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

1.目标RNA和Cas9构造和维修供体载体的制备<ol><li> gRNAs的克隆<ol><li>针对设计痘苗病毒按照先前25所列的原则N1L基因的引导-RNA靶序列。对准对牛痘病毒基因组中的导向RNA靶序列（在此情况下，痘苗病毒的利斯特株），以排除任何脱靶gRNA靶序列。 </li><li>合成和克隆gRNA寡成gRNA克隆载体如前面所述25。确认每个单独gRNA在所得载体用Sanger测序33的序列。命名产生gRNA建设作为gRNA N2 33。 注:gRNA目标站点是N1L基因内，并且是在左臂和右臂之间的区域中，该修供体载体盖（ 见图1）。 </li></ol></li><li>克隆Cas9基因的核定位信号（NLS）缺少进pST1374矢量并指定构建物PST-Cas9 33。 注意:任何哺乳动物表达克隆载体可用于Cas9表达载体的构建。 </li><li>人力资源修复捐助载体的构建<ol><li>使用VV的N1L基因作为目标区域进行修改33。确保左右臂的长度各自是约500-600碱基对，和侧翼目标区域。 注:左手臂/右手臂最多可以有与目标区域50个基点重叠（ 见图1）。 </li><li>克隆N1L区域修复供体载体如先前33,34描述和指定维修供体载体为PT-N1L。 注:请参阅图1的修为供体载体和它的目标区域。维维的其它区域可使用区域（基因）特异性gRNA（S）和特定区域的修复供体载体进行定位。任何克隆载体可用于修复供体载体的构建。 </li> &lt;/ OL&gt; </li><li>质粒的拓展<ol><li>变换质粒入化学感受态根据制造商的说明大肠杆菌 。纯化使用质粒提取试剂盒指的是由制造商提供的协议的质粒。 </li></ol></li></ol> 2.种子细胞（1天） <ol><li>保持CV-1细胞（猴肾成纤维细胞）在Dulbecco氏改良Eagle氏培养基（DMEM），补充有5％胎牛血清（FBS），100U / mL青霉素和100微克/毫升链霉素，在37℃用5％的CO 2。 </li><li> Trypsinize CV-1细胞是80-90％汇合<ol><li>除去从含有CV-1细胞在T-175烧瓶中的细胞培养基。冲洗用5mL无菌PBS的单层，以除去残留的血清和吸出PBS中。加2.5毫升1×胰蛋白酶-EDTA溶液，以覆盖细胞，并放置在烧瓶中37℃培养箱中约23分钟，以帮助细胞剥离。 </li><li>加10毫升细胞培养基（参见步骤2.1），以暂停通过温和移液动作的细胞。算使用血球细胞数。 </li><li>种子细胞在6孔板<ol><li>种子2×10 5个 CV-1细胞在2ml细胞培养基的成6孔板的每个孔中的转染的前一天。 注意:一个附加的离心步骤以除去胰蛋白酶是不需要的。 </li></ol></li></ol></li></ol> 3. Cas9质粒和gRNA质粒转染（2日） <ol><li>转染CV-1细胞用0.5微克PST-Cas9质粒和使用根据制造商的说明转染试剂0.5微克gRNA N 2质粒（在步骤2.2.3.1制备）。 </li><li>孵育24小时的细胞在培养箱（37℃，5％的CO 2），然后替换用2毫升的新鲜细胞培养基（在步骤2.1中所述）的介质。 </li></ol> 4.感染CV-1细胞及穿梭供体的V埃克特转染HR（3日） <ol><li>稀骨干VVL15病毒用DMEM细胞培养基以2×10 5 PFU / mL的浓度。 Cas9和gRNA质粒的24小时后转染，添加100微升稀释的病毒进入转染CV-1细胞的每个孔中。 </li><li> 2小时后的病毒感染，转染1.0μg的穿梭供体载体成使用根据制造商的说明转染试剂VVL15感染细胞的人力资源。放置转染的细胞在37℃培养箱，5％CO 2下24小时。 </li></ol> 5.收获重组病毒（4日） <ol><li>在步骤4.2的HR穿梭供体载体转染的24小时后，分离使用细胞刮转染CV-1细胞。收集细胞悬液到一个离心管，并在-80℃以供将来使用存储。 注:立即擦去消毒剂任何细胞培养液溢出，然后用70％乙再次擦拭醇。 </li></ol> 6.纯化改性VV（第一轮） <ol><li>在第1天，接种15 6孔板用3×10 5个 CV-1细胞在每孔中。 <ol><li>第2天，解冻来自步骤5.1的水浴中的冷冻的细胞悬浮液，在37℃，3分钟，并大力旋涡振荡冷冻管30秒，以获得细胞裂解物不去除细胞碎片。 </li></ol></li><li>感染CV-1细胞中的一个6孔板的，稀释细胞裂解物的1微升用3mL的DMEM，并添加0.5毫升稀释的细胞裂解物进6孔板的每个孔中，对媒体顶部已经当下。感染所有在步骤6.1中制备的6孔板中。 注:不需要的细胞碎片的去除。 </li><li>感染后两天（ 即第4天），发现RFP阳性斑块使用10X物镜在荧光显微镜下，并贴上标签斑块用记号笔由盘旋斑块位置板下方。 注:请参阅<st荣&gt;图3为代表的RFP阳性牌匾。 <ol><li>制备含有200微升无血清的DMEM细胞培养基中拾起六个不同的RFP阳性噬斑6标记的冷冻管。吸从井用标记斑块（S）（步骤6.3）的细胞培养基。附加200微升尖端P200的吸管，将音量设置为30μL，并采取〜从一个冷冻管10微升的细胞培养基。 </li><li>握住吸管按钮而不释放介质，并使用前端划伤一种标记斑块的面积。松开吸管按钮抬起脱落的细胞，并将其转移到含有无血清DMEM 190微升冷冻管。 </li><li>乘坐从与划伤细胞（从最后一步）小瓶另外10μL中，重复拿起相同的RFP阳性斑块三次，以收集尽可能多的划伤细胞尽可能的过程。传输所有细胞进入同一小瓶中，将小瓶储存于-80℃。 </ol></li></ol> 7.纯化改性VV（第二轮） <ol><li>种子3×10 5个 CV-1细胞到每个孔六6孔板并培养所述细胞24小时。 </li><li>解冻在37℃水浴冷冻冷冻管（从步骤6.3.3）3分钟，然后涡冷冻管剧烈30秒，以获得细胞裂解物。 <ol><li>添加混合细胞裂解物的0.5微升至6孔板的一个孔中。感染每个斑块一个6孔板中。用5％的CO 2 2天（第二轮噬斑纯化）孵育VV感染6孔板在37℃。然后重复第6.3节。 注:六点多RFP阳性斑块可以捡到;两个或更多个6孔板可用于各斑块纯化。 </li></ol></li></ol> 8.斑块普瑞更多轮fication <ol><li>进行进一步的三到五轮纯化的以下步骤7中所述，直到从一个阳性噬菌斑形成的所有的噬菌斑表现为RFP阳性显微镜下相同的协议。 注意:然后，这个斑块被认为是纯粹的。 </li><li>一旦斑块是纯粹的（ 图4，表达RFP所有被感染的细胞），如6.3节所述收获了积极的牌匾进入混和，存于-80℃。验证以下步骤9和10中描述的协议的斑块。 </li></ol> 9.展开纯化的斑块（S） <ol><li>种子3×10 5个 CV-1细胞到病毒感染前的6孔板一天每孔中。 </li><li>解冻从8.2冷冻冷冻管在37℃水浴中3分钟以获得细胞裂解物。添加所有溶胞产物（不除去细胞碎片）到CV-1细胞的6孔平板的一个孔中（在步骤9.1接种）。孵育感染CV-1细胞在37℃，5％CO 2的最多3天。 <ol><li>展开所有纯化的斑（至少三个噬斑）。 注:感染时间从1-3天，取决于病毒的裂解物的量而变化。 </li></ol></li><li>收获使用细胞刮刀VV感染的细胞时，50％以上的细胞显示在显微镜下观察细胞病变效应（细胞被四舍五入，容易脱落和RFP阳性）。用细胞培养基到15毫升管收集脱离的细胞一起。 <ol><li>离心以300g沉淀细胞3分钟。去除上清，并保持细胞沉淀在-80℃直至进一步使用或立即使用细胞沉淀为步骤10。 </li></ol></li></ol> 10.突变VV的利用PCR改性的验证<ol><li>提取用DNA提取试剂盒根据制造商的协议，在步骤9.3.1制备的细胞沉淀VV的DNA。洗脱用的双蒸水200微升的DNA。 注意:使用粒料的体积的一半以提取的DNA，并保持其余为病毒产生，如果克隆被验证为含有正确修饰/插入。 </li><li>所述N1L基因的缺失和RFP基因的插入N1L区域的验证。 <ol><li>扩增跨越N1L基因（L025），并使用设计针对N1L基因的引物的L026基因的DNA片段。使用放大RFP特异性引物RFP基因。扩增使用特定A52R-引物通过PCR跨越A52R一个对照DNA片段。 注:请参阅材料清单对特异性引物。 <ol><li>使用PCR主混合试剂盒进行PCR。设置25使用DNA模板的2微升PCR反应100μL，变性在94℃，PCR反应进行2分钟，然后运行下列步骤的PCR 30个循环:在94℃使DNA变性15秒，然后退火引物的DNA模板在52℃15秒延伸72℃30秒的PCR反应。 </li></ol></li></ol></li><li>解决使用1％琼脂糖凝胶33的PCR产物。捕获使用UV胶文档的凝胶图像。如果N1L PCR是阴性和A52R和RFP的PCR是阳性，那么斑块中正确的N1L区域进行修改。 </li></ol>

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Authors have no competing financial interests.

有关于用于治疗目的VV的修改两个主要目标。之一是要删除一个特定的基因，如胸苷激酶（TK）基因，以衰减病毒抗肿瘤的治疗用途。另一种是手臂VV与期望的治疗基因（如GM-CSF）或标记基因（如荧光素酶基因）。实现任一这些涉及的目标区域/基因的缺失。一个直接的DNA连接方法35和体外包装方法36先前已被用来产生的TK基因突变的修改痘苗病毒。然而，这些方法都对基因组中的其他区域的突变应用由于缺乏在整个基因组的独特限制酶位点的限制。当前的方法来创建突变的VV涉及用选择标记（RFP或GFP）到CV-1细胞感染VV后两小时的穿梭（供体）载体的转染以诱导同源重组incorporati纳克选择标记到目标区域。标记阳性噬斑的选择，随后可以通过纯化后24小时转染。斑块净化过程可能需要长达10个回合，最后3-4周，并常常导致不希望重组病毒插入在非目标区域选择标记。 DNA双链断裂能有效诱导同源重组在哺乳动物细胞37，以及通过利用这种机制，产生突变的VV的效率可以大大提高。所述gRNA引导Cas9系统已在基因组中的编辑被成功地并增强同源重组的效率在真核生物22,25。最近，在宿主细胞中我单纯疱疹病毒是由gRNA引导Cas9编辑腺病毒和类型的基因组系统24。最近已经证明，CRISPR Cas9系统是用于有效地编辑VV基因组，构建一个VV载体用于表达的有力工具特定基因。 既N1L gRNA制导Cas9和传统的同源重组（HR）方法导致在N1L的相同靶位点成功的人力资源的事件。然而有趣的是gRNA引导Cas9引起HR在更高水平的效率比传统的人力资源的方法。因此，使用gRNA制导Cas9的无需纯化许多斑块以获得突变的VV。在这项工作中，我们显著提高用于产生使用gRNA引导Cas9系统33突变的VV的效率和时限。值得注意的是，用于获得同源重组的一种高效率的一个关键步骤是在执行传统的同源重组之前转染CV-1细胞用表达Cas9和gRNA质粒24小时。在24小时的时间间隔允许Cas9和gRNA至在合理的水平来表示。此外，靶向特定基因的gRNA序列可能需要被优化的，因为我们发现，不同gRNA序列吨argeting N1L基因的效率33，34变化。 在编辑VV为CRISPR / Cas9的限制是在第一轮重组RFP阳性斑块的低速率。以获得足够数量的含有重组病毒的RFP阳性噬斑，通常至少十个，需要6孔板，这使得第一轮筛选乏味。因此，仍有需要优化来克服这种限制的协议。 RFP阳性噬斑的小尺寸是另一个潜在的问题，这是由于用于感染6孔板高容量裂解物。为了克服这个，裂解物的一个较小的体积，应用于感染6孔板，以获得更大的尺寸的RFP阳性噬菌斑。使用裂解物的一个更小的体积也将使从RFP-消极的RFP阳性噬斑的更好的分离。因此较少轮噬斑纯化的需要获得纯的RFP阳性噬菌斑。 <p类="jove_content"&gt;关靶效应总是在基因的编辑不需要的问题。 CRISPR-Cas9表明脱靶效应。然而，随着gRNA的精心设计，脱靶可以编辑VV基因组的33，34时避免了影响。这是使用gRNA引导Cas9系统用于编辑VV的基因组的特定益处。由于VV的承诺，使用CRISPR / Cas9系统将加快生物医学研究和转化医学突变些VV的发展。此外，这样的系统也将加速在细胞生物学的发现，例如用于其基于肌动蛋白的蠕动使用VV的信号通路的清扫。

痘苗病毒（VV）是属于痘病毒家族包膜DNA病毒，并且在消灭天花的药物最伟大的成就之一，起到了至关重要的作用。在消灭了天花后时代，维维已经发展成为通过插入由各种病原体基因导入VV载体基因交付防治艾滋病毒和其他传染性疾病的疫苗1-7的向量。 VV也被广泛地用作载体的癌症免疫治疗8-14特别是对于肿瘤靶向复制溶瘤痘苗病毒的发展。为了创建与癌细胞改进选择性的有效疫苗载体，病毒基因组内的修改是必要的，包括基因缺失或导入治疗基因。 用DNA技术的发展和更好地理解分子生物学和病毒学，外源DNA的插入到VV最初实现d。使用同源重组（HR）20世纪80年代15。这种方法仍然是广泛用于构建VV载体。遗传修饰的导入是通过使用了HR的穿梭载体，它与在预感染的细胞的基因组中的VV重组实现。然而，这种方法已被证明是非常低效的（小于1％的同源重组效率16）和通常导致选择标记的随机插入非目标区域和/或在病毒扩张所述标记的损失。 DNA的同源重组在基因组位点插入的外源DNA的效率可以在双链断裂（DNA双链断裂）17的存在而显着提高。因此，可以在目标位点DNA双链断裂诱导技术适用于VV基因组工程的巨大潜力。 最近开发的CRISPR-Cas9系统显示在任何VV基因区域触发DNA双链断裂的承诺。 CRISPR-Cas9是RNA-引导核酸酶参与打击入侵的噬菌体等外来遗传物质18-20适应性免疫。有在一个范围内的微生物物种21三CRISPR-CAS系统。 II型CRISPR-CAS系统广泛用于编辑真核细胞和大的病毒的基因组。它由RNA导向Cas9核酸内切酶（由化脓性链球菌 ）中，单个导的RNA（因组）和反式激活crRNA（tracrRNA）22-24。所述Cas9 /因组复杂识别在哺乳动物细胞22 5&#39;-NGG-3&#39;Protospacer相邻基序（PAM）的序列，23之前的互补20-核苷酸基因组序列，已被成功地用于有效产生遗传修饰的细胞，病毒和动物模型22-32。 所述CRISPR-Cas9系统已被证明是对基因组中的VV的细胞质与同源重组结合靶向的有效工具感染的CV-1细胞s至产生突变牛痘病毒33,34。为了延长该方法的潜在应用，我们提出有关此系统的方法的详细信息。所述协议可用于创建与特定的基因缺失的突变体VV和/或与治疗性转基因臂的突变病毒。

<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.

对于建设一个新的VV载体，关键出发点是设计和建造，可以针对基因组的特定区域供体穿梭载体。在这项研究中，VV N1L基因被用作例子的目标。施主穿梭载体的重组，并在VV目标区域的盒中示出在图1中，为了提高同源重组的效率，表达Cas9和N1L特异性gRNA质粒共转染入CV -1细胞48小时之前，执行所述同源重组33。一天（24小时）转染后，RFP在CV-1细胞（ 图2）表示。 CV-1细胞的感染与来自同源重组整个裂解物（步骤5）后，RFP阳性和阴性噬菌斑均在CV-1细胞（ 图3）观察到。以下所描述的纯化方案，一个纯粹的噬斑共<html>经过3-5轮的净化ULD获得。如在图4中所示，感染与来自与突变牛痘病毒纯菌斑的细胞裂解物后所有斑块提出了RFP信号。为了验证是否纯突变的VV具有正确的基因修饰，用PCR，确认没有靶N1L基因的， 如图5的改性病毒的PCR显示为RFP阳性信号和用于N1L缺席信号（ 图5车道AG），而N1L为控制病毒（CTR）具有完整N1L区域的PCR检测结果为阳性。 A52R的控制DNA片段呈阳性所有的重组病毒和CTR病毒。在RFP阳性斑块人力资源的效率是在这个实验中（这是高达94％，在以前的工作34）×100％（6/6）。本文所描述的方法已超过100倍相比传统协议33,34提高了复合效率。 内容"FO:保together.within页="1"&gt; <img alt="图1" src="/files/ftp_upload/54171/54171fig1.jpg" /> 图1:供体穿梭载体的同源重组的盒，并在VV基因组中的目标区域的纯化标记基因RFP由H5启动子驱动。针对在RFP基因N1L区域结果修复供体载体被纳入同源重组后N1L区域。 "X"表示在修复供体载体，将取代在牛痘病毒基因组相同的序列同源序列。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54171/54171fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54171/54171fig2.jpg" /> 图2:同源重组后的CV-1细胞一天成像一天AFTE- [R同源重组，感染VV和维修供体载体转染的细胞是RFP阳性答 :相衬图像（放大100X）B:荧光显微镜图像（放大100倍）。比例尺= 50微米。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54171/54171fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54171/54171fig3.jpg" /> 图3:在突变病毒的第一轮的纯化的RFP阳性噬菌斑在突变病毒的第一轮纯化，将RFP阳性斑块（用红线中圈出）与目标区缺失是通过由形成斑块包围野生型病毒（与黄线盘旋。）A:相衬图像（放大100X）B:荧光MICRoscopy图像（放大100倍）。比例尺= 50微米。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54171/54171fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54171/54171fig4.jpg" /> 图4:纯突变VV后3-5轮的净化，纯净的斑块获得所有斑块RFP阳性答 :相衬图像（放大100X）B:荧光显微镜图像（放大100X） 。比例尺= 50微米。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54171/54171fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54171/54171fig5.jpg" /> 图5:Verificatio纯的突变体VV的DNA从VV萃取的n个感染CV-1细胞。 N1L通过PCR使用引物N1L验证了目标区域的缺失，RFP的插入N1L区通过PCR用引物的RFP证实。泳道AF对应六个纯化的噬菌斑，泳道G是N1L缺失先前的工作33验证一个控制斑块，泳道CTR（对照）是野生型牛痘病毒（与N1L区完整）。 A52R基因扩增为所有样品对照基因。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54171/54171fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</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

牛痘病毒（VV）已被广泛用于生物医学研究和人体健康的改善。本文介绍使用CRISPR-Cas9系统编辑VV基因组的简单，高效的方法。

一种简单有效的方法构建突变牛痘病毒载体

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

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

11.1K 次观看.  Queen Mary University of London. 牛痘病毒（VV）已被广泛用于生物医学研究和人体健康的改善。本文介绍使用CRISPR-Cas9系统编辑VV基因组的简单，高效的方法。 

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

Packaging HIV- or FIV-based Lentivector Expression Constructs &amp; Transduction of VSV-G Pseudotyped Viral Particles

As with standard plasmid vectors, it is possible to transfect lentivectors in plasmid form into cells with low-to-medium efficiency to obtain transient expression of effectors. Packaging lentiviral expression constructs into pseudoviral particles, however, enables up to 100% transduction, even with difficult-to-transfect cells, such as primary, stem, and differentiated cells. Moreover, the lentiviral delivery does not produce the specific cellular responses typically associated with chemical transfections, such as cell death resulting from toxicity of the transfection reagent 1, 2. When transduced into target cells, the lentiviral construct integrates into genomic DNA and provides stable expression of the small hairpin RNA (shRNA), cDNA, microRNA or reporter gene 3, 4. Target cells stably expressing the effector molecule can be isolated using a selectable marker contained in the expression vector construct such as puromycin or GFP. After pseudoviral particles infect target cells, they cannot replicate within target cells because the viral structural genes are absent and the long terminal repeats (LTRs) are designed to be self-inactivating upon transduction 5, 6.
There are three main components necessary for efficient lentiviral packaging 1, 5, 6, 7. 
1. The lentiviral expression vector that contains some of the genetic elements required for packaging, stable integration of the viral expression construct into genomic DNA, and expression of the effector or reporter. 
2. The lentiviral packaging plasmids that provide the proteins essential for transcription and packaging of an RNA copy of the expression construct into recombinant pseudoviral particles. This protocol uses the pPACK plasmids (SBI) that encode for gag, pol, and rev from the HIV or FIV genome and Vesicular Stomatitis Virus g protein (VSV-G) for the viral coat protein. 
3. 293TN producer cells (derived from HEK293 cells) that express the SV40 large T antigen, which is required for high-titer lentiviral production and a neomycin resistance gene, useful for reselecting the cells for maintenance.
An overview of the viral production protocol can be seen in Figure 1. Viral production starts by co-transfecting 293TN producer cells with the lentiviral expression vector and the packaging plasmids. Viral particles are secreted into the media. After 48-72 hours the cell culture media is harvested. Cellular debris is removed from the cell culture media, and the viral particles are precipitated by centrifugation with PEG-it for concentration. Produced lentiviral particles are then titered and can be used to transduce target cells. Details of viral titering are not included in this protocol, but can be found at:
<a href="http://www.systembio.com/downloads/global_titer_kit_web_090710.pdf" target="_blank">http://www.systembio.com/downloads/global_titer_kit_web_090710.pdf</a> 8.
This protocol has been optimized using the specific products indicated. Other reagents may be substituted, but the same results cannot be guaranteed.

Packaging HIV- or FIV-based Lentivector Expression Constructs & Transduction of VSV-G Pseudotyped Viral Particles

Lentiviral expression vectors are the most effective vehicles for stably expressing different effector molecules or reporter constructs in dividing and non-dividing mammalian cells and whole organisms. Here we provide a protocol on how to package lentivector expression constructs in pseudoviral particles and to transduce target cells using the pseudoviral particles.

包装艾滋病毒或FIV的基于Lentivector表达结构与VSV-G假型病毒颗粒转导

As with standard plasmid vectors, it is possible to transfect lentivectors in plasmid form into cells with low-to-medium efficiency to obtain transient ...

科研

免疫与感染

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.

肿瘤靶向免疫调节副病毒的设计与评价

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

癌症研究

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.

工程和演变的合成腺相关病毒（AAV），基因治疗载体，通过DNA家族改组

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.

病毒遗传背景明确的神经回路跟踪

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

神经科学

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.

从救援的cDNA重组新城疫病毒

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.

有效生成荧光标记疫苗病毒蛋白的方法

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.

利用衣壳抗原掺入战略的腺病毒血清型5载体疫苗方法的发展

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.

用 crispr/cas9 基因编辑生成重组禽流感病毒载体

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.

使用主机范围和视觉选择快速、无缝地生成重组痘病毒

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 ...