alexa 568 or goat anti-chicken IgG Alexa 488 at 1:200 in blocking buffer, add 200 &#181;L per well, incubate at room temperature for 1 h, and wash the cells 3x with PBS. Check the protein expression under the fluorescence microscope. 
		NOTE: See Figure 4 for a representative VP2 staining result.</li>
	</ol>
	</li>
	<li>Sequencing of the VP2 gene
	<ol>
		<li>Amplify the DNA sequence spanning the UL45 and UL46 intergenic region with high-fidelity DNA polymerase and primers UL45-F1 and UL46-R1 (Table 1) to detect the whole insertion.</li>
		<li>Prepare the following 50 &#181;L reaction mix: 5 &#181;L of 10x Pfx Reaction Mix, 1.5 &#181;L of 10 &#181;M upstream and downstream primer mix, 1 &#181;L of 10 mM dNTP mix, 1 &#181;L of 50 mM MgSO4, 1 &#181;L of DNA template, and 0.5 &#181;L of Pfx DNA polymerase, and add nuclease-free water to 50 &#181;L. The amplification program is: 95 &#176;C for 2 min; 95 &#176;C for 15 s, 55 &#176;C for 30 s, and 68 &#176;C for 3 min for 35 cycles. Load all of the PCR product to 1% agarose gel for gel electrophoresis.</li>
		<li>Purify the PCR product following the instruction of a DNA gel purification kit. Send 10 &#181;L of the PCR product (30 ng/&#181;L) to a sequencing company to confirm the knock-in of the VP2 gene.</li>
	</ol>
	</li>
</ol>
7. Stability of the Recombinant Virus
<ol>
	<li>Seed 2.6 x 106 CEF cells into each T25 flask the day before the virus expansion.</li>
	<li>Thaw at least three positive clones from step 5.2; add one vial of cells/viruses into each T25 flask. Incubate at 38.5 &#176;C with 5% CO2 until a 50% cytopathic effect is observed.</li>
	<li>Harvest the cells in 2 mL of culture medium; infect 50 &#181;L to a new T25 flask preseeded with CEF cells for the next generation. Keep passaging the recombinant virus for at least 15 generations. Analyze each generation of viruses by PCR for the presence of the VP2 sequence and by indirect immunofluorescence assay (IFA) for VP2 expression to assess the stability of the recombinant viruses.</li>
</ol>

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The authors have nothing to disclose.

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.

Marek&#39;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.

<table><tbody><tr><td>M199 medium, Earle&#039;s Salts</td><td>Life technology</td><td>11150059</td><td>cell culture medium</td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma</td><td>F0926</td><td>cell culture medium</td></tr><tr><td>Penicillin and Streptomycin</td><td>Life technology</td><td>15140148</td><td>antibiotics</td></tr><tr><td>Fungizone</td><td>Sigma</td><td>1397-89-3</td><td>antifunigal</td></tr><tr><td>Tryptose Phosphate Broth</td><td>Sigma</td><td>T8782</td><td>cell culture medium</td></tr><tr><td>HVT Fc126 strain</td><td>Avian Disease and Oncology Laboratory</td><td></td><td>wildtype HVT virus</td></tr><tr><td>pX459-v2</td><td>Addgene</td><td>Plasmid #62988</td><td>Cas9 and gRNA expression vector</td></tr><tr><td>pGEM-T Easy Vector Systems</td><td>promega</td><td>A1360</td><td>donor vector cloning</td></tr><tr><td>Subcloning Efficiency DH5&amp;alpha; Competent Cells</td><td>Invitrogen</td><td>18265-017</td><td>transformation</td></tr><tr><td>PacI</td><td>NEB</td><td>rR0547S</td><td>donor vector cloning</td></tr><tr><td>SfiI</td><td>NEB</td><td>R0123S</td><td>donor vector cloning</td></tr><tr><td>T4 DNA Ligase</td><td>NEB</td><td>M0202S</td><td>cloning</td></tr><tr><td>QIAprep Spin Miniprep Kit</td><td>QIAGEN</td><td>27106</td><td>plasmid extraction</td></tr><tr><td>TransIT-X2</td><td>Mirus</td><td>MIR 6004</td><td>transfection</td></tr><tr><td>Cell Culture 6-well Plate</td><td>Thermofisher</td><td>140675</td><td>cell culture</td></tr><tr><td>GoTaq Master Mixes</td><td>Promega</td><td>M7123</td><td>junction PCR amplification</td></tr><tr><td>Platinum Pfx DNA Polymerase</td><td>Thermofisher</td><td>11708021</td><td>PCR amplification of full insert</td></tr><tr><td>Goat anti-Chicken IgY (H+L) Antibody, Alexa Fluor 488</td><td>Invitrogen</td><td>A-11039</td><td>immunoflourescence staining</td></tr><tr><td>Goat anti-Mouse IgG Antibody, Alexa Fluor 568</td><td>Invitrogen</td><td>A-11004</td><td>immunoflourescence staining</td></tr><tr><td>Confocal laser scanning microscope</td><td>Leica Microsystems</td><td>Leica TCS SP5 LASAF</td><td>immunoflourescence</td></tr><tr><td>FACS cell sorter</td><td>BD biosciences</td><td>BD FACSAria</td><td>single cell sorting</td></tr><tr><td>Paraformaldehyde (4% in PBS)</td><td>Santa cruz biotechnology</td><td>SC-281692</td><td>cell fixation</td></tr><tr><td>Triton X-100</td><td>GeneTex</td><td>GTX30960</td><td>permeabilization</td></tr></tbody></table>

generating recombinant avian herpesvirus vectors with crispr/cas9 gene editing

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.

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&#39; 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&#39; 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).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58193/58193fig1.jpg" /> 
Figure 1:&#160;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. <a href="https://www.jove.com/files/ftp_upload/58193/58193fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58193/58193fig2.jpg" /> 
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 &#38; UL46-R1 for the 3&#39; 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&#39; junction. <a href="https://www.jove.com/files/ftp_upload/58193/58193fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58193/58193fig3.jpg" /> 
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&#39; and 3&#39; junctions and a schematic presentation of HVT-VP2 with key elements with corresponding colors presented in sequences. <a href="https://www.jove.com/files/ftp_upload/58193/58193fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58193/58193fig4.jpg" /> 
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 &#181;m. <a href="https://www.jove.com/files/ftp_upload/58193/58193fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing at JoVE.com

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing

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 ...

generating-recombinant-avian-herpesvirus-vectors-with-crisprcas9-gene

Research

JoVE Journal

Immunology and Infection

13.2K Views.  The Pirbright Institute & UK-China Centre of Excellence for Research on Avian Diseases. Deze aanpak biedt een efficiënte manier om andere virale antigenen in het HVT-genoom te introduceren voor een snelle ontwikkeling van recombinante vaccins. Een voordeel van deze techniek is dat een GFP expressie cassette eerst aan de insert wordt bevestigd voor eenvoudige visualisatie en vervolgens verwijderd door Cre-LoxP systeem. Dezelfde aanpak kan worden gebruikt om meer virale genen in te voegen op verschillende locaties van het HVT-genoom, of andere vogelherpesvirussen voor de ontwikke...

Video: Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing

Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts

Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation of gene functions. Their therapeutic potential in the heart can also be determined. There are limited approaches for in vivo molecular intervention in the mouse heart. Recombinant adeno-associated virus (rAAV)-based genome engineering has been utilized as an essential tool for in vivo cardiac gene manipulation. The specific advantages of this technology include high efficiency, high specificity, low genomic integration rate, minimal immunogenicity, and minimal pathogenicity. Here, a detailed procedure to construct, package, and purify the rAAV9 vectors is described. Subcutaneous injection of rAAV9 into neonatal pups results in robust expression or efficient knockdown of the gene(s) of interest in the mouse heart, but not in the liver and other tissues. Using the cardiac-specific TnnT2 promoter, high expression of GFP gene in the heart was obtained. Additionally, target mRNA was inhibited in the heart when a rAAV9-U6-shRNA was utilized. Working knowledge of rAAV9 technology may be useful for cardiovascular investigations.

In this manuscript, a method to prepare recombinant adeno-associated virus 9 (rAAV9) vectors to manipulate gene expression in the mouse heart is described.

Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation ...

Genetics

A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells

Lentiviral vectors are an ideal choice for delivering gene-editing components to cells due to their capacity for stably transducing a broad range of cells and mediating high levels of gene expression. However, their ability to integrate into the host cell genome enhances the risk of insertional mutagenicity and thus raises safety concerns and limits their usage in clinical settings. Further, the persistent expression of gene-editing components delivered by these integration-competent lentiviral vectors (ICLVs) increases the probability of promiscuous gene targeting. As an alternative, a new generation of integrase-deficient lentiviral vectors (IDLVs) has been developed that addresses many of these concerns. Here the production protocol of a new and improved IDLV platform for CRISPR-mediated gene editing and list the steps involved in the purification and concentration of such vectors is described and their transduction and gene-editing efficiency using HEK-293T cells was demonstrated. This protocol is easily scalable and can be used to generate high titer IDLVs that are capable of transducing cells in vitro and in vivo. Moreover, this protocol can be easily adapted for the production of ICLVs.

We describe the production strategy of integrase-deficient lentiviral vectors (IDLVs) as vehicles for delivering CRISPR/Cas9 to cells. With an ability to mediate quick and robust gene editing in cells, IDLVs present a safer and equally effective vector platform for gene delivery compared to integrase-competent vectors.

Lentiviral vectors are an ideal choice for delivering gene-editing components to cells due to their capacity for stably transducing a broad range of cells ...

In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line using an Adeno-Associated Virus-Cas9 System

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by introducing specific mutations in oncogenes and tumor suppressor genes, using clustered regulatory interspaced short palindromic repeat (CRISPR)-based genome editing technology in mice. This technology allows us to accurately mimic the genetic changes that occur in human cancers using mice. By genetically transforming murine primary cells, we can better study cancer development, progression, treatment, and diagnosis. In this study, we used Cre-inducible Cas9 mouse tongue epithelial cells to enable genome editing using adeno-associated virus (AAV) in vitro. Specifically, by altering KRAS, p53, and APC in normal tongue epithelial cells, we generated a murine head and neck cancer (HNC) cell line in vitro,which is tumorigenic in syngeneic mice. The method presented here describes in detail how to generate HNC cell lines with specific genomic alterations and explains their suitability for predicting tumor progression in syngeneic mice. We envision that this promising method will be informative and useful to study tumor biology and therapy of HNC.

Development of murine models with specific genes mutated in head and neck cancer patients is required for understanding of neoplasia. Here, we present a protocol for in vitro transformation of primary murine tongue cells using an adeno-associated virus-Cas9 system to generate murine HNC cell lines with specific genomic alterations.

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by ...

Cancer Research

CRISPR-Cas9 Genome Editing of Rat Embryos using Adeno-Associated Virus (AAV) and 2-Cell Embryo Electroporation

Genome editing technology is widely used to produce genetically modified animals, including rats. Cytoplasmic or pronuclear injection of DNA repair templates and CRISPR-Cas reagents is the most common delivery method into embryos. However, this type of micromanipulation necessitates access to specialized equipment, is laborious, and requires a certain level of technical skill. Moreover, microinjection techniques often result in lower embryo survival due to the mechanical stress on the embryo. In this protocol, we developed an optimized method to deliver large DNA repair templates to work in conjunction with CRISPR-Cas9 genome editing without the need for microinjection. This protocol combines AAV-mediated DNA delivery of single-stranded DNA donor templates along with the delivery of CRISPR-Cas9 ribonucleoprotein (RNP) by electroporation to modify 2-cell embryos. Using this novel strategy, we have successfully produced targeted knock-in rat models carrying insertion of DNA sequences from 1.2 to 3.0 kb in size with efficiencies between 42% and 90%.

CRISPR-Cas9 Genome Editing of Rat Embryos using Adeno-Associated Virus AAV and 2-Cell Embryo Electroporation

This protocol presents an optimized approach for producing genetically modified rat models. Adeno-associated virus (AAV) is used to deliver a DNA repair template, and electroporation is used to deliver CRISPR-Cas9 reagents to complete the genome editing process in 2-cell embryo.

Genome editing technology is widely used to produce genetically modified animals, including rats. Cytoplasmic or pronuclear injection of DNA repair ...

Engineering Oncogenic Heterozygous Gain-of-Function Mutations in Human Hematopoietic Stem and Progenitor Cells

Throughout their lifetime, hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations. Some of these mutations alter HSPC functional properties such as proliferation and differentiation, thereby promoting the development of hematologic malignancies. Efficient and precise genetic manipulation of HSPCs is required to model, characterize, and better understand the functional consequences of recurrent somatic mutations. Mutations can have a deleterious effect on a gene and result in loss-of-function (LOF) or, in stark contrast, may enhance function or even lead to novel characteristics of a particular gene, termed gain-of-function (GOF). In contrast to LOF mutations, GOF mutations almost exclusively occur in a heterozygous fashion. Current genome-editing protocols do not allow for the selective targeting of individual alleles, hampering the ability to model heterozygous GOF mutations. Here, we provide a detailed protocol on how to engineer heterozygous GOF hotspot mutations in human HSPCs by combining CRISPR/Cas9-mediated homology-directed repair and recombinant AAV6 technology for efficient DNA donor template transfer. Importantly, this strategy makes use of a dual fluorescent reporter system to allow for the tracking and purification of successfully heterozygously edited HSPCs. This strategy can be employed to precisely investigate how GOF mutations affect HSPC function and their progression toward hematological malignancies.

Novel strategies to faithfully model somatic mutations in hematopoietic stem and progenitor cells (HSPCs) are necessary to better study hematopoietic stem cell biology and hematological malignancies. Here, a protocol to model heterozygous gain-of-function mutations in HSPCs by combining the use of CRISPR/Cas9 and dual rAAV donor transduction is described.

Throughout their lifetime, hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations. Some of these mutations alter HSPC functional ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Designing, Packaging, and Delivery of High Titer CRISPR Retro and Lentiviruses via Stereotaxic Injection

Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique has been widely used to encode fluorescent proteins, opto- or chemo-genetic controllers of cell activity, or heterologous expression of human genes in model organisms. These viruses have also successfully been used to deliver recombinases to relevant target sites in transgenic animals, or even deliver small hairpin or micro RNAs in order to manipulate gene expression. While these techniques have been fruitful, they rely on transgenic animals (recombinases) or frequently lack high efficacy and specificity (shRNA/miRNA). In contrast, the CRISPR/Cas system uses an exogenous Cas nuclease which targets specific sites in an organism's genome via an exogenous guide RNA in order to induce double stranded breaks in DNA. These breaks are then repaired by non-homologous end joining (NHEJ), producing insertion and deletion (indel) mutations that can result in deleterious missense or nonsense mutations. This manuscript provides detailed methods for the design, production, injection, and validation of single lenti/retro virus particles that can stably transduce neurons to express a fluorescent reporter, Cas9, and sgRNAs to knockout genes in a model organism.

The CRISPR/Cas9 system offers the potential to make targeted genome editing accessible and affordable to the scientific community. This protocol is intended to demonstrate how to create viruses that will knockout a gene of interest using the CRISPR/Cas9 system, and then inject them stereotaxically into the adult mouse brain.

Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique ...

Neuroscience

Lentiviral CRISPR/Cas9-Mediated Genome Editing for the Study of Hematopoietic Cells in Disease Models

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting from advances in genome editing technology and lentivirus-mediated transgene delivery systems, an efficient and economical method is described here that establishes mice in which genes are manipulated specifically in hematopoietic stem cells. Lentiviruses are used to transduce Cas9-expressing lineage-negative bone marrow cells with a guide RNA (gRNA) targeting specific genes and a red fluorescence reporter gene (RFP), then these cells are transplanted into lethally-irradiated C57BL/6 mice. Mice transplanted with lentivirus expressing non-targeting gRNA are used as controls. Engraftment of transduced hematopoietic stem cells are evaluated by flow cytometric analysis of RFP-positive leukocytes of peripheral blood. Using this method, ~90% transduction of myeloid cells and ~70% of lymphoid cells at 4 weeks after transplantation can be achieved. Genomic DNA is isolated from RFP-positive blood cells, and portions of the targeted site DNA are amplified by PCR to validate the genome editing. This protocol provides a high-throughput evaluation of hematopoiesis-regulatory genes and can be extended to a variety of mouse disease models with hematopoietic cell involvement.

Described are protocols for the highly efficient genome editing of murine hematopoietic stem and progenitor cells (HSPC) by the CRISPR/Cas9 system to rapidly develop mouse model systems with hematopoietic system-specific gene modifications.

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting ...

Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and living organisms from a wide variety of species. The CRISPR technology has also been explored as novel therapeutics for a number of human diseases. Proof-of-concept data are highly encouraging as exemplified by recent studies that demonstrate the feasibility and efficacy of gene editing-based therapeutic approach for Duchenne muscular dystrophy (DMD) using a murine model. In particular, intravenous and intraperitoneal injection of the recombinant adeno-associated virus (rAAV) serotype rh.74 (rAAVrh.74) has enabled efficient cardiac delivery of the Staphylococcus aureus CRISPR-associated protein 9 (SaCas9) and two guide RNAs (gRNA) to delete a genomic region with a mutant codon in exon 23 of mouse Dmd gene. This same approach can also be used to knock out the gene-of-interest and study their cardiac function in postnatal mice when the gRNA is designed to target the coding region of the gene. In this protocol, we show in detail how to engineer rAAVrh.74-CRISPR vector and how to achieve highly efficient cardiac delivery in neonatal mice.

Here we provide a detailed protocol to carry out in vivo cardiac gene editing in mice using recombinant Adeno-Associated Virus(rAAV)-mediated delivery of CRISPR. This protocol offers a promising therapeutic strategy to treat dystrophic cardiomyopathy in Duchenne muscular dystrophy and can be used to generate cardiac-specific knockout in postnatal mice.

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and ...

Medicine

Rapid, Seamless Generation of Recombinant Poxviruses using Host Range and Visual Selection

Vaccinia virus (VACV) was instrumental in eradicating variola virus (VARV), the causative agent of smallpox, from nature. Since its first use as a vaccine, VACV has been developed as a vector for therapeutic vaccines and as an oncolytic virus. These applications take advantage of VACV&#8217;s easily manipulated genome and broad host range as an outstanding platform to generate recombinant viruses with a variety of therapeutic applications. Several methods have been developed to generate recombinant VACV, including marker selection methods and transient dominant selection. Here, we present a refinement of a host range selection method coupled with visual identification of recombinant viruses. Our method takes advantage of selective pressure generated by the host antiviral protein kinase R (PKR) coupled with a fluorescent fusion gene expressing mCherry-tagged E3L, one of two VACV PKR antagonists. The cassette, including the gene of interest and the mCherry-E3L fusion is flanked by sequences derived from the VACV genome. Between the gene of interest and mCherry-E3L is a smaller region that is identical to the first ~150 nucleotides of the 3&#8217; arm, to promote homologous recombination and loss of the mCherry-E3L gene after selection. We demonstrate that this method permits efficient, seamless generation of rVACV in a variety of cell types without requiring drug selection or extensive screening for mutant viruses.

This is a method to generate &#8220;scarless&#8221; recombinant vaccinia viruses using host-range selection and visual identification of recombinant viruses.

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing

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