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.

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			<td height="20" style="height:20px;width:343px;">Plasmid #41824</td>
			<td style="width:212px;">41824</td>
			<td style="width:137px;">Addgene</td>
			<td style="width:225px;">gRNA cloning vector</td>
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			<td>13426</td>
			<td>Addgene</td>
			<td>Cas9 cloning vector</td>
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			<td height="20" style="height:20px;">pGEMT easy</td>
			<td>A1360</td>
			<td>promega</td>
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			<td height="20" style="height:20px;">One Shot TOP10 Chemically Competent&#160;E .Coli</td>
			<td>C4040-10</td>
			<td>Invitrogen</td>
			<td>transformation</td>
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			<td height="20" style="height:20px;">QIAprep Spin Miniprep Kit&#160;</td>
			<td>27106</td>
			<td>Qiagen</td>
			<td>plasmid extraction</td>
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			<td height="22" style="height:22px;">Dulbecco&#8217;s Eagle&#8217;s Medium (DMEM)</td>
			<td>11965-092</td>
			<td>Life Technologies</td>
			<td>cell culture medium</td>
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			<td height="20" style="height:20px;">fetal bovine serum (FBS)&#160;</td>
			<td>SH30088.03HI</td>
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			<td>15070-063</td>
			<td>Thermo Scientific</td>
			<td>antibiotics</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Effectene</td>
			<td>301425</td>
			<td>Qiagen</td>
			<td>transfection</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Thermo Scientific Nalgene Cryogenic vial; 2.0 mL</td>
			<td>W-06754-96</td>
			<td>Thermo Scientific</td>
			<td>collect virus</td>
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		<tr height="21">
			<td height="21" style="height:21px;">fluorescence microscope&#160;</td>
			<td>Olympus BX51 Fluorescence&#160;</td>
			<td>Olympus</td>
			<td>find RFP-positive plque</td>
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		<tr height="20">
			<td height="20" style="height:20px;">DNeasy Blood &#38; Tissue Kit</td>
			<td>69506</td>
			<td>Qiagen</td>
			<td>DNA extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Extensor Long PCR ReddyMix Master Mix</td>
			<td>AB-0794/B</td>
			<td>Thermo Scientific</td>
			<td>PCR reagent</td>
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		<tr height="21">
			<td height="21" style="height:21px;">10cm culture dish</td>
			<td>Z688819-6EA</td>
			<td>sigma</td>
			<td>cell culture&#160;</td>
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			<td height="21" style="height:21px;">6-well plate</td>
			<td>Z707759</td>
			<td>sigma</td>
			<td>cell culture&#160;</td>
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			<td height="20" style="height:20px;">cell scraper</td>
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			<td>sigma</td>
			<td>scrap cells</td>
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			<td>T4299</td>
			<td>sigma</td>
			<td>trypsinize cells</td>
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			<td height="21" style="height:21px;">Virkon</td>
			<td>95015661</td>
			<td>Anachem Ltd</td>
			<td>desinfectant</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Falcon tube</td>
			<td>CLS430791-500EA</td>
			<td>Sigma</td>
			<td>hold cell suspension</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N1L forward 5&#8217;-TATCTAGCAATGGACCGT-3&#8217;</td>
			<td></td>
			<td>Sigma</td>
			<td>PCR primer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N1L reverse&#160; 5&#8217;-CCGAAGGTAGTAGCATGGA-3&#39;</td>
			<td></td>
			<td>Sigma</td>
			<td>PCR primer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">A52R forward 5&#8217;-ATAGGATTGTGTGCATGC-3&#8217;</td>
			<td></td>
			<td>Sigma</td>
			<td>PCR primer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">A52R reverse 5&#8217;-TTGCGGTATATGTATGAGGTG-3&#8217;</td>
			<td></td>
			<td>Sigma</td>
			<td>PCR primer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RFP forward 5&#8217;- GCTACCGGACTCAGATCCA-3&#8217;&#160;&#160;</td>
			<td></td>
			<td>Sigma</td>
			<td>PCR primer</td>
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		<tr height="20">
			<td height="20" style="height:20px;">RFP reverse 5&#8217;-CGCCTTAAGATACATTGATGAG-3&#8217;</td>
			<td></td>
			<td>Sigma</td>
			<td>PCR primer</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Argarose</td>
			<td>A7431</td>
			<td>Sigma</td>
			<td>resolve PCR product</td>
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		<tr height="20">
			<td height="20" style="height:20px;">G:BOX F3</td>
			<td>G:BOX F3</td>
			<td>Syngene</td>
			<td>UV gel doc</td>
		</tr>
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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="https://cloudflare-jove-com.remotexs.ntu.edu.sg/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="https://cloudflare-jove-com.remotexs.ntu.edu.sg/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="https://cloudflare-jove-com.remotexs.ntu.edu.sg/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="https://cloudflare-jove-com.remotexs.ntu.edu.sg/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="https://cloudflare-jove-com.remotexs.ntu.edu.sg/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

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Immunology and Infection

11.1K Views.  Queen Mary University of London. 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.

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

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in bioterrorism is also a concern. A better understanding of the cellular factors that impact infection would facilitate the development of much-needed therapeutics. Recent advances in RNA interference (RNAi) technology coupled with complete genome sequencing of several organisms has led to the optimization of genome-wide, cell-based loss-of-function screens. Drosophila cells are particularly amenable to genome-scale screens because of the ease and efficiency of RNAi in this system 1. Importantly, a wide variety of viruses can infect Drosophila cells, including a number of mammalian viruses of medical and agricultural importance 2,3,4. Previous RNAi screens in Drosophila have identified host factors that are required for various steps in virus infection including entry, translation and RNA replication 5. Moreover, many of the cellular factors required for viral replication in Drosophila cell culture are also limiting in human cells infected with these viruses 4,6,7,8, 9. Therefore, the identification of host factors co-opted during viral infection presents novel targets for antiviral therapeutics. Here we present a generalized protocol for a high-throughput RNAi screen to identify cellular factors involved in viral infection, using vaccinia virus as an example.

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Novel host factors involved in viral infection can be identified through cell-based genome-wide loss of function RNAi screening. A Drosophila cell culture model is particularly amenable to this approach due to the ease and efficiency of RNAi. Here we demonstrate this technique using vaccinia virus as an example.

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in ...

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal phase during establishment of infection in a given host. Consequently, study of encapsidation deciphers the information regarding the know-how of the mechanism regulating virus assembly to form infectious virions. Such information is vital in formulating novel methods of curbing virus spread and disease control. Virus encapsidation can be studied in vivo and in vitro. Genome encapsidation in vivo is a highly regulated selective process involving macromolecular interactions and subcellular compartmentalization. Therefore, study leading to dissect events encompassing virus encapsidation in vivo would provide basic knowledge to understand how viruses proliferate and assemble. Recently in vitro encapsidation has been exploited for the research in the area of biomedical imaging and therapeutic applications. Non-enveloped plant viruses stand far ahead in the venture of in vitro encapsidation of the negatively charged foreign material. Brome mosaic virus (BMV), a non-enveloped multicomponent RNA virus pathogenic to plants, has been used as a model system for studying genome packaging in vivo and in vitro. For encapsidation assays in Nicotiana benthamiana plants, Agrobacterium -mediated transient expression, refer to as agroinfiltration, is an efficient and robust technique for the synchronized delivery and expression of multiple components to the same cell. In this approach, a suspension of Agrobacterium tumefaciens cells carrying binary plasmid vectors carrying cDNAs of desiredviral mRNAs is infiltrated into the intercellular space withina leaf using nothing more sophisticated than a 1 ml disposable syringe (without needle). This process results in the transfer of DNA insert into plant cells; the T-DNA insert remains transiently in the nucleus and is then transcribed by the host polymerase II, leading to the transient expression. The resulting mRNA transcript (capped and polyadenylated) is then exported to the cytoplasm for translation. After approximately 24 to 48 hours of incubation,sections of infiltrated leaves can be sampled for microscopyor biochemical analyses. Agroinfiltration permits large numbers (hundreds to thousands) of cells to be transfected simultaneously. For in vitro encapsidation, purified virions of BMV are dissociated into capsid protein by dialyzing against dissociation buffer containing calcium chloride followed by removal of RNA and un-dissociated virions by centrifugation. Genome depleted capsid protein subunits are then reassembled with desired viral genome components or non-viral components such as indocyanine dye.

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

A simple, efficient and robust way to synchronize the delivery of multiple viral components to plant cells via Agrobacterium-mediated transient expression is described. This approach is amenable for studying replication, encapsidation followed by in vitro reassembly of non-viral components into genome depleted optical viral ghosts suitable for biomedical applications.

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal ...

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 ...

A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

Neutrophils are the most abundant leukocytes in peripheral blood. These cells are the first to appear at sites of inflammation and infection, thus becoming the first line of defense against invading microorganisms. Neutrophils possess important antimicrobial functions such as phagocytosis, release of lytic enzymes, and production of reactive oxygen species. In addition to these important defense functions, neutrophils perform other tasks in response to infection such as production of proinflammatory cytokines and inhibition of apoptosis. Cytokines recruit other leukocytes that help clear the infection, and inhibition of apoptosis allows the neutrophil to live longer at the site of infection. These functions are regulated at the level of transcription. However, because neutrophils are short-lived cells, the study of transcriptionally regulated responses in these cells cannot be performed with conventional reporter gene methods since there are no efficient techniques for neutrophil transfection. Here, we present a simple and efficient method that allows detection and quantification of nuclear factors in isolated and immunolabeled nuclei by flow cytometry. We describe techniques to isolate pure neutrophils from human peripheral blood, stimulate these cells with anti-receptor antibodies, isolate and immunolabel nuclei, and analyze nuclei by flow cytometry. The method has been successfully used to detect NF-&kappa;B and Elk-1 nuclear factors in nuclei from neutrophils and other cell types. Thus, this method represents an option for analyzing activation of transcription factors in isolated nuclei from a variety of cell types.

Neutrophils are the most abundant leukocytes in blood. Neutrophils possess transcriptionally regulated functions such as production of proinflammatory cytokines and inhibition of apoptosis. These functions can be studied with the method presented here, which allows detection and quantification of nuclear factors by flow cytometry in isolated nuclei

Neutrophils  are the most abundant leukocytes in peripheral blood. These cells are the first  to appear at sites of inflammation and infection, thus ...

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. ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Simple and Efficient Production and Purification of Mouse Myelin Oligodendrocyte Glycoprotein for Experimental Autoimmune Encephalomyelitis Studies

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), thought to occur as a result of autoimmune responses targeting myelin. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model of CNS autoimmune disease, and is typically induced via immunization with short peptides representing immunodominant CD4+ T cell epitopes of myelin proteins. However, B cells recognize unprocessed protein directly, and immunization with short peptide does not activate B cells that recognize the native protein. As recent clinical trials of B cell-depleting therapies in MS have suggested a role for B cells in driving disease in humans, there is an urgent need for animal models that incorporate B cell-recognition of autoantigen. To this end, we have generated a new fusion protein containing the extracellular domain of the mouse version of myelin oligodendrocyte glycoprotein (MOG) as well as N-terminal fusions of a His-tag for purification purposes and the thioredoxin protein to improve solubility (MOGtag). A tobacco etch virus (TEV) protease cleavage site was incorporated to allow the removal of all tag sequences, leaving only the pure MOG1-125 extracellular domain. Here, we describe a simple protocol using only standard laboratory equipment to produce large quantities of pure MOGtag or MOG1-125. This protocol consistently generates over 200 mg of MOGtag protein. Immunization with either MOGtag or MOG1-125 generates an autoimmune response that includes pathogenic B cells that recognize the native mouse MOG.

We describe a simple protocol using only basic lab equipment to generate and purify large quantities of a fusion protein that contains mouse Myelin Oligodendrocyte Glycoprotein. This protein can be used to induce experimental autoimmune encephalomyelitis driven by both T and B cells.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), thought to occur as a result of autoimmune responses ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed feeder cells, purified NK or T cells with as much as 10 mL of blood, or a high-dose of IL-2. This study presents a new method with a powerful and simple strategy to establish NK and T cell lines by culturing the peripheral blood mononuclear cells (PBMC) with the addition of recombinant human IL-2 (rhIL-2), and uses as little as 2 mL of whole blood. The cells can proliferate quickly in two weeks and be maintained for more than 3 months. With this method, 7 NK or T cell lines have been established with a high success rate. This method is simple, reliable, and applicable to establishing cell lines from more cases of CAEBV or NK/T cell lymphoma.

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...