This work was supported by The MRC DPFS grant (MR/M015696/1) and Ministry of Sciences and Technology of China (2013DFG32080).

1. Preparation of Target RNA and Cas9 Constructs and Repair Donor Vector
<ol>
	<li>Cloning of gRNAs
	<ol>
		<li>Design a guide-RNA target sequence targeting the N1L gene of vaccinia virus following the principle stated previously25. Align the guide-RNA target sequence against the genome of the vaccinia virus (in this case, Lister strain of vaccinia virus) to rule out any off-target gRNA target sequences.</li>
		<li>Synthesize and clone gRNA oligos into a gRNA cloning vector as described previously25. Confirm the sequence of each individual gRNA in the resulting vector by Sanger sequencing33. Name the resulting gRNA construct as gRNA N233. 
		NOTE: The gRNA target site is within the N1L gene, and is in the region between the left arm and right arm that the repair donor vector covers (see Figure 1).</li>
	</ol>
	</li>
	<li>Clone the Cas9 gene lacking the nuclear localization signal (NLS) into the pST1374 vector and designate the construct as pST-Cas933. 
	NOTE: Any mammalian expression cloning vector can be used for the construction of the Cas9 expression vector.</li>
	<li>Construction of repair donor vector for HR
	<ol>
		<li>Use the N1L gene of VV as the target region for modification33. Ensure that the lengths of the left and right arms are about 500-600 bp each, and flanking the target region. 
		NOTE: The left arm/right arm can have up to 50 bp overlap with the target region (See Figure 1).</li>
		<li>Clone the N1L region repair donor vector as described previously33, 34 and designate the repair donor vector as pT-N1L. 
		NOTE: See Figure 1 for the repair donor vector and its target region. Other regions of the VV may be targeted using region (gene)-specific gRNA(s) and region-specific repair donor vector. Any cloning vector can be used for the construction of the repair donor vector.</li>
	</ol>
	</li>
	<li>Expansion of plasmid
	<ol>
		<li>Transform the plasmid into chemically competent E. coli according to the manufacturer&#39;s instructions. Purify the plasmid using a plasmid extraction kit referring to the protocol supplied by the manufacturer.</li>
	</ol>
	</li>
</ol>
2. Seeding Cells (Day 1)
<ol>
	<li>Maintain CV-1 cells (Monkey kidney fibroblasts) in Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) supplemented with 5% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 &#181;g/mL streptomycin, at 37 &#176;C with 5% CO2.</li>
	<li>Trypsinize CV-1 cells that are 80-90% confluent
	<ol>
		<li>Remove the cell culture medium from the T-175 flask containing CV-1 cells. Rinse the monolayer with 5 mL of sterile PBS to remove residual serum and aspirate the PBS. Add 2.5 mL 1&#215; Trypsin-EDTA solution to cover the cells and place the flask in 37 &#176;C incubator for approximately 5 min to aid cell detachment.</li>
		<li>Add 10 mL of cell culture medium (see step 2.1) to suspend the cells by gentle pipette action. Count the cell numbers using a hemocytometer.</li>
		<li>Seeding cells in 6-well plates
		<ol>
			<li>Seed 2 &#215; 105 CV-1 cells in 2 mL of cell culture medium into each well of a 6-well plate the day before transfection. 
			NOTE: An additional centrifugation step to remove trypsin is not required.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Transfection of Cas9 Plasmid and gRNA Plasmid (Day 2)
<ol>
	<li>Transfect CV-1 cells (prepared in step 2.2.3.1) with 0.5 &#181;g pST-Cas9 plasmid and 0.5 &#181;g gRNA N2 plasmid using a transfection reagent according to the manufacturer&#39;s instructions.</li>
	<li>Incubate the cells for 24 h in an incubator (37 &#176;C with 5% CO2), and then replace the medium with 2 mL of fresh cell culture medium (described in step 2.1).</li>
</ol>
4. Infection of CV-1 Cells and Shuttle Donor Vector Transfection for HR (Day 3)
<ol>
	<li>Dilute the backbone VVL15 virus with DMEM cell culture medium to a concentration of 2 &#215; 105 pfu/mL. 24 h post-transfection of Cas9 and gRNA plasmids, add 100 &#181;L of the diluted virus into each well of the transfected CV-1 cells.</li>
	<li>2 h post-virus infection, transfect 1.0 &#181;g shuttle donor vector into VVL15-infected cells for HR using a transfection reagent according to the manufacturer&#39;s instructions. Place the transfected cells in a 37 &#176;C incubator with 5% CO2 for 24 h.</li>
</ol>
5. Harvest the Recombinant Virus (Day 4)
<ol>
	<li>After 24 h of transfection with the shuttle donor vector for HR in step 4.2, detach the transfected CV-1 cells using a cell scraper. Collect the cell suspension into a cryovial and store at -80 &#176;C for future use. 
	NOTE: Wipe off any cell culture medium spillage with disinfectant immediately and then wipe again with 70% ethanol.</li>
</ol>
6. Purification of the Modified VV (First Round)
<ol>
	<li>On day 1, seed 15 6-well plates with 3 &#215; 105 CV-1 cells in each well.
	<ol>
		<li>On day 2, thaw the frozen cell suspension from step 5.1 in a water bath at 37 &#176;C for 3 min and vortex the cryovials vigorously for 30 s to obtain cell lysate without removing cell debris.</li>
	</ol>
	</li>
	<li>For the infection of one 6-well plate of CV-1 cells, dilute 1 &#181;L of cell lysate with 3 mL of DMEM and add 0.5 mL of the diluted cell lysate into each well of the 6-well plate, on top of media already present. Infect all the 6-well plates prepared in step 6.1. 
	NOTE: Removal of cell debris is not required.</li>
	<li>After two days of infection (i.e., on day 4), identify RFP-positive plaques under a fluorescence microscope using 10X objective lens and label the plaques underneath the plate using a marker pen by circling the plaque location. 
	NOTE: See Figure 3 for representative RFP-positive plaque.
	<ol>
		<li>Prepare six labelled cryovials containing 200 &#181;L serum-free DMEM cell culture medium to pick up six different RFP-positive plaques. Aspirate the cell culture medium from the well with labelled plaque(s) (step 6.3). Attach a 200 &#181;L tip to a P200 pipette, set the volume at 30 &#181;L and take ~ 10 &#181;L cell culture medium from one cryovial.</li>
		<li>Hold the pipette push button without releasing the medium and use the tip to scratch the area of one labelled plaque. Release the pipette push button to lift up the detached cells and transfer it into the cryovial containing 190 &#181;L of serum-free DMEM.</li>
		<li>Take another 10 &#181;L medium from the vial with the scratched cells (from last step) and repeat the procedure of picking up the same RFP-positive plaque three times to collect as many scratched cells as possible. Transfer all cells into the same vial and store the vial at -80 &#176;C. 
		NOTE: Alternatively, freeze the cryovials on dry ice for 10 min if the second round of purification is performed immediately.</li>
	</ol>
	</li>
</ol>
7. Purification of the Modified VV (Second Round)
<ol>
	<li>Seed 3 &#215; 105 CV-1 cells into each well of six 6-well plates and culture the cells for 24 h.</li>
	<li>Thaw the frozen cryovials (from step 6.3.3) in 37 &#176;C water bath for 3 min, then vortex the cryovials vigorously for 30 sec to obtain cell lysate.
	<ol>
		<li>Add 0.5 &#181;L of the mixed cell lysate to one well of a 6-well plate. Infect one 6-well plate for each plaque. Incubate the VV-infected 6-well plates at 37 &#176;C with 5% CO2 for 2 days (Second round plaque purification). Then repeat section 6.3. 
		NOTE: More than six RFP-positive plaques can be picked up; two or more 6-well plates can be used for purification of each plaque.</li>
	</ol>
	</li>
</ol>
8. Further Rounds of Plaque Purification
<ol>
	<li>Carry on a further three to five rounds of purification following the same protocol described in step 7 until all the plaques formed from one positive plaque appear to be RFP-positive under a microscope. 
	NOTE: This plaque then is considered pure.</li>
	<li>Once the plaque is pure (Figure 4, all infected cells expressing RFP), harvest a positive plaque into a cryovial and store at -80 &#176;C as described in section 6.3. Verify the plaque following the protocol described in step 9 and 10.</li>
</ol>
9. Expand the Purified Plaque(s)
<ol>
	<li>Seed 3 &#215; 105 CV-1 cells into each well of a 6-well plate one day before virus infection.</li>
	<li>Thaw the frozen cryovial from 8.2 in a 37 &#176;C water bath for 3 min to obtain cell lysate. Add all the lysate (without removing cell debris) into one well of a 6-well plate of CV-1 cells (seeded in step 9.1). Incubate the infected CV-1 cells at 37 &#176;C with 5% CO2 for up to 3 days.
	<ol>
		<li>Expand all the purified plaques (at least three plaques). 
		NOTE: The infection time varies from 1-3 days depending on the amount of virus in the lysate.</li>
	</ol>
	</li>
	<li>Harvest the VV-infected cells using a cell scraper when more than 50% cells show cytopathic effect observed under a microscope (cells are rounded, easily detached and RFP positive). Collect the detached cells together with cell culture medium into a 15 mL tube.
	<ol>
		<li>Pellet the cells by centrifugation at 300 g for 3 min. Remove the supernatant and keep the cell pellet at -80 &#176;C until further use or use the cell pellet immediately for step 10.</li>
	</ol>
	</li>
</ol>
10. Verification of the Modification of Mutant VV Using PCR
<ol>
	<li>Extract VV DNA from the cell pellet prepared in step 9.3.1 using a DNA extraction kit following the manufacturer&#39;s protocol. Elute DNA with 200 &#181;L of double-distilled water. 
	NOTE: Use half of the volume of the pellet to extract DNA and keep the rest for viral production if the clone is verified as containing the correct modification/insertion.</li>
	<li>Verification of the deletion of the N1L gene and the insertion of RFP gene into the N1L region.
	<ol>
		<li>Amplify a DNA fragment spanning the N1L gene (L025) and the L026 gene using primers designed against the N1L gene. Amplify the RFP gene using RFP -specific primers. Amplify a control DNA fragment spanning A52R by PCR using A52R-specific primers. 
		NOTE: See List of Materials for specific primers.
		<ol>
			<li>Perform the PCR using a PCR master mix kit. Set up 25 &#181;L of PCR reaction using 2 &#181;L of DNA template, denature the PCR reaction at 94 &#176;C for 2 min, and then run 30 cycles of PCR following these steps: denature the DNA at 94 &#176;C for 15 s, then anneal the primers to the DNA templates at 52 &#176;C for 15 s extend the PCR reaction at 72 &#176;C for 30 s.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Resolve the PCR products using a 1% agarose gel 33. Capture the gel image using a UV gel doc. If the N1L PCR is negative and A52R and RFP PCRs are positive, then the plaque is correctly modified in the N1L region.</li>
</ol>

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M., Chen, B., Fodor, N., Fodor, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+recombinant+vaccinia+viruses+using+PUV-inactivated+virus+as+a+helper.">Construction of recombinant vaccinia viruses using PUV-inactivated virus as a helper.</a> Biotechniques. 31, 534-540 (2001).</li><li>Johnson, R. D., Jasin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-strand-break-induced+homologous+recombination+in+mammalian+cells.">Double-strand-break-induced homologous recombination in mammalian cells.</a> Biochem Soc Trans. 29, 196-201 (2001).</li></ol>

Authors have no competing financial interests.

There are two main goals regarding modification of VV for therapeutic purposes. One is to delete a particular gene, such as the thymidine kinase (TK) gene to attenuate virus for anti-tumor therapeutic use. The other is to arm VV with a desired therapeutic gene (such as GM-CSF) or a marker gene (such as luciferase gene). Achieving either of these involves the deletion of a target region/gene. A direct DNA ligation method35 and an in vitro packaging method36 have been used previously to generate mutant vaccinia viruses with TK gene modifications. However, these methods have limited application regarding mutation of other regions in the genome due to a lack of unique restriction enzyme sites across the genome. The current approach to creating mutant VV involves transfection of a shuttle (donor) vector with a selection marker (RFP or GFP) into CV-1 cells two hours after infection with VV to induce a homologous recombination incorporating the selection marker into the target region. Selection of marker-positive plaques is followed by their purification 24 h post-transfection. The plaque purification process can take up to 10 rounds, last 3-4 weeks and often leads to undesired recombinant viruses with selection markers inserted in off-target regions. DNA double-strand breaks can effectively induce homologous recombination in mammalian cells 37, and by harnessing this mechanism, the efficiency of generating mutant VV can be vastly improved. The gRNA-guided Cas9 system has been successfully employed in genome editing and enhances the efficiency of homologous recombination in eukaryotic organisms22, 25. Recently, in host cells the genomes of adenovirus and type I herpes simplex virus were edited by the gRNA-guided Cas9 system 24. It has been recently demonstrated that CRISPR Cas9 system is a powerful tool for efficiently editing the VV genome and constructing a VV vector for expressing a particular gene.
Both N1L gRNA-guided Cas9 and the traditional homologous recombination (HR) method resulted in successful HR events at the same target site of N1L. Interestingly, however gRNA-guided Cas9 induced HR at much higher levels of efficiency than the traditional HR method. Thus, the use of gRNA-guided Cas9 eliminates the need to purify as many plaques to obtain mutant VVs. In this work, we significantly improved the efficiency and time-frame for generation of mutant VV using the gRNA-guided Cas9 system33. Of note, one critical step for obtaining a high efficiency of homologous recombination is to transfect CV-1 cells with the plasmids expressing Cas9 and the gRNA for 24 h before performing the conventional homologous recombination. The 24 h interval allows Cas9 and gRNA to be expressed at a reasonable level. Furthermore, the gRNA sequence targeting a particular gene may need to be optimized as we found that the different gRNA sequences targeting N1L gene varied in their efficiency33, 34.
The limitation for the CRISPR/Cas9 in editing VV is the low rate of RFP-positive plaques in the first round of recombination. To obtain a sufficient number of RFP-positive plaques containing recombinant virus, usually at least ten 6-well plates are needed, which makes first-round screening tedious. Hence, there is still a need to optimize the protocol to overcome this limitation.
The small size of RFP-positive plaques is another potential problem, which is due to a high volume of lysate used to infect a 6-well plate. To overcome this, a smaller volume of lysate should be used to infect a 6-well plate to obtain larger size RFP-positive plaques. Using a smaller volume of lysate will also enable for better separation of RFP-positive plaques from the RFP-negative ones. Consequently fewer rounds of plaque purification are required to obtain pure RFP-positive plaques.
Off-target effects are always an unwanted issue in gene editing. CRISPR-Cas9 has shown off-target effects. However, with careful design of gRNA, off-target effects can be avoided when editing the VV genomes33, 34. This is the particular benefit of using the gRNA-guided Cas9 system for editing the genome of VV. Given the promises of VV, using the CRISPR/Cas9 system will speed up the development of mutant VVs for biomedical research and in translational medicine. In addition, such a system would also expedite discoveries in cell biology, such as dissection of the signaling pathways used by VV for its actin-based motility.

Vaccinia virus (VV) is an enveloped DNA virus belonging to the poxvirus family and has played a crucial role in one of the greatest achievements in medicine of the eradication of smallpox. In the post-eradication era of smallpox, VV has been developed as a vector for delivering genes for vaccines against HIV and other infectious diseases1-7 by inserting genes from various pathogens into VV vectors. VV has also been extensively used as a vector for cancer immunotherapy8-14 especially for the development of tumor-targeted replicating oncolytic vaccinia virus. In order to create an efficient vaccine vector with improved selectivity for cancer cells, modifications within the viral genome are required, including gene deletions or introduction of therapeutic genes.
With the development of DNA technology and a better understanding of molecular biology and virology, insertion of foreign DNA into VV was originally achieved using homologous recombination (HR) in 1980s 15. This method is still widely used for constructing VV vectors. Introduction of the genetic modification is achieved by using a shuttle vector for HR, which recombines with the VV genome in pre-infected cells. However, this method has proven to be highly inefficient (less than 1% homologous recombination efficiency16) and often results in random insertion of the selection marker into non-targeted regions and/or loss of the marker upon virus expansion.
The efficiency of DNA homologous recombination for inserting exogenous DNA at genomic loci can be dramatically enhanced in the presence of double-strand breaks (DSBs) 17. Therefore, the technology that can induce DSBs at target loci holds great potential for genome engineering of VV.
The recently developed CRISPR-Cas9 system shows promise for triggering DSBs in any VV gene region. CRISPR-Cas9 is an RNA-guided nuclease involved in adaptive immunity against invading phages and other foreign genetic materials18-20. There are three CRISPR-Cas systems in a range of microbial species21. The type II CRISPR-Cas system is widely used for editing the genome of eukaryotic cells and large viruses. It consists of the RNA-guided Cas9 endonuclease (from Streptococcus pyogenes), a single guide RNA (sgRNA) and the trans-activating crRNA (tracrRNA) 22-24. The Cas9/sgRNA complex recognizes the complementary 20-nucleotide genomic sequence preceding a 5&#39;-NGG-3&#39; Protospacer adjacent motif (PAM) sequence in mammalian cells22, 23. It has been successfully used for effective generation of genetically modified cells, viruses and animal models22-32.
The CRISPR-Cas9 system has proven to be an efficient tool for genome targeting in combination with homologous recombination in the cytoplasm of VV infected CV-1 cells to generate mutant vaccinia viruses33, 34. In order to extend the potential application of this method, we present detailed information about this system&#39;s methodology. The described protocol can be used to create a mutant VV with a particular gene deletion and/or arm the mutant virus with a therapeutic transgene.

<table><tbody><tr><td>Plasmid #41824</td><td>41824</td><td>Addgene</td><td>gRNA cloning vector</td></tr><tr><td>pST1374</td><td>13426</td><td>Addgene</td><td>Cas9 cloning vector</td></tr><tr><td>pGEMT easy</td><td>A1360</td><td>promega</td><td>repair donor vector cloning</td></tr><tr><td>One Shot TOP10 Chemically Competent &lt;em&gt;E. coli&lt;/em&gt;</td><td>C4040-10</td><td>Invitrogen</td><td>transformation</td></tr><tr><td>QIAprep Spin Miniprep Kit&amp;nbsp;</td><td>27106</td><td>Qiagen</td><td>plasmid extraction</td></tr><tr><td>Dulbecco&amp;rsquo;s Eagle&amp;rsquo;s Medium (DMEM)</td><td>11965-092</td><td>Life Technologies</td><td>cell culture medium</td></tr><tr><td>fetal bovine serum (FBS)&amp;nbsp;</td><td>SH30088.03HI</td><td>Hyclone</td><td>cell culture serum</td></tr><tr><td>penicillin, streptomycin</td><td>15070-063</td><td>Thermo Scientific</td><td>antibiotics</td></tr><tr><td>Effectene</td><td>301425</td><td>Qiagen</td><td>transfection</td></tr><tr><td>Thermo Scientific Nalgene Cryogenic vial; 2.0&amp;nbsp;mL</td><td>W-06754-96</td><td>Thermo Scientific</td><td>collect virus</td></tr><tr><td>fluorescence microscope&amp;nbsp;</td><td>Olympus BX51 Fluorescence&amp;nbsp;</td><td>Olympus</td><td>find RFP-positive plque</td></tr><tr><td>DNeasy Blood &amp;amp; Tissue Kit</td><td>69506</td><td>Qiagen</td><td>DNA extraction</td></tr><tr><td>Extensor Long PCR ReddyMix Master Mix</td><td>AB-0794/B</td><td>Thermo Scientific</td><td>PCR reagent</td></tr><tr><td>10 cm culture dish</td><td>Z688819-6EA</td><td>sigma</td><td>cell culture&amp;nbsp;</td></tr><tr><td>6-well plate</td><td>Z707759</td><td>sigma</td><td>cell culture&amp;nbsp;</td></tr><tr><td>cell scraper</td><td>C5981</td><td>sigma</td><td>scrap cells</td></tr><tr><td>1x Trypsin-EDTA solution</td><td>T4299</td><td>sigma</td><td>trypsinize cells</td></tr><tr><td>Virkon</td><td>95015661</td><td>Anachem Ltd</td><td>desinfectant</td></tr><tr><td>Falcon tube</td><td>CLS430791-500EA</td><td>Sigma</td><td>hold cell suspension</td></tr><tr><td>N1L forward 5&amp;rsquo;-TATCTAGCAATGGACCGT-3&amp;rsquo;</td><td></td><td>Sigma</td><td>PCR primer</td></tr><tr><td>N1L reverse&amp;nbsp; 5&amp;rsquo;-CCGAAGGTAGTAGCATGGA-3&#039;</td><td></td><td>Sigma</td><td>PCR primer</td></tr><tr><td>A52R forward 5&amp;rsquo;-ATAGGATTGTGTGCATGC-3&amp;rsquo;</td><td></td><td>Sigma</td><td>PCR primer</td></tr><tr><td>A52R reverse 5&amp;rsquo;-TTGCGGTATATGTATGAGGTG-3&amp;rsquo;</td><td></td><td>Sigma</td><td>PCR primer</td></tr><tr><td>RFP forward 5&amp;rsquo;- GCTACCGGACTCAGATCCA-3&amp;rsquo;&amp;nbsp;&amp;nbsp;</td><td></td><td>Sigma</td><td>PCR primer</td></tr><tr><td>RFP reverse 5&amp;rsquo;-CGCCTTAAGATACATTGATGAG-3&amp;rsquo;</td><td></td><td>Sigma</td><td>PCR primer</td></tr><tr><td>Argarose</td><td>A7431</td><td>Sigma</td><td>resolve PCR product</td></tr><tr><td>G:BOX F3</td><td>G:BOX F3</td><td>Syngene</td><td>UV gel doc</td></tr></tbody></table>

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.

For construction of a new VV vector, the key starting point is to design and construct a donor shuttle vector that can target a particular region of the genome. In this study, the VV N1L gene was used as an example target. The cassette of the donor shuttle vector for the recombination and the targeted region in the VV are shown in Figure 1. In order to enhance the efficiency of the homologous recombination, plasmids expressing Cas9 and a N1L-specific gRNA were co-transfected into the CV-1 cells 48 h prior to performing the homologous recombination33. One day (24 h) post-transfection, RFP was expressed in the CV-1 cells (Figure 2). After infection of CV-1 cells with the whole lysate from the homologous recombination (step 5), RFP-positive and negative plaques were both observed in the CV-1 cells (Figure 3). Following the purification protocol described, a pure plaque could be obtained after 3-5 rounds of purification. As shown in Figure 4, all plaques after infection with cell lysate derived from a pure plaque with the mutant vaccinia virus presented an RFP signal. To verify whether the pure mutant VV had the correct gene modification, PCR was used to confirm the absence of the targeted N1L gene, as shown in Figure 5. PCR of the modified virus showed a positive signal for RFP and an absent signal for N1L (Figure 5 lane A-G), while the PCR results of N1L for the control virus (Ctr) with an intact N1L region is positive. The control DNA fragments of A52R were positive for all recombinant viruses and the Ctr virus. The HR efficiency in RFP-positive plaques is 100% (6/6) in this experiment (it was up to 94% in the previous work34). The method described herein has improved the recombination efficiency by more than 100 fold compared to conventional protocols33, 34.
<img alt="Figure 1" src="/files/ftp_upload/54171/54171fig1.jpg" /> 
Figure 1: The cassette of the donor shuttle vector for the homologous recombination, and the targeted region in the VV genome. The purification marker gene RFP is driven by the H5 promoter. The repair donor vector targeting the N1L region results in the RFP gene being incorporated into the N1L region after homologous recombination. &#39;X&#39; indicates the homologous sequence in the repair donor vector that will replace the same sequence on the vaccinia virus genome. <a href="http://cloudflare.jove.com/files/ftp_upload/54171/54171fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54171/54171fig2.jpg" /> 
Figure 2: Imaging of the CV-1 cells one day after homologous recombination. One day after homologous recombination, the cells infected with VV and transfected by repair donor vector are RFP-positive. A: Phase contrast image (original magnification 100X). B: Fluorescence microscopy image (original magnification 100X). Scale bar = 50 &#181;m. <a href="http://cloudflare.jove.com/files/ftp_upload/54171/54171fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54171/54171fig3.jpg" /> 
Figure 3: An RFP-positive plaque in the first round purification of mutant virus. In the first-round purification of the mutant virus, the RFP-positive plaque (circled with red line) with the target region deletion are surrounded by plaques formed by wild type virus (circled with yellow line). A: Phase contrast image (original magnification 100X). B: Fluorescence microscopy image (original magnification 100X). Scale bar = 50 &#181;m. <a href="http://cloudflare.jove.com/files/ftp_upload/54171/54171fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54171/54171fig4.jpg" /> 
Figure 4: The pure mutant VV. After 3-5 rounds of purification, pure plaques were obtained as all the plaques were RFP-positive. A: Phase contrast image (original magnification 100X). B: Fluorescence microscopy image (original magnification 100X). Scale bar = 50 &#181;m. <a href="http://cloudflare.jove.com/files/ftp_upload/54171/54171fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54171/54171fig5.jpg" /> 
Figure 5: Verification of the pure mutant VV. DNA was extracted from VV infected CV-1 cells. The deletion of the target region N1L was verified by PCR using N1L primers, the insertion of RFP into N1L region was confirmed by PCR using RFP primers. Lanes A-F correspond to six purified plaques, lane G is a control plaque with N1L deletion verified in previous work33, lane Ctr (control) is the wild type vaccinia virus (with N1L region intact). A52R gene was amplified as the control gene for all the samples. <a href="http://cloudflare.jove.com/files/ftp_upload/54171/54171fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors at JoVE.com

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

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 ...

a-simple-efficient-approach-to-construct-mutant-vaccinia-virus

Research

JoVE Journal

Immunology and Infection

11.1K Views.  Queen Mary University of London.  Vacciniavirus (VV) is op grote schaal gebruikt in biomedisch onderzoek en de verbetering van de volksgezondheid. Dit artikel beschrijft een eenvoudige, zeer efficiënte methode om de VV genoom bewerken met een CRISPR-Cas9 systeem. 

Video: A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for safe and effective therapies tailored to individual tumor immune profiles. Oncolytic viruses enable the induction of anti-tumor immune responses as well as tumor-restricted gene expression. This protocol describes the generation and ex vivo analysis of immunomodulatory oncolytic vectors. Focusing on measles vaccine viruses encoding bispecific T cell engagers as an example, the general methodology can be adapted to other virus species and transgenes. The presented workflow includes the design, cloning, rescue, and propagation of recombinant viruses. Assays to analyze replication kinetics and lytic activity of the vector as well as functionality of the isolated immunomodulator ex vivo are included, thus facilitating the generation of novel agents for further development in preclinical models and ultimately clinical translation.

This protocol describes a detailed workflow for the generation and ex vivo characterization of oncolytic viruses for expression of immunomodulators, using measles viruses encoding bispecific T cell engagers as an example. Application and adaptation to other vector platforms and transgenes will accelerate the development of novel immunovirotherapeutics for clinical translation.

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for ...

Cancer Research

Engineering and Evolution of Synthetic Adeno-Associated Virus (AAV) Gene Therapy Vectors via DNA Family Shuffling

Adeno-associated viral (AAV) vectors represent some of the most potent and promising vehicles for therapeutic human gene transfer due to a unique combination of beneficial properties1. These include the apathogenicity of the underlying wildtype viruses and the highly advanced methodologies for production of high-titer, high-purity and clinical-grade recombinant vectors2. A further particular advantage of the AAV system over other viruses is the availability of a wealth of naturally occurring serotypes which differ in essential properties yet can all be easily engineered as vectors using a common protocol1,2. Moreover, a number of groups including our own have recently devised strategies to use these natural viruses as templates for the creation of synthetic vectors which either combine the assets of multiple input serotypes, or which enhance the properties of a single isolate. The respective technologies to achieve these goals are either DNA family shuffling3, i.e. fragmentation of various AAV capsid genes followed by their re-assembly based on partial homologies (typically &gt;80% for most AAV serotypes), or peptide display4,5, i.e. insertion of usually seven amino acids into an exposed loop of the viral capsid where the peptide ideally mediates re-targeting to a desired cell type. For maximum success, both methods are applied in a high-throughput fashion whereby the protocols are up-scaled to yield libraries of around one million distinct capsid variants. Each clone is then comprised of a unique combination of numerous parental viruses (DNA shuffling approach) or contains a distinctive peptide within the same viral backbone (peptide display approach). The subsequent final step is iterative selection of such a library on target cells in order to enrich for individual capsids fulfilling most or ideally all requirements of the selection process. The latter preferably combines positive pressure, such as growth on a certain cell type of interest, with negative selection, for instance elimination of all capsids reacting with anti-AAV antibodies. This combination increases chances that synthetic capsids surviving the selection match the needs of the given application in a manner that would probably not have been found in any naturally occurring AAV isolate. Here, we focus on the DNA family shuffling method as the theoretically and experimentally more challenging of the two technologies. We describe and demonstrate all essential steps for the generation and selection of shuffled AAV libraries (Fig. 1), and then discuss the pitfalls and critical aspects of the protocols that one needs to be aware of in order to succeed with molecular AAV evolution.

Engineering and Evolution of Synthetic Adeno-Associated Virus AAV Gene Therapy Vectors via DNA Family Shuffling

We demonstrate the basic technique to molecularly engineer and evolve synthetic Adeno-associated viral (AAV) gene therapy vectors via DNA family shuffling. Moreover, we provide general guidelines and representative examples for selection and analysis of individual chimeric capsids with enhanced properties on target cells in culture or in mice.

Adeno-associated viral (AAV) vectors represent some of the most potent and promising vehicles for therapeutic human gene transfer due to a unique ...

Viral Tracing of Genetically Defined Neural Circuitry

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus (RABV), and more recently vesicular stomatitis virus (VSV), for studying circuitry, is becoming increasingly popular. These higher throughput methods use viruses that transmit between neurons in either the anterograde or retrograde direction.
Recently, a modified RABV for monosynaptic retrograde tracing was developed. (Figure 1A). In this method, the glycoprotein (G) gene is deleted from the viral genome, and resupplied only in targeted neurons. Infection specificity is achieved by substituting a chimeric G, composed of the extracellular domain of the ASLV-A glycoprotein and the cytoplasmic domain of the RABV-G (A/RG), for the normal RABV-G1. This chimeric G specifically infects cells expressing the TVA receptor1. The gene encoding TVA can been delivered by various methods2-8. Following RABV-G infection of a TVA-expressing neuron, the RABV can transmit to other, synaptically connected neurons in a retrograde direction by nature of its own G which was co-delivered with the TVA receptor. This technique labels a relatively large number of inputs (5-10%)2 onto a defined cell type, providing a sampling of all of the inputs onto a defined starter cell type.
We recently modified this technique to use VSV as a transsynaptic tracer9. VSV has several advantages, including the rapidity of gene expression. Here we detail a new viral tracing system using VSV useful for probing microcircuitry with increased resolution. While the original published strategies by Wickersham et al.4 and Beier et al.9 permit labeling of any neurons that project onto initially-infected TVA-expressing-cells, here VSV was engineered to transmit only to TVA-expressing cells (Figure 1B). The virus is first pseudotyped with RABV-G to permit infection of neurons downstream of TVA-expressing neurons. After infecting this first population of cells, the virus released can only infect TVA-expressing cells. Because the transsynaptic viral spread is limited to TVA-expressing cells, presence of absence of connectivity from defined cell types can be explored with high resolution. An experimental flow chart of these experiments is shown in Figure 2. Here we show a model circuit, that of direction-selectivity in the mouse retina. We examine the connectivity of starburst amacrine cells (SACs) to retinal ganglion cells (RGCs).

A method of tracing synaptically connected neurons is described. We use TVA specificity of an upstream cell to probe whether a cell population of interest receives synaptic input from genetically defined cell types.

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus ...

Neuroscience

Rescue of Recombinant Newcastle Disease Virus from cDNA

Newcastle disease virus (NDV), the prototype member of the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented, negative-sense, single-stranded, enveloped RNA virus (Figure 1) with potential applications as a vector for vaccination and treatment of human diseases. In-depth exploration of these applications has only become possible after the establishment of reverse genetics techniques to rescue recombinant viruses from plasmids encoding their complete genomes as cDNA2-5. Viral cDNA can be conveniently modified in vitro by using standard cloning procedures to alter the genotype of the virus and/or to include new transcriptional units. Rescue of such genetically modified viruses provides a valuable tool to understand factors affecting multiple stages of infection, as well as allows for the development and improvement of vectors for the expression and delivery of antigens for vaccination and therapy. Here we describe a protocol for the rescue of recombinant NDVs.

Newcastle disease virus (NDV) has been extensively studied in the last few years in order to develop new vectors for vaccination and therapy, among others. These studies have been possible due to techniques to rescue recombinant virus from cDNA, such as those we describe here.

Newcastle disease virus (NDV), the prototype member of  the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented,  negative-sense, ...

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

Tagging of viral proteins with fluorescent proteins has proven an indispensable approach to furthering our understanding of virus-host interactions. Vaccinia virus (VACV), the live vaccine used in the eradication of smallpox, is particularly amenable to fluorescent live-cell microscopy owing to its large virion size and the ease with which it can be engineered at the genome level. We report here an optimized protocol for generating recombinant viruses. The minimal requirements for targeted homologous recombination during vaccinia replication were determined, which allows the simplification of construct generation. This enabled the alliance of transient dominant selection (TDS) with a fluorescent reporter and metabolic selection to provide a rapid and modular approach to fluorescently label viral proteins. By streamlining the generation of fluorescent recombinant viruses, we are able to facilitate downstream applications such as advanced imaging analysis of many aspects of the virus-host interplay that occurs during virus replication.

A rapid and modular protocol for the generation of recombinant vaccinia viruses expressing fluorescently&#160;tagged proteins simultaneously using the method of transient dominant selection is described here.

Tagging of viral proteins with fluorescent  proteins has proven an indispensable approach to furthering our understanding  of virus-host interactions. ...

Utilizing the Antigen Capsid-Incorporation Strategy for the Development of Adenovirus Serotype 5-Vectored Vaccine Approaches

Adenovirus serotype 5 (Ad5) has been extensively modified with traditional transgene methods for the vaccine development. The reduced efficacies of these traditionally modified Ad5 vectors in clinical trials could be primarily correlated with Ad5 pre-existing immunity (PEI) among the majority of the population. To promote Ad5-vectored vaccine development by solving the concern of Ad5 PEI, the innovative Antigen Capsid-Incorporation strategy has been employed. By merit of this strategy, Ad5-vectored we first constructed the hexon shuttle plasmid HVR1-KWAS-HVR5-His6/pH5S by subcloning the hypervariable region (HVR) 1 of hexon into a previously constructed shuttle plasmid HVR5-His6/pH5S, which had His6 tag incorporated into the HVR5. This HVR1 DNA fragment containing a HIV epitope ELDKWAS was synthesized. HVR1-KWAS-HVR5-His6/pH5S was then linearized and co-transformed with linearized backbone plasmid pAd5/&#8710;H5 (GL) , for homologous recombination. This recombined plasmid pAd5/H5-HVR1-KWAS-HVR5-His6 was transfected into cells to generate the viral vector Ad5/H5-HVR1-KWAS-HVR5-His6. This vector was validated to have qualitative fitness indicated by viral physical titer (VP/ml), infectious titer (IP/ml) and corresponding VP/IP ratio. Both the HIV epitope and His6 tag were surface-exposed on the Ad5 capsid, and retained epitope-specific antigenicity of their own. A neutralization assay indicated the ability of this divalent vector to circumvent neutralization by Ad5-positive sera in vitro. Mice immunization demonstrated the generation of robust humoral immunity specific to the HIV epitope and His6. This proof-of-principle study suggested that the protocol associated with the Antigen Capsid-Incorporation strategy could be feasibly utilized for the generation of Ad5-vectored vaccines by modifying different capsid proteins. This protocol could even be further modified for the generation of rare-serotype adenovirus-vectored vaccines.

Here, we present a protocol to generate a proof-of-principle divalent adenovirus type 5 (Ad5) vector Ad5/H5-HVR1-KWAS-HVR5-His6 by utilizing the Antigen Capsid-Incorporation strategy. This vector was demonstrated to exhibit qualitative fitness, the capability to escape Ad5-positive sera in vitro, and the antigenicity as well as immunogenicity to the incorporated antigens.

Adenovirus serotype 5 (Ad5) has been extensively modified with traditional transgene methods for the vaccine development. The reduced efficacies of these ...

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.

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 ...

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.

Vaccinia virus (VACV) was instrumental in eradicating variola virus (VARV), the causative agent of smallpox, from nature. Since its first use as a ...

Construction of Adenoviral Vectors using DNA Assembly Technology

Adenoviral vectors have been used as a gene transfer tool in gene therapy for more than three decades. Here, we introduce a protocol to construct an adenoviral vector by manipulating the genomic DNA of wild-type HAdV-7 by using a DNA assembly method. First, an infectious clone of HAdV-7, pKan-Ad7, was generated by fusing the viral genomic DNA with a PCR product from plasmid backbone, comprising of the kanamycin-resistant gene and the origin of replication (Kan-Ori), through DNA assembly. This was done by designing a pair of PCR primers, that contained ~25 nucleotides of the terminal sequence of HAdV-7 inverted terminal repeat (ITR) at the 5' end, a non-cutter restriction enzyme site for HAdV-7 genome in the middle, and a template-specific sequence for PCR priming at the 3' end. Second, an intermediate plasmid-based strategy was employed to replace the E3 region with transgene-expressing elements in the infectious clone to generate an adenoviral vector. Briefly, pKan-Ad7 was digested with dual-cutter restriction enzyme Hpa I, and the fragment containing the E3 region was ligated to another PCR product of plasmid backbone by Gibson assembly to construct an intermediate plasmid pKan-Ad7HpaI. For convenience, restriction-assembly was used to designate the plasmid cloning method of combined restriction digestion and assembly. Using restriction-assembly, the E3 genes in pKan-Ad7HpaI was replaced with a green fluorescent protein (GFP) expression cassette, and the modified E3 region was released from the intermediate plasmid and restored to the infectious clone to generate an adenoviral plasmid pKAd7-E3GFP. Finally, pKAd7-E3GFP was linearized by Pme I digestion and used to transfect HEK293 packaging cells to rescue recombinant HAdV-7 virus. To conclude, a DNA assembly-based strategy was introduced here for constructing adenoviral vectors in general laboratories of molecular biology without the need of specialized materials and instruments.

In this study, an infectious clone of human adenovirus type 7 (HAdV-7) was constructed, and an E3-deleted HAdV-7 vector system was established by modifying the infectious clone. This strategy used here can be generalized to make gene transfer vectors from other wild-type adenoviruses.

Adenoviral vectors have been used as a gene transfer tool in gene therapy for more than three decades. Here, we introduce a protocol to construct an ...

AAV Deployment of Enhancer-Based Expression Constructs In Vivo in Mouse Brain

Enhancers are binding platforms for a diverse array of transcription factors that drive specific expression patterns of tissue- and cell-type-specific genes. Multiple means of assessing non-coding DNA and various chromatin states have proven useful in predicting the presence of enhancer sequences in the genome, but validating the activity of these sequences and finding the organs and developmental stages they are active in is a labor-intensive process. Recent advances in adeno-associated virus (AAV) vectors have enabled the widespread delivery of transgenes to mouse tissues, enabling in vivo enhancer testing without necessitating a transgenic animal. This protocol shows how a reporter construct that expresses EGFP under the control of a minimal promoter, which does not drive significant expression on its own, can be used to study the activity patterns of candidate enhancer sequences in the mouse brain. An AAV-packaged reporter construct is delivered to the mouse brain and incubated for 1-4 weeks, after which the animal is sacrificed, and brain sections are observed under a microscope. EGFP appears in cells in which the tested enhancer is sufficient to initiate gene expression, pinpointing the location and developmental stage in which the enhancer is active in the brain. Standard cloning methods, low-cost AAV packaging, and expanding AAV serotypes and methods for in vivo delivery and standard imaging readout make this an accessible approach for the study of how gene expression is regulated in the brain.

AAV Deployment of Enhancer-Based Expression Constructs In Vivo in Mouse Brain

This protocol describes a novel rAAV-based transient enhancer-reporter assay. This assay can be used to induce enhancer-driven expression in vivo in the mouse brain.

Enhancers are binding platforms for a diverse array of transcription factors that drive specific expression patterns of tissue- and cell-type-specific ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

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