这项工作得到了日本促进科学协会的支持 [授予数字 16K15860 T.M. 和15K15777 新罕布什尔州]。

1. 入门设计 <ol> <li> 准备2步融合 PCR 的引物和基因破坏的验证. 
	注意: 表 1 显示了本协议中使用的入门序列。 图 1 显示了用于生成 变形链球菌 和 #916 的2步融合 PCR 方法的示意图; gtfC 。
	<ol> <li> 设计用于在基因组中替换目标区域的霉抗性基因 ( spc r ) 7 。此协议将 gtfC 基因的整个编码区域替换为 spc r 。
		<li> 在5和 #39 中包括与 "spc" r 的上、下两端互补的15基, 分别为上反转和下向引物的区域。同样, 还包括与 gtfC 的上游侧翼区域的下端以及在5和 #39 上的 gtfC 的下游侧翼区域的上端互补的15基地; spc r 前向和 spc 的区域 -分别反转底漆 ( 图 1A )。
		注意: 上反转、下向前、 spc r 前向和 spc r 反向底漆的顺序将根据中断构造的合并站点自动确定. </li>
		<li> 设计向上和向下反转引物以构成和 #62; gtfC 基因的上游和下游侧翼区域分别为 1 kb。根据上反和下向引物的熔化温度分别设置前向和向下反转底漆的熔化温度 ( T m ) ( 图 1A ). 
		注意: 请参阅以下公式以确定 T m : T m (and #176; C) = 2 (* NA + * NT) + 4 (* NC + NG)-5, 其中 ** 表示具有指定标识 (A、T、C 或 G) 的入门核苷酸的数量. </li>
		<li> 设计 2 nd PCR ( 图 1B ) 的嵌套底漆 (N_up 前向和 N_up). 
		注: 虽然最外层的底漆对 (向前和向下反转) 可用作 2 nd PCR 的引物, 但在本协议中设计了嵌套底漆 (N_up 和 N_up 反转)。2 nd PCR 通常需要嵌套的底漆, 详见 代表性结果 。
		<li> 设计特定于 spc r 的底漆。该引物用于菌落 PCR 筛选 gtfC 中断. </li>
		<li> 设计用于验证 gtfC 中断的底漆. 
		注: 使用这些引物扩增的 PCR 产品横跨 gtfC 或 spc r 和 gtfC 的上游或下游侧翼区域之间的边界。入门集和 #39; gtfC 前向和 gtfC 上-反向和 #39; 和 #39; gtfC 下向前和 gtfC 向下反转和 #39; 特定于 gtfC 和 #39; gtfC 前向和 spc r 2-反转和 #39; 和 #39; spc r 2 向前和 gtfC 下反转和 #39; spc r 的特定项。
	</ol> </li> </ol> 2。基因组 DNA 从 s. 变形 <ol> <li> 条纹储存牡丹 s UA159 到脑心输液 (BHI) 琼脂板上。在厌氧条件下, 在37和 #176 的夜间孵育盘. </li>
	<li> 用一根消毒过的牙签拿起一个菌落, 将它接种到5毫升的 BHI 汤中。将 s. 牡丹 的区域性一夜之间孵化为37和 #176; C 在厌氧条件下. </li>
	<li> 在2000和 #215 离心细菌细胞培养15分钟; 重5毫升磷酸盐缓冲盐水 (PBS) 中的细胞颗粒. </li>
	<li> 在2000和 #215 离心15分钟; 重200和 #181 中的细胞小球; PBS 的 L. </li>
	<li> 根据制造商提供的说明从悬浮液中提取基因组 dna, 使用珠 beating-base 基因组 dna 提取试剂盒. 
	<ol> <li> 将悬浮液传输到包含玻璃珠的2.0 毫升样品管 (包括在套件中)。在细胞悬浮液中加入750和 #181; L 溶解溶液 (包括在试剂盒中). </li>
		<li> 盖紧并将管子对称地放在磁珠跳动的破坏装置的管架上。在最高速度为5分钟的过程。在1万和 #215 离心1分钟的微珠样品; g. 将上清液转移到旋转过滤器 (包括在套件中) 在2.0 毫升收集管和离心机为 1 min 在7000和 #215; g . </li>
		<li> 添加1200和 #181; L 的 DNA 结合缓冲 (包括在工具箱中) 到滤液。将800和 #181 的混合物转到自旋柱 (包括在套件中), 以2.0 毫升的收集管和离心机1分钟, 在1万和 #215; g. </li>
		<li> 丢弃流式, 添加200和 #181; l 清洗缓冲器 (包括在套件中), 以洗涤柱矩阵, 离心1分钟, 在1万和 #215; g. 丢弃流, 添加500和 #181; 洗涤缓冲器 (包括在套件中) 的旋转柱洗柱矩阵, 离心1分钟, 在1万和 #215; g. </li>
		<li> 将自旋列放入新的 1.5 mL 离心管中, 并将100和 #181 的洗脱缓冲器 (包括在套件中) 添加到柱形矩阵中. </li>
		<li> 离心三十年代在1万和 #215; g 洗基因组 DNA。通过测量 260 nm 和 280 nmusing 分光光度计的吸光度来估计 DNA 浓度和纯度, 并确认 260 / 280 比率大于 1.8. </li>
	</ol> </li> </ol> 3。pcr 放大 <ol> <li> 执行 1 st pcr 使用牡丹 s 基因组和合成的 spc r 基因作为 pcr 模板 (参见 表 1 )。使用上面描述的三套引物 ( 图 1A ), 放大 gtfC 基因的上游侧翼区域、 gtfC 基因的下游侧翼区域以及 spc r 基因, 按 表 2 和 表 3 . 
	注: PCR 引物、试剂和扩增周期分别在 表 1 、 表 2 和 表 3 中进行了总结. </li>
	<li> 在1% 琼脂糖凝胶上分级每个 PCR 产品, 并使用凝胶带切割器对相应的频段进行切除. </li>
	<li> 使用自旋柱和硅胶膜凝胶萃取试剂盒从凝胶中纯化每个放大的 DNA 产物. 
	<ol> <li> 将凝胶切片转换为干净的离心管, 并将其与500和 #181; L 绑定缓冲区 (包括在套件中)。将混合物孵育在55和 #176; C 为15分钟, 直到凝胶切片完全溶解. </li>
		<li> 将自旋列 (包括在套件中) 放入收集管 (包括在套件中), 将样品加载到自旋柱上, 并在1.1万和 #215 上离心三十年代; g 。放弃流动, 添加600和 #181; 洗涤缓冲器 (包括在套件中), 以洗涤硅胶膜, 并离心三十年代在1.1万和 #215; g . </li>
		<li> 在1.1万和 #215 丢弃流经和离心机2分钟; g 干燥二氧化硅膜. </li>
		<li> 将自旋列放入一个新的 1.5 mL 离心管中, 添加50和 #181; L 洗脱缓冲液 (包括在套件中), 室温孵育2分钟. </li>
		<li> 在1.1万和 #215 离心2分钟; g 洗放大的 DNA。通过测量 260 nm 和 280 nmusing 分光光度计的吸光度来估计 DNA 浓度和纯度, 并确认 260 / 280 比率大于 1.8. </li>
	</ol> </li> <li> 使用三近似摩尔片段作为 pcr 模板执行2步融合 pcr 的 2 nd pcr, 按 表 2/st荣 > 和 表 3 . 
	注意: 这些片段被放大和拼接使用嵌套的引物和 #39; N_up 向前和 N_down-反向和 #39; 或最外层的底漆和 #39; 向上和向下反转和 #39; ( 图 1B )。PCR 引物、试剂和扩增周期分别在 表 1 、 表 2 和 表 3 中进行了总结. </li>
	<li> 在0.8% 琼脂糖凝胶上加载1/10 的 PCR 反应混合物 (5 和 #181; L), 并将放大的带大小与预期的带大小进行比较, 以确认适当的增 ( 图 1C ) 的生成. </li>
	<li> 将剩余的最终 PCR 产物浓缩为无纯化的乙醇沉淀. 
	<ol> <li> 添加55和 #181; 超纯水到剩余的 PCR 混合物调整总容量到100和 #181; l. 添加1/10 卷的3米醋酸钠 (10 和 #181; l), pH 5.2, 其次是2.5 量的100% 乙醇 (250 和 #181; l)。在-80 和 #176 中混合和冻结30分钟; C. </li>
		<li> 离心20分钟, 在1.5万和 #215; g 在4和 #176; C, 检查管底部的颗粒, 并丢弃上清液。用1毫升70% 乙醇洗涤颗粒, 离心5分钟, 在1.5万和 #215; g 在4和 #176; C, 并丢弃上清。空气干燥颗粒约30分钟, 溶解与10和 #181; 超纯水的 L. </li>
	</ol> </li> </ol> 4。合格的细胞制备和细胞转化 <ol> <li> 条纹股票 牡丹 s UA159 到 BHI 琼脂板上。在厌氧条件下, 在37和 #176 的夜间孵育盘. </li>
	<li> 用一根消毒过的牙签拿起一个菌落, 将它接种到5毫升的 BHI 汤中。将 s. 牡丹 的区域性一夜之间孵化为37和 #176; C 在厌氧条件下. </li>
	<li> 将2毫升的 S. 牡丹 s 培养到50毫升的 BHI 汤, 并在37和 #176 孵育6-8 小时; C 到光学密度在600毫微米 (OD 600 ) 0.2-0.4 在厌氧条件下. </li>
	<li> 在2000和 #215 离心细胞15分钟; g at 4 和 #176; C、丢弃上清, 然后用40毫升的 ice-cold 水冲洗两次细胞, 然后40毫升10% 的甘油. 
	注: 残余物质会影响随后的电穿孔. </li>
	<li> 用巴斯德吸管将上清液仔细吸出, 因为在10% 甘油中, 细胞团的粘附量减少了。重1毫升新鲜10% 甘油, 免除50和 #181; L 悬浮入每1.5 毫升离心管, 并且存放在-80 和 #176; C 直到使用前. </li>
	<li> 将50和 #181 混合在一起; l 分 ice-cold 5 和 #181 的合格细胞; 上文3节所述的破坏结构 l。将混合物添加到电穿孔试管 (电极之间的距离为0.2 厘米)。采用 金黄色葡萄球菌 模式 (1.8 伏、600、#937、10、#181、1脉) 的电穿孔装置 electroporate. </li>
	<li> 将500和 #181 中的单元格挂起, 并在电穿孔后立即 BHI 肉汤, 并添加50和 #181; 悬浮物的 l 霉 BHI 琼脂板. 
	注: 电穿孔后额外的孵育时间不需要。但是, 在电穿孔后1-2 小时的孵化可以提高转化效率. </li>
	<li> 在37和 #176 下孵育盘2-6 天; C. 在厌氧条件下. 
	注意: S 变形 和 #916; gtfB 是按照上面的描述构造的。将 gtfB 基因的整个编码区域替换为 变形链球菌 和 #916 中的红霉素抵抗基因 8 ; gtfB. 每个 S. 变形菌 菌株在37和 #176 的 BHI 汤中培养; C. 在厌氧条件下. </li>
</ol> 5。基因中断和存储的验证 <ol> <li> 选取随机菌落并将其接种到 pcr 混合物中, 以按 表 2 和 表 3 进行菌落 pcr。PCR 引物、试剂和扩增周期分别在 表 1 、 表 2 和 表 3 中进行了总结. </li>
	<li> 在1% 琼脂糖凝胶上加载 PCR 反应混合物以确认 spc r 特定的 DNA 带。
	<li> 在10毫升含霉的 BHI 汤中, 用无菌牙签和亚文化细胞选择阳性菌落, 2 天在37和 #176; C 在厌氧条件下. </li>
	<li> 验证 S. 变形 和 #916 的生成; gtfC 
	<ol> <li> 平均划分单元格挂起。从上面描述的细胞中提取基因组 DNA (步骤 2.3. 2.5.5.)。以基因组 DNA 为 pcr 模板, 使用引物来验证 gtfC 中断 ( 表 1 ) 按 表 2 和 表 3 . 
		注: PCR 引物、试剂和扩增周期分别在 表 1 、 表 2 和 表 3 中进行了总结. </li>
		<li> 在2% 琼脂糖凝胶上加载 PCR 混合物, 并将放大的带大小与预期的带大小进行比较, 以确认适当增的生成 . </li>
		<li> 将剩余的电池悬浮液离心15分钟, 在2000和 #215; g at 4 和 #176; c. 重5毫升的 PBS 中的细胞团. </li>
		<li> 离心15分钟, 在2000和 #215; g at 4 和 #176; c. 重1.5 毫升的 BHI 汤中含有25% 甘油的细胞团, 贮存于-80 和 #176; 林-20 和 #176; c. </li>
	</ol> </li> </ol> 6。表型分析 <ol> <li> 将每个 的牡丹 s 的应变置于 BHI 琼脂板上。在厌氧条件下, 在37和 #176 的夜间孵育盘. </li>
	<li> 使用无菌牙签取一个单一的菌落, 并将其接种到2毫升的 BHI 汤中, 或没有适当的抗生素。在37和 #176 的夜间孵育; C. 在厌氧条件下. </li>
	<li> 接种20和 #181; 夜间养殖的 L 在玻璃试管中含有1% 或5% 蔗糖, 不含抗生素的 BHI 肉汤; 在无氧条件下, 在37和 #176 的情况下, 在一夜之间培养与试管在倾斜位置的细胞;
	<li> 涡流玻璃试管十年代和醒酒文化悬浮。用蒸馏水冲洗试管三次。添加1毫升的0.25% 马斯亮辉煌蓝色 (CBB), 以染色的生物膜在管壁上, 然后孵化1分钟. </li>
	<li> 醒酒染色液, 用蒸馏水冲洗试管三次, 并烘干试管. </li>
	<li> 以视觉方式评估 CBB 染色生物膜的颜色密度和延伸度, 以确定表型分析中的生物膜形成能力. </li>
</ol>

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作者没有什么可透露的。

在本议定书中, 1st PCR 的引物必须被设计为在基因组中放大大约 1 kb 的目标区域的上游和下游侧翼区域。这样长的侧翼序列对于提高同源重组的效率是必要的。
本协议中的引物序列 (向上反转、下向前、 spcr前向和spcr反转) 都是根据该中断构造的合并站点自动确定的。每个引物包括15基地补充到 2nd PCR 模板在 5 ' 地区的结束。更长的...

基因功能分析通常涉及一种表型比较的野生类型菌株和应变, 其中一个特定的基因的利益已被打乱。这种基因破坏技术已被用于基因功能分析的各种分类群, 从细菌到哺乳动物。基因中断的构造是产生基因中断的菌株所必需的;这种脱氧核糖核酸 (脱氧核糖核酸) 构造包括与目标基因的上游和下游侧翼区域融合的抗生素抗性标记基因, 并通过同源重组将其纳入基因组。使用含有抗生素的选择性培养基可以分离出基因紊乱的菌株。传统的方法用于构建的基因中断的应变需要复杂的步骤, 如放大的目标基因的聚合酶链反应 (PCR), 结扎基因成合适的质粒, 消化的限制酶, 并 religation 插入抗生素抗性标记基因。这个过程很耗时, 需要几天到几个星期, 取决于每个步骤的成功与否。此外, 破坏构造的设计也依赖于靶基因的限制性酶位点。为了解决这些问题, pcr 的 DNA 剪接方法1,2为设计基因中断的变种人网柄3和Synechocystis sp. PCC68034已报告。在本研究中, 利用一种改进的 pcr 方法 (指定的2步融合 PCR 技术) 来建立一个以相对快速、简便和廉价的方式产生基因中断的链球菌菌株的方法。遗传转化的构造和电穿孔。
2步融合 pcr 需要基因组 DNA, 抗生素抗性标记基因作为 pcr 模板, 和四对适当设计的寡核苷酸引物。在第一步 (1st pcr), 目标基因的一个 1 kb 的上游侧翼区域, 目标基因的一个 1 kb 的下游侧翼区域和抗生素抗性标记基因通过 pcr 放大。反向底漆和前向引物的5个区域分别为上游和下游侧翼区域的放大, 包括与标记基因末端互补的15碱基。标记基因扩增的前、反向引物的5个区域, 类似地包括目标基因上游侧翼区域的下端和目标基因下游侧翼区域的上端的15基。分别.因此, 三放大的片段在融合点包含重叠的30基对区域。在第二步 (2nd pcr) 中, 采用嵌套 pcr 引物进行 pcr, 使用 1st pcr 作为模板放大的三片段。模板的互补区域通过 2nd PCR 相互连接。最后, 采用电穿孔法对野生型菌株进行同源重组的构造。成功的基因破坏可以通过 PCR 方法通过特异的引物来验证。虽然破坏结构使用高保真 dna 聚合酶放大, 但额外的酶反应, 如 dna 结扎或 dna 消化, 是不需要生成的结构。此外, 在基因组上的一个被删除或保留的区域可以根据底漆的设计灵活地选择。
在目前的工作中, 删除的整个编码区域的gtfC基因, 编码葡萄糖在变形链球菌, 是为了证明快速, 容易的基因破坏方法的链球菌物种。此外, 以相同的方式生成了gtfB中断的应变。由gtfC和gtfB编码的 glucosyltransferases 有助于龋牙科生物膜的开发5,6。对野生型菌株的生物膜形成能力 (变形量)、 gtfC-被破坏的应变 (变形者δgtfC) 和gtfB中断的应变 (变形者δgtfB) 进行了评估用于比较表型分析。这个协议扩展了 two-step 方法的应用为基因中断对一个另外的种类和可能被修改对基因功能的研究在更大的种类分类群。

<table>
	<tbody>
		<tr>
			<td>Streptococus mutans</td>
			<td>Stock strain in the lab.</td>
			<td></td>
			<td>Wild-type strain (Strain UA159)</td>
		</tr>
		<tr>
			<td>Genomic DNA extraction kit</td>
			<td>Zymo Research</td>
			<td>D6005</td>
			<td></td>
		</tr>
		<tr>
			<td>Bead-beating disruption apparatus</td>
			<td>Taitec</td>
			<td>GM-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Synthetic genes</td>
			<td>Eurofins Genomics</td>
			<td>Custom-ordered</td>
			<td>Spectinomycin- and erythromycin-resistance gene</td>
		</tr>
		<tr>
			<td>Thermal cycler</td>
			<td>Astec</td>
			<td>PC-708</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR primers</td>
			<td>Eurofins Genomics</td>
			<td>Custom-ordered</td>
			<td>35 bases in length</td>
		</tr>
		<tr>
			<td>DNA polymerase</td>
			<td>Takara</td>
			<td>R045A</td>
			<td>High-fidelity DNA polymerase</td>
		</tr>
		<tr>
			<td>Gel extraction kit</td>
			<td>Nippon Genetics</td>
			<td>FG-91202</td>
			<td>DNA extraction from agarose gel</td>
		</tr>
		<tr>
			<td>Gel band cutter</td>
			<td>Nippon Genetics</td>
			<td>FG-830</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Beckman Coulter</td>
			<td>DU 800</td>
			<td></td>
		</tr>
		<tr>
			<td>Electroporator</td>
			<td>Bio-rad</td>
			<td>1652100</td>
			<td></td>
		</tr>
		<tr>
			<td>Electroporation cuvette</td>
			<td>Bio-rad</td>
			<td>1652086</td>
			<td>0.2-cm gap</td>
		</tr>
		<tr>
			<td>Anaeropack</td>
			<td>Mitsubishi Gas Chemical</td>
			<td>A-03</td>
			<td>Anaerobic culture system</td>
		</tr>
		<tr>
			<td>Brain heart infusion broth</td>
			<td>Becton, Dickinson</td>
			<td>237500</td>
			<td>Bacterial culture media</td>
		</tr>
		<tr>
			<td>Spectinomycin</td>
			<td>Wako</td>
			<td>195-11531</td>
			<td>Antibiotics</td>
		</tr>
		<tr>
			<td>Erythromycin</td>
			<td>Wako</td>
			<td>057-07151</td>
			<td>Antibiotics</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Wako</td>
			<td>196-00015</td>
			<td>For biofilm development</td>
		</tr>
		<tr>
			<td>CBB R-250</td>
			<td>Wako</td>
			<td>031-17922</td>
			<td>For biofilm staining</td>
		</tr>
	</tbody>
</table>

generation of a gene-disrupted streptococcus mutans strain without gene cloning

典型的解释特定基因的功能的方法包括对野生型菌株和一种被破坏的基因的应变的比较表性分析。基因破坏 DNA 结构包含一个合适的抗生素抗性标记基因是有用的产生基因中断菌株的细菌。然而, 传统的建设方法, 需要基因克隆步骤, 涉及复杂和耗时的协议。在这里, 一个相对简便, 快速, 和 cost-effective 的方法为目标基因的破坏在变形链球菌描述。该方法利用2步融合聚合酶链反应 (PCR) 产生的破坏结构和电穿孔的遗传转化。这种方法不需要酶反应, 除了 PCR, 并提供更大的灵活性, 从设计的破坏结构。电穿孔的使用有利于制备合格的细胞, 提高转化效率。本方法可适用于不同品种的基因阻断菌株的生成。

图 2显示了 1st PCR 中的每个产品的大小, 如在1% 琼脂糖凝胶上观察到的, 与预测的大约 1 kb 的大小一致。图 3显示了 2nd PCR 产物的凝胶电泳分析。图 4显示了由破坏构造转化而来的变形人菌落, 以及菌落 PCR 产物的凝胶电泳分析。所有殖民地的增都是预测的大小。 <strong class...

Watch this Scientific Journal Video about Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning at JoVE.com

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

我们描述了一种简便, 快速, 和相对廉价的方法, 产生基因中断的变形链球菌菌株;这种技术可以适应于各种物种的基因中断菌株的产生。

无基因克隆的基因破坏的变形链球菌菌株的生成

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in ...

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JoVE Journal

Genetics

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

7.6K 次观看.  Tsurumi University School of Dental Medicine. 我们描述了一种简便, 快速, 和相对廉价的方法, 产生基因中断的变形链球菌菌株;这种技术可以适应于各种物种的基因中断菌株的产生。

视频: Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

Generation of Fluorescent Protein Fusions in Candida Species

Candida species, prevalent colonizers of the intestinal and genitourinary tracts, are the cause of the majority of invasive fungal infections in humans. Thus, molecular and genetic tools are needed to facilitate the study of their pathogenesis mechanisms. PCR-mediated gene modification is a straightforward and quick approach to generate epitope-tagged proteins to facilitate their detection. In particular, fluorescent protein (FP) fusions are powerful tools that allow visualization and quantitation of both yeast cells and proteins by fluorescence microscopy and immunoblotting, respectively. Plasmids containing FP encoding sequences, along with nutritional marker genes that facilitate the transformation of Candida species, have been generated for the purpose of FP construction and expression in Candida. Herein, we present a strategy for constructing a FP fusion in a Candida species. Plasmids containing the nourseothricin resistance transformation marker gene (NAT1) along with sequences for either green, yellow, or cherry FPs (GFP, YFP, mCherry) are used along with primers that include gene-specific sequences in a polymerase chain reaction (PCR) to generate a FP cassette. This gene-specific cassette has the ability to integrate into the 3'-end of the corresponding gene locus via homologous recombination. Successful in-frame fusion of the FP sequence into the gene locus of interest is verified genetically, followed by analysis of fusion protein expression by microscopy and/or immuno-detection methods. In addition, for the case of highly expressed proteins, successful fusions can be screened for primarily by fluorescence imaging techniques.

Generation of Fluorescent Protein Fusions in Candida Species

PCR-mediated gene modification can be used to generate fluorescent protein fusions in Candida species, which facilitates visualization and quantitation of yeast cells and proteins. Herein, we present a strategy for constructing a fluorescent protein fusion (Eno1-FP) in Candida parapsilosis.

在荧光蛋白融合的一代<em&gt;念珠菌</em&gt;物种

Candida species, prevalent colonizers of the intestinal and genitourinary tracts, are the cause of the majority of invasive fungal infections in humans. ...

科研

遗传学

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 ...

Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an excellent model for rapid assessment of whole transcriptome from whole animal or individual cell populations due to the ease of isolation of RNA from large numbers of animals. Here a protocol for global gene expression analysis in zebrafish embryos using RNA sequencing (RNASeq) is presented. We describe preparation of RNA from whole embryos or from cell populations obtained using cell sorting in transgenic animals. We also describe an approach for analysis of RNASeq data to identify enriched pathways and Gene Ontology (GO) terms in global gene expression data sets. Finally, we provide a protocol for validation of gene expression changes using quantitative reverse transcriptase PCR (qRT-PCR). These protocols can be used for comparative analysis of control and experimental sets of zebrafish to identify novel gene expression changes, and provide molecular insight into phenotypes of interest.

This protocol presents an approach for whole transcriptome analysis from zebrafish embryos, larvae, or sorted cells. We include isolation of RNA, pathway analysis of RNASeq data, and qRT-PCR-based validation of gene expression changes.

斑马鱼 RNASeq-based 基因表达数据的样品制备与分析

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an ...

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site&#8212;in essence, analyzing onsite mutagenesis.

CRISPR/Cas9 和 SsODN 在人细胞中催化点突变修复功能的标准方法研究

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development of a customizable platform to rapidly generate gene modifications in a wide variety of organisms, including zebrafish. CRISPR-based genome editing uses a single guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to a genomic DNA (gDNA) target of interest, where the Cas endonuclease generates a double-strand break (DSB). Repair of DSBs by error-prone mechanisms lead to insertions and/or deletions (indels). This can cause frameshift mutations that often introduce a premature stop codon within the coding sequence, thus creating a protein-null allele. CRISPR-based genome engineering requires only a few molecular components and is easily introduced into zebrafish embryos by microinjection. This protocol describes the methods used to generate CRISPR reagents for zebrafish microinjection and to identify fish exhibiting germline transmission of CRISPR-modified genes. These methods include in vitro transcription of sgRNAs, microinjection of CRISPR reagents, identification of indels induced at the target site using a PCR-based method called a heteroduplex mobility assay (HMA), and characterization of the indels using both a low throughput and a powerful next-generation sequencing (NGS)-based approach that can analyze multiple PCR products collected from heterozygous fish. This protocol is streamlined to minimize both the number of fish required and the types of equipment needed to perform the analyses. Furthermore, this protocol is designed to be amenable for use by laboratory personal of all levels of experience including undergraduates, enabling this powerful tool to be economically employed by any research group interested in performing CRISPR-based genomic modification in zebrafish.

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Targeted genome editing in the model system Danio rerio (zebrafish) has been greatly facilitated by the emergence of CRISPR-based approaches. Herein, we describe a streamlined, robust protocol for generation and identification of CRISPR-derived nonsense alleles that incorporates the heteroduplex mobility assay and identification of mutations using next-generation sequencing.

斑马斑马模型系统中 CRISPR/Cas9-generated 基因挖空的高效生产与鉴定

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development ...

Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective tag single nucleotide polymorphism (SNP) approach using a multiplexed genotyping assay with mass spectrometry, to investigate gene pathway associations in clinical cohorts. We investigate the food allergy candidate locus Interleukin13 (IL13) as an example. This method efficiently maximizes the coverage by taking advantage of shared linkage disequilibrium (LD) within a region. Selected LD SNPs are then designed into a multiplexed assay, enabling up to 40 different SNPs to be analyzed simultaneously, boosting cost-effectiveness. Polymerase chain reaction (PCR) is used to amplify the target loci, followed by single nucleotide extension, and the amplicons are then measured using matrix-assisted laser desorption/ionization-time of flight(MALDI-TOF) mass spectrometry. The raw output is analyzed with the genotype calling software, using stringent quality control definitions and cut-offs, and high probability genotypes are determined and output for data analysis.

Identification of genetic variants contributing to complex human disease allows us to identify novel mechanisms. Here, we demonstrate a multiplex genotyping approach to candidate genes or gene pathway analysis that maximizes the coverage at low cost and is amenable to cohort-based studies.

应用多重基因分型和质谱法进行临床队列研究的候选基因检测

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective ...

CRISPR Guide RNA Cloning for Mammalian Systems

The outlined protocol describes streamlined methods for the efficient and cost-effective generation of Cas9-associated guide RNAs. Two alternative strategies for guide RNA (gRNA) cloning are outlined based on the usage of the Type IIS restriction enzyme BsmBI in combination with a set of compatible vectors. Outside of the access to Sanger sequencing services to validate the generated vectors, no special equipment or reagents are required aside from those that are standard to modern molecular biology laboratories. The outlined method is primarily intended for cloning one single gRNA or one paired gRNA-expressing vector at a time. This procedure does not scale well for the generation of libraries containing thousands of gRNAs. For those purposes, alternative sources of oligonucleotide synthesis such as oligo-chip synthesis are recommended. Finally, while this protocol focuses on a set of mammalian vectors, the general strategy is plastic and is applicable to any organism if the appropriate gRNA vector is available.

Here, a simple, efficient, and cost-effective method of sgRNA cloning is outlined.

哺乳动物系统 CRISPR 指南 RNA 克隆

The outlined protocol describes streamlined methods for the efficient and cost-effective generation of Cas9-associated guide RNAs. Two alternative ...

Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near full-length (intact and defective), HIV-1 proviruses. FLIPS allows determination of the genetic composition of integrated HIV-1 within a cell population. Through identifying defects within HIV-1 proviral sequences that arise during reverse transcription, such as large internal deletions, deleterious stop codons/hypermutation, frameshift mutations, and mutations/deletions in cis acting elements required for virion maturation, FLIPS can identify integrated proviruses incapable of replication. The FLIPS assay can be utilized to identify HIV-1 proviruses that lack these defects and are&#160;therefore potentially replication-competent. The FLIPS protocol involves: lysis of HIV-1 infected cells, nested PCR of near full-length HIV-1 proviruses (using primers targeted to the HIV-1 5&#39; and 3&#39; LTR), DNA purification and quantification, library preparation for Next-generation Sequencing (NGS), NGS, de novo assembly of proviral contigs, and a simple process of elimination for identifying replication-competent proviruses. FLIPS provides advantages over traditional methods designed to sequence integrated HIV-1 proviruses, such as single-proviral sequencing. FLIPS amplifies and sequences near full-length proviruses enabling replication competency to be determined, and also uses fewer amplification primers, preventing the consequences of primer mismatches. FLIPS is a useful tool for understanding the genetic landscape of integrated HIV-1 proviruses, especially within the latent reservoir, however, its utilization can extend to any application in which the genetic composition of integrated HIV-1 is required.

Full-length individual proviral sequencing (FLIPS) provides an efficient and high-throughput method for the amplification and sequencing of single, near full-length (intact and defective) HIV-1 proviruses and allows for determination of their potential replication-competency. FLIPS overcomes limitations of previous assays designed to sequence the latent HIV-1 reservoir.

用于下一代测序的近全长 HIV-1 Proviruses 的扩增

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near ...

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 ...

A Customizable Protocol for String Assembly gRNA Cloning (STAgR)

The bacterial CRISPR/Cas9 system has substantially increased methodological options for life scientists. Due to its utilization, genetic and genomic engineering became applicable to a large range of systems. Moreover, many transcriptional and epigenomic engineering approaches are now generally feasible for the first time. One reason for the broad applicability of CRISPR lies in its bipartite nature. Small gRNAs determine the genomic targets of the complex, variants of the protein Cas9, and the local molecular consequences. However, many CRISPR approaches depend on the simultaneous delivery of multiple gRNAs into individual cells. Here, we present a customizable protocol for string assembly gRNA cloning (STAgR), a method that allows the simple, fast and efficient generation of multiplexed gRNA expression vectors in a single cloning step. STAgR is cost-effective, since (in this protocol) the individual targeting sequences are introduced by short overhang primers while the long DNA templates of the gRNA expression cassettes can be re-used multiple times. Moreover, STAgR allows single step incorporation of a large number of gRNAs, as well as combinations of different gRNA variants, vectors and promoters.

A Customizable Protocol for String Assembly gRNA Cloning STAgR

Here, we present string assembly gRNA cloning (STAgR), a method to easily multiplex gRNA vectors for CRISPR/Cas9 approaches. STAgR makes gRNA multiplexing simple, efficient and customizable.

字符串装配 grna 克隆 (stacr) 的可自定义协议

The bacterial CRISPR/Cas9 system has substantially increased methodological options for life scientists. Due to its utilization, genetic and genomic ...