笔者想感谢谭氏闵女士，海伦·Ong女士和越·Yi博士与毛细管电泳实验帮助。这项工作是由NMRC / IRG授予NMRC /二千零十一分之一千三百一十四和教育部ACRF二级基金赠款MOE2011-T2-1-051支持。

1.获取CRISPR / Cas9靶向单细胞克隆<ol><li>在6孔板种子HepG2细胞以每孔500,000个细胞在2mL不含抗生素的Dulbecco补充有10％胎牛血清（FBS）改良的Eagle培养基（DMEM）的。孵育在37℃下24小时和5％CO 2。 </li><li>转染细胞用质粒共表达使用适当的转染试剂，根据制造商的说明书和Cas9针对目标基因特异性因组。 注意:例如，因组可以克隆到pSpCas9（BB）-2A-GFP载体，如先前4所述。 </li><li>更换培养基4 - 用2mL新鲜的无抗生素的培养基的转染后16小时。 </li><li>转染后约48小时，通过胰蛋白酶将细胞收集单细胞悬浮液。 <ol><li>胰蛋白酶化将细胞在0.2毫升0.25％胰蛋白酶-EDTA，并在37℃下孵育5分钟。加入1毫升培养基中并悬浮thoroughl年。 </li></ol></li><li>排序为GFP阳性克隆细胞，如前所述12，并收集约3,500细胞。 </li><li>板10-cm培养皿中GFP阳性分选的细胞在每皿500，1000和2000细胞在8毫升青霉素/链霉素补充的培养基。孵育细胞在37℃和5％CO 2。 </li><li>保持在37℃和5％CO 2的细胞，更换培养基每五天，直到它们长成单细胞集落大到足以可见肉眼。对于大多数癌细胞系，这需要从电镀每天约两个星期。 </li><li>当菌落具有适当的大小（ 即，可见肉眼）的，单独的菌落转移到含DMEM的200μL96孔培养板的孔补充有10％FBS。 <ol><li>使用200-μL吸管介质的小体积吸出单细胞集落。悬浮细胞彻底在井灵敏度独立通过研磨几次。 </li></ol></li><li>保持在37℃和5％CO 2的细胞，更换培养基每五天，直到它们达到50 - 90％汇合。对于大多数癌细胞系，这需要约24 - 48小时。 </li></ol> 2.原油提取基因组DNA，使用直接裂解法<ol><li>当细胞达到50 - 90％汇合，从孔中尽可能使用多通道真空抽吸或者多通道移液管除去尽可能多的培养基。 </li><li>添加0.05％胰蛋白酶-EDTA（不含酚红）的25μL到每个孔中，并在37℃下孵育7分钟。 </li><li>移液器向上和向下几次重悬胰酶消化细胞彻底。在显微镜下检查细胞以确保它们从塑料表面分离。 </li><li>通过转移约5的单细胞悬液的μL到一个空的96孔培养高原创建各克隆的复制即添加培养基的200微升到每个孔中并保持细胞直到阳性克隆使用荧光PCR-毛细管凝胶电泳（见下文）来识别。连续展开的细胞至10厘米培养皿或选择​​的任何其他比例（见第7节）。 </li><li>从步骤2.3加入5μL的单细胞悬液，以10μL自制直接裂解缓冲液（10毫摩尔Tris pH 8.0中，2.5毫摩尔EDTA，0.2M NaCl的，0.15％SDS，和0.3％吐温-20）12以96孔PCR板和移液器向上和向下几次调匀。短暂离心（把液体倒在井的底部）。 </li><li>从步骤2.3添加培养基的200微升细胞悬浮液的剩余〜15μL，并且在37℃和5％CO 2孵育，与来自步骤2.4的复制在一起。 </li><li>受试者步骤2.5以下热循环程序中的裂解物中，以确保细胞完全裂解和基因组DNA的释放:65℃30秒，8°C，30秒，65℃1.5分钟，97℃3分钟，8℃下1分钟，65℃3分钟，97℃1分钟，65℃1分钟，和80℃10分钟。离心裂解液短暂。 </li><li>通过加入不含核酸酶的水40微升稀释裂解物，并使用涡旋混合器调匀。短暂离心。将稀释的裂解物可以立即使用或储存于-20℃下数月而无质量的显著损失。 </li></ol> 3.执行荧光PCR扩增到CRISPR / Cas9目标区域<ol><li>设计中的两个荧光团标记的正向引物（都标记在5&#39; 端），用于每个CRISPR / Cas9目标区域;切割是比300bp的彼此应当被认为是两个分开的目标区域（ 例如，绿色荧光团标记的引物用于非靶向野生型对照和用于CRISPR / Cas9靶向克隆蓝色荧光团标记的引物进一步的网站;参见本草的表<html> LS）。 <ol><li>促使这些标记的正向引物和未标记的反向引物相应。需要注意的是引物可以使用所选择的任何工具来设计和扩增子应为200 - 500布普·隆恩。 </li></ol></li><li>如先前描述的12以扩增使用标记的引物的目标区域进行PCR。 <ol><li>从步骤2.8在20-μL反应使用稀释裂解物的3微升（ 见表1）和下面的热循环程序:94℃10分钟（1个循环）; 94℃10秒，64℃30秒，68℃1分钟（4个循环）; 94℃10秒，61℃30秒，68℃1分钟（4个循环）; 94℃10秒，58℃30秒，68℃1分钟（4个循环）; 94℃10秒，55℃30秒，和68℃1分钟（35个循环）。 </li></ol></li><li>解决的PCR扩增子5μL在1％琼脂糖凝胶上以检查尺寸和扩增子的相对量SS = "外部参照"&gt; 12。 注意:在执行此步骤的所有样品被鼓励，但不是必需的;解决样品中所选择的数量足以估计存在于一般的样品中的扩增子的量。 </li></ol> 4.准备样品毛细管电泳<ol><li>稀野生型（非靶向亲本细胞）和CRISPR / Cas9靶向DNA在无核酸酶水的扩增子以约2.5纳克/微升。确保足够稀释的野生型DNA的样品要被添加到每个目标DNA样品（最小每靶向样品稀释野生型样品的0.5μL的是必需的）。 </li><li>混合在等于比率稀释的野生型和有针对性的DNA样品（ 例如，野生型混合样品的1微升1微升样品靶向的）。 </li><li>加入1微升的混合的扩增子的870μL的去离子甲酰胺和的染料标记的大小标准品0.3μL在96孔PCR板，其与遗传的兼容alyzer。 注意:使用甲酰胺的主混合物和尺寸标准的（ 即，甲酰胺的预混物的制备，并在29尺寸标准:在加入扩增子之前1的比例，以确保规范化的量）被推荐。 </li><li>紧密地密封所述板，并使用PCR热循环在95℃下加热样品3分钟。 </li><li>置于冰上的板在加热步骤之后，立即并孵育至少3分钟。 </li></ol> 5.上的遗传分析仪执行毛细管凝胶电泳<ol><li>设置测定参数，仪器协议，以及与之相连的遗传分析仪毛细管电泳软件大小呼协议。 注:此步骤只需要对第一电泳运行;该程序可以保存以备将来使用。对于随后的运行中，请直接执行步骤5.2。 <ol><li>点击该软件的仪表板"创建新盘"图标。 </li><li>写出R联合国一个虚拟名称，然后选择下列选项:96井的数量;板型，片段;毛细管长度50厘米;和聚合物，POP7。点击"分配板内容"按钮。 </li><li>在"试验"，单击"创建新分析";一个新的面板将出现。 </li><li>命名法"FPCR-CGE分析"，检查"应用型"正确设置为"片段"。 </li><li>点击下的"新建"按钮设置仪器协议"仪器协议。" <ol><li>设置或选择合适的领域中，以下选项和参数:应用类型，片段;毛细管长度50厘米;聚合物，POP7;染料组，G5;运行模块，FragmentAnalysis;协议名称，FPCR-CGE仪器协议;烘箱温度，60℃;运行电压19.5千伏;预运行电压15千伏;注射电压，1.6千伏;运行时间，1330秒;预运行时间，180秒;注射时间15秒;和数据延迟，1秒。 </li></ol></li><li>氯在"应用到分析"按钮，然后单击"保存到库"按钮ICK保存程序。关闭面板继续。 </li><li>点击下的"新建"按钮，设置一个大小调用协议"Sizecalling协议。" <ol><li>设置或选择合适的领域中，以下选项和参数:协议名称，FPCR-CGE Sizecalling协议;尺寸标准，GS500（-250）LIZ;大小呼叫者，SizeCaller V1.1.0;分析设置， - ;分析范围，全面;浆纱程，全;大小调用方法，局部南;引物峰，现状;蓝，绿，橙频道，（检查）;最小峰高，175对所有信道;使用平滑，无;使用基线（基线窗口（PTS）），（检查）51;最小半峰宽，2;峰值窗口大小，15;多项式度，3;斜率阈值峰开始，0.0;斜率阈值峰结束，0.0; QC设置， - ;尺寸画质， - ;如果失败值&lt;0.25;通过如果值&lt;0.75;从0基点至80假定线性BP 0;如果Actuate的上拉标志上拉率≤0.1和上拉扫描≤1。 </li></ol></li><li>点击"应用到分析"按钮，然后单击"保存到库"按钮保存程序。关闭面板继续。 </li><li>确保试验是通过点击"保存到库"按钮即可保存，并通过点击"关闭"按钮退出面板。 </li><li>回到"分配板内容"页上，单击下的"创建新的文件名公约"链接"文件名称约定。" </li><li>命名方案"FPCR-CGE文件名。" </li><li>在"可用属性"，选择将显示在文件名中所需的属性（如"运行日期"，"运行时间"，"好位置"和"样品名称"）。选择在何处运行的结果将保存所需文件的位置。 </li><li>点击"应用到分析"按钮，然后"保存到库"按钮保存程序。关闭面板继续。 </li><li>回到"分配板内容"页上，单击下的"创建新的结果组"链接"结果组"。 </li><li>命名方案"FPCR-CGE结果组"。 </li><li>在"可用属性"，选择将显示在文件名中所需的属性（如"分析名称"）。选择在何处运行的结果将保存所需文件的位置。 </li><li>点击"应用到分析"按钮，然后单击"保存到库"按钮保存程序。关闭面板继续。 </li></ol></li><li>建立程序按以下步骤运行电泳。 <ol><li>转至控制台，然后单击"创建新盘"图标。 </li><li>命名如所期望的运行（以便于参考，包括日期，细胞系，和基因名称）。选择下列选项:井数，96;板型，片段;毛细管长度50厘米;和聚合物，POP7。点击"分配板内容"按钮。 </li><li>作为所需的标签样品的每个孔中（ 例如，样品可以被命名为"NC"，以指示该阱是阴性对照）。 </li><li>在"试验"，"文件名称约定"和"结果组"框中，单击"添加从库"链接，然后选择在步骤5.1.3创建5.1.17的程序。 </li><li>选中所有要分析的井和下选择相关项目"试验"，"文件名称约定"和"结果组"通过检查旁边的框。 </li></ol></li><li>遗传分析仪的托盘上装载板之前，可在96孔板的橡胶密封件，把所述密封板在随附基因分析仪塑料外壳。 </li><li>按下"托盘"按钮在遗传分析仪的前部，并在托盘重新时酸痛的设备的前部，打开门并加载包裹板在托盘上。确保板锁定到位，然后关上门。 </li><li>点击"链板的运行"按钮。 </li><li>在"加载板的运行"页面，做最后的检查，以确保所有的试剂和条件是正确的，为了。 </li><li>点击"开始→运行"按钮。的24个样品（96孔板的第一3×8孔）中每个运行时间不超过55分钟来完成。 </li></ol> 6.电泳的分析，以确定碱基对的差异<ol><li>当毛细管电泳运行完成后，打开分析软件分析结果。 </li><li>点击"添加样品到项目"图标，然后搜索包含运行文件的文件夹。从步骤5.1.12结果文件的指定地点回忆。 </li><li>在完成的游程和CL选择每次注射的所有结果文件ICK"添加到列表"按钮;这些文件的名称以"汽化室"，并包含运行的详细信息。 </li><li>点击"添加和分析"按钮进行分析。 </li><li>通过点击第一个样品，拖动光标到最后样本选择运行中所有的样本，然后点击"显示绘制"图标。 </li><li>要检查尺寸标准的质量，通过检查橙色图标，取消选中的彩色图标，其余开结果的橙色通道;这一点很重要，以确保尺寸通话是准确可靠的。 </li><li>要查看对应于从非目标控制得到的片段和靶向克隆的峰，检查蓝色和绿色图标从这些通道打开读数。 </li><li>将光标放在第一的成绩积，然后右键单击选择的横轴"完整视图。"向下滚动一目了然查看所有结果，以确定如果存在与运行的任何重大问题。要放大值的特定范围，用鼠标右键单击该地块的相关轴移动鼠标，选择"缩放到..."，并在相关值的范围键。 注:一个潜在的主要问题是，所有的样品给低强度峰。这通常是由于之前的毛细管电泳运行或扩增子的过度稀释，其可容易地通过重复PCR步骤或通过稀释使用较低的稀释因子，分别扩增子进行整流荧光团标记的扩增子的长期储存。 </li><li>要保存结果，请按照以下的步骤。 <ol><li>确保蓝色和绿色通道的选择和点击"选型表"图标。 </li><li>通过打开"文件"，然后选择保存在制表符分隔文本（.txt）格式值表"导出表"。因此命名该文件，并选择所选择的文件位置。 </li><li>为了节省剧情PDF格式运行的S，转至"文件"，然后点击"打印"选项。选择相关的PDF作家，然后按"打印"。因此命名该文件，并选择所选择的文件位置。 </li></ol></li><li>为了计算在从非目标野生型对照和CRISPR / Cas9靶向克隆的片段的大小的差，按照以下描述的步骤。 <ol><li>从步骤6.9.2使用电子表格程序中打开制表符分隔的文本（.txt）文件。该表应包括这四个重要的列:"染料/取样峰值"（表示蓝色或绿色通道），"样本的文件名"（第一个字符表示好或样品名称），"大小"（表示的大小片段称为），和"高度"（表示该峰的荧光强度）。 </li><li>挑出相关峰，排除那些没有在预期的大小范围。 注:通常情况下，插入缺失突变很少导致更多比尺寸的100-bp的差值;因此，峰的大小由来自野生型对照峰（主峰在绿色通道）多于100bp的不同除外。这可以通过使用下的"条件格式"功能"之间"选项，在电子表格中轻松完成。 </li><li>为了除去非特异性峰，这是从背景水平区分，使用"小于"选项下的"条件格式"功能的电子表格程序，以排除峰其高度超过2000台低。该截止值被经验地确定，因此可以调节在认为合适的。 </li><li>通过从前者中减去后者计算每个在蓝色通道（CRISPR / Cas9靶向克隆），并在绿色信道（非靶向野生型对照）的唯一峰所得到的峰值点间的片段大小的差异。 注意:要使用归因于每个毛细管electrophor值是非常重要的ESIS样品，因为可能存在在野生型对照的片段大小样本间的差异。 </li><li>舍入的值到最接近的整数，以确定已被插入或从（如果有），所讨论的基因组序列中删除碱基对的数目。当敲除基因是兴趣，选择克隆，其插入缺失突变是不是3基点的倍数，以确保有在编码序列移。 </li></ol></li></ol> 7.克隆的基因敲除状态的验证<ol><li>通过使用0.25％胰蛋白酶-EDTA的25μL胰蛋白酶处理细胞，并在37℃下温育5分钟，扩大从96孔板（来自步骤2.4和2.6）单独敲除克隆到一个24孔板中。 </li><li>添加培养基的125微升（DMEM补充有10％FBS）的胰蛋白酶处理细胞，并重新悬浮彻底。 </li><li>细胞悬液转移到15毫升管中并离心以400xg在室温下5分钟。 </li><li>除去尽可能多的介质的尽可能使用真空抽吸或吸移管，重悬在培养基中的500μL细胞沉淀，将细胞悬浮液转移到24孔平板的孔中。孵育在37℃和5％CO 2，直到细胞达到80-90％汇合。 </li><li>展开单个克隆串联从24孔板的6孔板并从6孔板时，他们达到80 10-cm培养皿 - 通过重复步骤7.1至7.4，使用下列体积的90％汇合:的0.25％50μL胰蛋白酶-EDTA和介质的150μL（从24孔板扩展到6孔板）和0.25％200μL胰蛋白酶-EDTA和1mL培养基（用于从6-扩大孔板的10-cm培养皿）。 </li><li>收获的克隆当他们到达80 - 通过用1ml 0.25％胰蛋白酶-EDTA，并在37℃下孵育5分钟胰蛋白酶处理细胞90％汇合。 </li><li>加入5毫升培养基（DMEM补充有10％FBS）的胰蛋白酶处理细胞，并resuspeND彻底。 </li><li>传送同样的细胞悬浮液分成两个15-mL管，离心分离机，在400×g下在室温下5分钟，并尽可能多的去除介质成为可能。其中一个单元部分是用于基因组DNA提取，另一种是对总蛋白质的提取。 </li><li>对于通过Sanger测序验证，按照下面描述的步骤。 <ol><li>如先前描述的12从克隆提取基因组DNA。 </li><li>进行PCR扩增跨越CRISPR / Cas9目标位点的区域，如在第3.2节描述的那样，但使​​用未标记的引物。不要使用标记引物进行这一步骤，从标签中的荧光将与随后的Sanger测序一步干扰。 </li><li>纯化使用PCR清洁试剂盒扩增子，如先前描述的12。 </li><li>序列使用在步骤7.9.2中使用的PCR引物来确定克隆的基因型纯化的扩增子，如所描述previouslY 12。 </li></ol></li><li>对于通过蛋白质印迹分析验证，从野生型细胞和靶向克隆提取总蛋白，如先前描述的12。 <ol><li>使用合适的抗体针对靶基因的蛋白质，如先前所描述的12进行Western印迹分析;一个真正敲除克隆缺乏所述蛋白质的表达。 </li></ol></li></ol>

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作者宣称，他们没有竞争的经济利益。

中选择的模型细胞系敲除特定基因已成为阐明的作用，该基因在特定的细胞环境起着程序。事实上，一些全基因组的屏幕是目前可使用的CRISPR / Cas9系统基因组中的14，15，16靶向几乎所有已知的人类基因。随着这些大型屏幕（甚至小规模的利益个体基因靶向），它设计并利用sgRNAs针对同一基因的不同位点是非常重要的。整个使?...

反向遗传方法已使科学家能够阐明对细胞或整个生物体的基因组中特异性改变的影响。例如，特定基因的表达可通过基因敲减1，2（部分还原）或基因敲除3，4（完全消融），以便确定这对细胞的或对功能的影响减弱有机体的发展。 基因敲除实验已经成为自从引入序列特异性核酸酶的可编程，如锌指核酸酶（ZFN）和转录激活样效应核酸酶（TALEN的）的更容易。然而，比较近的聚集经常穿插短回文重复（CRISPR）表征/ Cas9系统使它非常容易为世界各地的任何实验室进行根基因敲除实验。在本质上，CRISPR / Cas9系统由两个基本组分-单个引导RNA（因组），其识别与通过碱基互补结合到基因组中的特定序列，并称为Cas9核酸内切酶。基因组DNA上的因组-Cas9复合物的特异性结合和行动的后果是DNA的双链切割。这，反过来，触发了细胞，这是通过非同源末端连接（NHEJ）或同源重组（HR）途径随后修复DNA损伤反应的机制。由于NHEJ修复机制（但不是HR机制）通常导致随机插入或缺失核苷酸在修复位点，从而导致插入/缺失（INDEL）突变，它可能会导致外显子的阅读框移位。然后这可能导致在该基因的敲除由于翻译和无义介导衰变5,6的过早终止，7。 尽管通过引入CRISPR / Cas9系统中敲除基因的所提供的便利，有针对性的细胞的克隆的基因分型仍然是一个瓶颈，尤其是在高通量设定8,9。现有技术或者遭受重大固有的局限性或在经济上昂贵。例如，调查或T7E1测定，这是一种酶测定法，其检测DNA的错配双链体10，是不能够野生型克隆，纯合突变体之间进行区分（克隆其等位基因相同的突变），因为这些克隆具有相同的等位基因，因此不会在其DNA序列11存在的错配。此外，采用Sanger测序，这被认为在基因分型突变体克隆，在高通量设置的金标准是不希望的，由于其成本较高。在这里，我们提出了一个DET荧光PCR-毛细管凝胶电泳技术，可以绕过的其他现有基因分型技术的局限性和是在执行核酸酶介导基因敲除克隆的高通量筛选中特别有用的ailed协议。这种方法在技术上简单执行并节省了时间和成本。

<table border="0" cellpadding="0" cellspacing="0" width="1548">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:401px;">HEPG2 cells</td>
			<td style="width:196px;">ATCC</td>
			<td style="width:135px;">HB-8065</td>
			<td style="width:816px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HyClone Dulbecco&#39;s Modified Eagles Medium (DMEM)</td>
			<td>Thermo Fisher Scientific</td>
			<td>SH30022.01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HyClone Fetal Bovine Serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>SV30160.03</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pSpCas9(BB)-2A-GFP plasmid</td>
			<td>Addgene</td>
			<td>PX458</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lipofectamine 2000</td>
			<td>Thermo Fisher Scientific</td>
			<td>11668027</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin-EDTA (0.25%), phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin-EDTA (0.5%), no phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>15400054</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HyClone Water, Molecular Biology Grade</td>
			<td>GE Healthcare</td>
			<td>SH30538.02</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CRISPR sgRNA insert oligonucleotide (sense)</td>
			<td>AITbiotech</td>
			<td>None</td>
			<td>Sequence: 5&#39;-CACCGCTAACCTTTCAGCCTGCCTA-3&#39;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CRISPR sgRNA insert oligonucleotide (anti-sense)</td>
			<td>AITbiotech</td>
			<td>None</td>
			<td>Sequence: 5&#39;-AAACTAGGCAGGCTGAAAGGTTAGC-3&#39;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Unlabeled PCR amplification forward primer</td>
			<td>AITbiotech</td>
			<td>None</td>
			<td>Sequence: 5&#39;-CACTAACTCCAATGCTTCAGTTTC-3&#39;; this primer is also used to sequence PCR amplified alleles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6-FAM-labeled fluorescent PCR forward primer</td>
			<td>AITbiotech</td>
			<td>None</td>
			<td>Sequence: 5&#39;-6-FAM-CACTAACTCCAATGCTTCAGTTTC-3&#39;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEX-labeled fluorescent PCR forward primer</td>
			<td>AITbiotech</td>
			<td>None</td>
			<td>Sequence: 5&#39;-HEX-CACTAACTCCAATGCTTCAGTTTC-3&#39;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Unlabeled PCR reverse primer</td>
			<td>AITbiotech</td>
			<td>None</td>
			<td>Sequence: 5&#39;-CCTCTTCCAAGTCTGCTTATGT-3&#39;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Taq PCR Core Kit</td>
			<td>QIAGEN</td>
			<td>201223</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hi-Di Formamide</td>
			<td>Thermo Fisher Scientific</td>
			<td>4311320</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GeneScan 500 LIZ Dye Size Standard</td>
			<td>Thermo Fisher Scientific</td>
			<td>4322682</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MicroAmp Optical 96-Well Reaction Plate</td>
			<td>Thermo Fisher Scientific</td>
			<td>4306737</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3500xL Genetic Analyzer</td>
			<td>Thermo Fisher Scientific</td>
			<td>4405633</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3500 Series 2 program</td>
			<td>Thermo Fisher Scientific</td>
			<td>4476988</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gene Mapper 5 program</td>
			<td>Thermo Fisher Scientific</td>
			<td>4475073</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gentra Puregene Cell Kit</td>
			<td>QIAGEN</td>
			<td>1045696</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Wizard SV Gel and PCR Clean-Up System</td>
			<td>Promega</td>
			<td>A9282</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NAP1L1 Antibody (N-term)</td>
			<td>Abgent</td>
			<td>AP1920b</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nuclear Matrix Protein p84 antibody [5E10]</td>
			<td>GeneTex</td>
			<td>GTX70220</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Peroxidase AffiniPure Goat Anti-Rabbit IgG</td>
			<td>Jackson ImmunoResearch</td>
			<td>111-035-144</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Peroxidase AffiniPure Sheep Anti-Mouse IgG</td>
			<td>Jackson ImmunoResearch</td>
			<td>515-035-003</td>
			<td></td>
		</tr>
	</tbody>
</table>

using a fluorescent pcr-capillary gel electrophoresis technique to genotype crispr/cas9-mediated knockout mutants in a high-throughput format

可编程基因组编辑工具的发展促进了利用反向遗传学的理解角色的特定基因组序列在细胞和整个生物体的功能发挥。该事业由近期出台的CRISPR的/ Cas9系统一个多功能的工具，使研究人员能够以操纵基因组和转录组除其他事项外，敲，敲下来，或有针对性的基因敲了极大的帮助方式。用于敲除基因的目的，CRISPR / Cas9介导的双链断裂招收非同源末端连接的DNA修复途径在断裂位点上引入核苷酸的移码 - 导致插入或缺失。然而，一个单独的导向RNA可以导致不期望的脱靶效应，并排除这些出来，使用多个引导RNA的是必要的。的目标这种多重性也意味着克隆的高容量筛选是必需的，这又引出了一个使用的EF的够的高通量技术基因型的敲除克隆。目前的基因分型技术无论是从内在的局限性遭受或招致成本高，因此使得它们不适合高通量的目的。在这里，我们详细协议用于使用荧光PCR，其使用基因组DNA从粗细胞裂解物作为模板，然后通过解析毛细管凝胶电泳的PCR片段。这种技术是不够准确区分片段之间的一个碱基对差异，因此是在指示移码的在目标基因的编码序列的存在或缺乏足够的。这种精确的知识有效地排除了一个确定的顺序步骤的需求，并允许用户以节省时间和成本的过程。此外，该技术已被证明是通过基因分型对许多基因的RNA指导有针对性的各种组织器官的各种哺乳动物细胞多才多艺，因为这里和其他地方所示。

这里描述的荧光PCR-毛细管凝胶电泳技术被预期在几乎任何细胞系是适于外源DNA递送到适用于基因组中的任何靶向区域。之前我们已经通过在结肠癌细胞株12靶向三个基因显示出它的应用。在这里，我们将展示其基因分型肝癌细胞系HepG2，针对性与有效性CRISPR / Cas9构建针对核小体装配蛋白1样1（NAP1L1）基因。事实上，我们已经成功地利用荧光PCR-毛细管凝胶?...

Watch this Scientific Journal Video about Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format at JoVE.com

Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format

这里所描述的基因分型技术，荧光聚合酶链式反应（PCR），以毛细管凝胶电泳，其耦合，允许核酸酶介导敲除克隆的高通量基因分型。它规避所面临的其他基因分型技术的限制，并且比测序方法更具成本效益。

用荧光PCR毛细管电泳技术在基因型CRISPR / Cas9介导的敲除突变体的高通量形式

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play ...

using-fluorescent-pcr-capillary-gel-electrophoresis-technique-to

Research

JoVE Journal

Genetics

13.7K 次观看.  Duke-NUS Medical School. 这里所描述的基因分型技术，荧光聚合酶链式反应（PCR），以毛细管凝胶电泳，其耦合，允许核酸酶介导敲除克隆的高通量基因分型。它规避所面临的其他基因分型技术的限制，并且比测序方法更具成本效益。 

视频: Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format

Perturbations of Circulating miRNAs in Irritable Bowel Syndrome Detected Using a Multiplexed High-throughput Gene Expression Platform

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in a single reaction. The miRNA assay counts transcripts by directly imaging and digitally counting miRNA molecules that are labeled with color-coded fluorescent barcoded probe sets (a reporter probe and a capture probe). Barcodes are hybridized directly to mature miRNAs that have been elongated by ligating a unique oligonucleotide tag (miRtag) to the 3&#39; end. Reverse transcription and amplification of the transcripts are not required. Reporter probes contain a sequence of six color positions populated using a combination of four fluorescent colors. The four colors over six positions are used to construct a gene-specific color barcode sequence. Post-hybridization processing is automated on a robotic prep station. After hybridization, the excess probes are washed away, and the tripartite structures (capture probe-miRNA-reporter probe) are fixed to a streptavidin-coated slide via the biotin-labeled capture probe. Imaging and barcode counting is done using a digital analyzer. The immobilized barcoded miRNAs are visualized and imaged using a microscope and camera, and the unique barcodes are decoded and counted. Data quality control (QC), normalization, and analysis are facilitated by a custom-designed data processing and analysis software that accompanies the assay software. The assay demonstrates high linearity over a broad range of expression, as well as high sensitivity. Sample and assay preparation does not involve enzymatic reactions, reverse transcription, or amplification; has few steps; and is largely automated, reducing investigator effects and resulting in high consistency and technical reproducibility. Here, we describe the application of this technology to identifying circulating miRNA perturbations in irritable bowel syndrome.

We describe the use of a multiplexed high-throughput gene expression platform that quantitates gene expression by barcoding and counting molecules in biological substrates without the need for amplification. We used the platform to quantitate microRNA (miRNA) expression in whole blood in subjects with and without irritable bowel syndrome.

肠易激综合征的循环miRNA的扰动检测使用复用高通量基因表达平台

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in ...

科研

遗传学

High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii

Systematic identification and characterization of genetic perturbations have proven useful to decipher gene function and cellular pathways. However, the conventional approaches of permanent gene deletion cannot be applied to essential genes. We have pioneered a unique collection of ~70 temperature-sensitive (ts) lethal mutants for studying cell cycle regulation in the unicellular green algae Chlamydomonas reinhardtii1. These mutations identify essential genes, and the ts alleles can be conditionally inactivated by temperature shift, providing valuable tools to identify and analyze essential functions. Mutant collections are much more valuable if they are close to comprehensive, since scattershot collections can miss important components. However, this requires the efficient collection of a large number of mutants, especially in a wide-target screen. Here, we describe a robotics-based pipeline for generating ts lethal mutants and analyzing their phenotype in Chlamydomonas. This technique can be applied to any microorganism that grows on agar. We have collected over 3000 ts mutants, probably including mutations in most or all cell-essential pathways, including about 200 new candidate cell cycle mutations. Subsequent molecular and cellular characterization of these mutants should provide new insights in plant cell biology; a comprehensive mutant collection is an essential prerequisite to ensure coverage of a broad range of biological pathways. These methods are integrated with downstream genetics and bioinformatics procedures for efficient mapping and identification of the causative mutations that are beyond the scope of this manuscript.

High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii

Temperature-sensitive (ts) lethal mutants are valuable tools to identify and analyze essential functions. Here we describe methods to generate and classify ts lethal mutants in high throughput.

温度敏感致死突变体的高通量机器人辅助隔离<em&gt;莱茵衣藻</em

Systematic identification and characterization of genetic perturbations have proven useful to decipher gene function and cellular pathways. However, the ...

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide RNA (sgRNA) that confers specificity, the Cas9 protein cleaves both DNA strands at the targeted locus. The DNA break can trigger either non-homologous end joining (NHEJ) or homology directed repair (HDR). NHEJ can introduce small deletions or insertions which lead to frame-shift mutations, while HDR allows for larger and more precise perturbations. Here, we present protocols for generating knockout cell lines by coupling established CRISPR/Cas9 methods with two options for downstream selection/screening. The NHEJ approach uses a single sgRNA cut site and selection-independent screening, where protein production is assessed by dot immunoblot in a high-throughput manner. The HDR approach uses two sgRNA cut sites that span the gene of interest. Together with a provided HDR template, this method can achieve deletion of tens of kb, aided by the inserted selectable resistance marker. The appropriate applications and advantages of each method are discussed.

Recent advances in the ability to genetically manipulate somatic cell lines hold great potential for basic and applied research. Here, we present two approaches for CRISPR/Cas9 generated knockout production and screening in mammalian cell lines, with and without the use of selectable markers.

选择依赖和独立生成CRISPR / Cas9介导的哺乳动物细胞基因敲除

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide ...

G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have been directed toward elucidation of the location and timing of initiation of genome duplication rather than its completion. However, it is critical that we understand regions of the genome that are last to complete replication, because these regions suffer elevated levels of chromosomal breakage and mutation, and they have been associated with both disease and aging. Here we describe how we have extended a technique that has been used to monitor replication initiation to instead identify those regions of the genome last to complete replication. This approach is based on a combination of flow cytometry and high throughput sequencing. Although this report focuses on the application of this technique to yeast, the approach can be used with any cells that can be sorted in a flow cytometer according to DNA content.

We describe a technique for combining flow cytometry and high throughput sequencing to identify late replicating regions of the genome.

G2-seq: 一种基于高通量测序技术的基因组晚期复制区域识别方法

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have ...

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration of transgenes. However, due to the low efficiency of homologous recombination (HR) and various indel mutations of non-homologous end joining (NHEJ)-based strategies in non-dividing cells, in vivo genome editing remains a great challenge. Here, we describe a homology-mediated end joining (HMEJ)-based CRISPR/Cas9 system for efficient in vivo precise targeted integration. In this system, the targeted genome and the donor vector containing homology arms (~800 bp) flanked by single guide RNA (sgRNA) target sequences are cleaved by CRISPR/Cas9. This HMEJ-based strategy achieves efficient transgene integration in mouse zygotes, as well as in hepatocytes in vivo. Moreover, a HMEJ-based strategy offers an efficient approach for correction of fumarylacetoacetate hydrolase (Fah) mutation in the hepatocytes and rescues Fah-deficiency induced liver failure mice. Taken together, focusing on targeted integration, this HMEJ-based strategy provides a promising tool for a variety of applications, including generation of genetically modified animal models and targeted gene therapies.

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system provides a promising tool for genetic engineering, and opens up the possibility of targeted integration of transgenes. We describe a homology-mediated end joining (HMEJ)-based strategy for efficient DNA targeted integration in vivo and targeted gene therapies using CRISPR/Cas9.

CRISPR/Cas9-mediated 目标集成在体内使用基于同源介导的端接连接策略

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration ...

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

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

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

整合酶缺陷慢病毒载体载体的制备及其在细胞 CRISPR/Cas9-mediated 基因敲除中的表达

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

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our entire society's ability to treat systemic infections. In the United States alone, antibiotic-resistant infections kill approximately 23,000 people a year and cost around 20 billion USD in additional healthcare. One approach to combat antimicrobial resistance is combination therapy, which is particularly useful in the critical early stage of infection, before the infecting organism and its drug resistance profile have been identified. Many antimicrobial treatments use combination therapies. However, most of these combinations are additive, meaning that the combined efficacy is the same as the sum of the individual antibiotic efficacy. Some combination therapies are synergistic: the combined efficacy is much greater than additive. Synergistic combinations are particularly useful because they can inhibit the growth of antimicrobial drug resistant strains. However, these combinations are rare and difficult to identify. This is due to the sheer number of molecules needed to be tested in a pairwise manner: a library of 1,000 molecules has 1 million potential combinations. Thus, efforts have been made to predict molecules for synergy. This article describes our high-throughput method for predicting synergistic small molecule pairs known as the Overlap2 Method (O2M). O2M uses patterns from chemical-genetic datasets to identify mutants that are hypersensitive to each molecule in a synergistic pair but not to other molecules. The Brown lab exploits this growth difference by performing a high-throughput screen for molecules that inhibit the growth of mutant but not wild-type cells. The lab's work previously identified molecules that synergize with the antibiotic trimethoprim and the antifungal drug fluconazole using this strategy. Here, the authors present a method to screen for novel synergistic combinations, which can be altered for multiple microorganisms.

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Synergistic drug combinations are difficult and time-consuming to identify empirically. Here, we describe a method for identifying and validating synergistic small molecules.

通过重叠2方法高通量识别协同药物组合

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our ...

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been co-opted as a powerful tool for precise eukaryotic genome engineering. Here, we present a rapid and simple method using chimeric single guide RNAs (sgRNA) and CRISPR-Cas9 Ribonucleoproteins (RNPs) for the efficient and precise generation of genomic point mutations in C. elegans. We describe a pipeline for sgRNA target selection, homology-directed repair (HDR) template design, CRISPR-Cas9-RNP complexing and delivery, and a genotyping strategy that enables the robust and rapid identification of correctly edited animals. Our approach not only permits the facile generation and identification of desired genomic point mutant animals, but also facilitates the detection of other complex indel alleles in approximately 4 - 5 days with high efficiency and a reduced screening workload.

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

Here, we present a method to engineer the genome of C. elegans using CRISPR-Cas9 ribonucleoproteins and homology dependent repair templates.

利用 CRISPR/Cas9 Ribonucleoproteins 快速简便地生成线虫基因组点突变体的管道

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

利用下一代序列染色体构象捕获 (4 c 序列) 的高通量识别基因调控数列

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to perform and its ability to standardize the fitness of many strains to a wild-type organism. The technique is limited, however, by available phenotypic markers and the number of strains that can be assessed simultaneously, creating the need for a great number of replicate experiments. Concurrent with large numbers of experiments, the labor and material costs for quantifying bacteria based on phenotypic markers are not insignificant. To overcome these negative aspects while retaining the positive aspects, we have developed a molecular-based approach to directly quantify microorganisms after engineering genetic markers onto bacterial chromosomes. Unique, 25 base pair DNA barcodes were inserted at an innocuous locus on the chromosome of wild-type and mutant strains of Salmonella. In vitro competition experiments were performed using inocula consisting of pooled strains. Following the competition, the absolute numbers of each strain were quantified using digital PCR and the competitive indices for each strain were calculated from those values. Our data indicate that this approach to quantifying Salmonella is extremely sensitive, accurate, and precise for detecting both highly abundant (high fitness) and rare (low fitness) microorganisms. Additionally, this technique is easily adaptable to nearly any organism with chromosomes capable of modification, as well as to various experimental designs that require absolute quantification of microorganisms.

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

This molecular-based approach for determining bacterial fitness facilitates precise and accurate detection of microorganisms using unique genomic DNA barcodes that are quantified via digital PCR. The protocol describes calculating the competitive index for Salmonella strains; however, the technology is readily adaptable to protocols requiring absolute quantification of any genetically-malleable organism.

基于数字PCR的沙门氏菌健康高通量分析竞争指数

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to ...