这项研究是由美国农业部-NIFA 奖2015-67015-23488 到罗杰锥。作者感谢 Dr. 的描述在视频中的育雏股票选择标准。艾哈迈德 Elaswad 和卡里姆希望感谢埃及文化和教育局在华盛顿特区的资助他们的博士研究。

这项试验是在奥本大学 e.w. 贝壳渔业研究中心的鱼类遗传学研究组进行的。在实验开始之前, 这项实验所使用的所有实验协议都得到了奥本大学动物保育和使用委员会 (AU IACUC) 的批准。本试验所使用的设备和耗材清单可在材料表中找到。下面是在图 1中所示的对沟鲶单胞胚胎的制备和显微注射的步骤和程序。
1. 育雏选股和产卵
<ol><li>选择健康的鲶鱼幼鱼种群的应变或遗传线的利益。沟鲶雄性和雌性应表现出良好的次生性特征。</li>
	<li>选择男性有一个宽广的肌肉头, 比他们的身体其余的发达生殖器官宽。深色, 疤痕和领土战斗的伤口也是性准备的迹象。选择女性的头, 比他们的身体的其他部分更狭窄, 并有柔软, 可扪, 圆润的腹部。为了准确和避免在最初的处理过程中强调鱼, 在检查它们是否准备产卵之前, 停止喂养雌性2-3 天。</li>
	<li>快速执行鱼的操作, 但要小心。为了减少搬运压力, 在产卵袋 (32 毫米大小的网眼) 中, 执行所有需要处理雌性的过程。</li>
	<li>就在激素注射前诱导排卵, 测量雌性的体重。</li>
	<li>将产卵袋悬挂在流经水箱内的雌性体内, 并有连续的水流和充足的通气。</li>
	<li>菌, 载有14克针与85µg/千克促黄体激素释放激素模拟 (LHRHa) 植入物的注入。在注射前用0.9% 无菌生理盐水冲洗注射部位 (背部肌组织)。在45°角度插入注入并释放植入物。取出针头, 然后用前70% 乙醇浸泡过的棉球将注射部位刷卡。</li>
	<li>将产卵袋放在保温水箱中, 使鱼完全浸入水中15-20 厘米以下的水面。充足的曝气 (超过 5 ppm 溶解氧) 和良好的水质是重要的排卵高品质的鸡蛋。</li>
	<li>利用度-小时 (h) 关系35预测基于水温的排卵时间。度-h 反应时间 = 激素注射液与排卵时间 (h) * 水温 (° c)。例如, 对于一个900-1、000的度 h 响应时间, 雌激素在25° c 的荷尔蒙注射时间会产生36-40 小时。通常, 第一次检查的鸡蛋是在大约36小时后, 激素注射, 然后在4小时间隔。</li>
	<li>要检查排卵, 在你寻找产卵袋内的卵时, 轻轻地提起水面上的产卵袋。看到至少10鸡蛋表明, 这名女性是排卵和准备手剥。</li>
</ol>2. 精子制备
注意: 精子可以在预期排卵时间前准备几小时。
<ol><li>由于麻醉可能对精子的运动产生负面影响, 安乐男性通过撞击头部而不受麻醉。</li>
	<li>在一个小的称重锅中收集睾丸, 取出任何组织, 用50毫升的0.9% 盐水冲洗, 必要时取出血液。发育良好的睾丸是白色的, 有许多绒毛的预测 (图 2)。</li>
	<li>确定睾丸的重量。</li>
	<li>为了准备卵子受精的精子溶液, 用镊子将睾丸转移到100厘米2 100 µm 网的中心。折叠的网与睾丸内, 粉碎睾丸和过滤的精子成一个50毫升收集管。这可以通过施加压力, 对睾丸内的网格对收集管的边缘通过拇指。用0.9% 盐水溶液将精子从筛网中洗净成收集管。盐水溶液可加到10毫升/克的睾丸。</li>
	<li>确定精子的浓度, 检查其活力和活力。 注意: 浓度可以通过计数的精子细胞在已知稀释量的米特使用例。另外, 通过用分光光度计评价米特的光学密度可以估计精子密度。在这种方法中, 将 505 nm 波长的吸收与先前为吸收不同浓度的精子36而准备的控制图进行比较。精子运动可以检查显微镜下的时间从激活精子直到停止精子活动 (称为运动持续时间)37。精子的生存能力可以通过染色的选择染料, 如台盼蓝, 这将只染色非可行的精子细胞, 而可行的精子将保持不。然而, 对于这些微注射程序, 这是没有必要的, 如果睾丸是很好的发展。</li>
	<li>在4° c 的步骤2.5 中储存0.9% 盐水中的精子, 并在准备后24小时内使用。</li>
</ol>3. 蛋的收集和受精
<ol><li>应用一层薄薄的蔬菜缩短到20厘米直径清洁干燥产卵锅。</li>
	<li>将雌性放入含有 100 ppm 缓冲的 tricaine 甲烷磺酸钠的麻醉溶液中 (最终溶液的 pH 值应为 7.0), 直到鱼完全被麻醉38。 注意: Tricaine 甲烷磺酸可能会刺激, 如果吸入, 摄入或通过皮肤吸收。</li>
	<li>小心, 将鱼从产卵袋中取出, 浸在0.9% 盐水中, 冲洗掉麻醉液。用干净的毛巾将鱼完全晾干, 避免在鱼体表面去除粘液层。</li>
	<li>将蔬菜缩短到通风口周围的区域, 包括骨盆鳍, 以防止在手剥时附着鸡蛋。</li>
	<li>通过对卵巢施加轻柔的压力, 将卵带入油脂的产卵锅中。好的鸡蛋应自由流动, 颜色金黄, 并有最小或没有结块或血块 (图 2)。在受精前避免鸡蛋和水之间的接触, 因为水可以刺激孔关闭。</li>
	<li>为了使卵受精以进行微注射, 将300xg 的卵转移到油脂的产卵锅中。油脂塑料植物标签可以用来作为勺子转移鸡蛋到油脂产卵锅。加入1-2 毫升的精子溶液 (从步骤 2.5) 到鸡蛋和混合轻轻。精子与卵子的比例约为1.1万精子/卵子。</li>
	<li>将淡水添加到卵子中, 以激活精子和卵子。添加足够的水, 以稍微覆盖的鸡蛋。轻轻地将卵旋入三十年代. 施肥应在1-2 分钟内发生。这是很重要的受精鸡蛋是安排在一个单一的一层。鲶鱼卵相互粘在一起, 使得 microinject 多层胚胎变得困难。</li>
	<li>在受精卵中加入更多的淡水, 让卵硬化10-15 分钟。</li>
	<li>用湿毛巾覆盖含有剩余未受精卵的产卵锅, 防止在几个小时内干燥和施肥。受精可以以交错的方式进行, 例如,一组300xg 卵可以每30-60 分钟受精, 以确保在一个细胞阶段连续供应胚胎, 用于注射和控制非注射胚胎。避免湿毛巾和鸡蛋之间的接触, 因为鸡蛋可能会粘在湿毛巾上, 使其难以去除。在这个协议中, 卵子在2-3 小时内受精后被有效微量, 并与卵子在剥离后立即受精的成功率相似。</li>
</ol>4. 拔针和装载
<ol><li>将1.0 毫米的硼硅玻璃毛细管拉入2针 (图 3)。把针放在一个纸盒子里, 里面有一块海绵, 里面有几个切口。为了避免断针, 海绵的直径应小于针杆的长度。</li>
	<li>用锋利的物体打破针的最薄区域的一小部分, 打开针尖。另外, 在显微镜下用镊子掐掉尖端最薄的区域, 打开针头。</li>
	<li>将 gRNA (s) 与 Cas9 蛋白混合, 准备注射液。其他类型的 RNA 或蛋白质也可以注射相同的程序。在本协议中, gRNAs 的目的是针对两个通道鲶鱼免疫相关基因, 收费/白细胞介素1受体域的适配器分子 (TICAM1) 基因和李结合凝集蛋白 (RBL) 基因和准备根据沙阿et al。使用高保真度 Taq 聚合酶32,39。gRNAs 的基因组靶点为 5 " GGA GGT GAA 增值税1基因 (图 4) 和 5" GGA 3 "TICAM GGA GAA GTG G 3 ' 对 RBL 基因的外显子1和 protospacer 相邻的主题 (PAM) 序列下划线 (图 6)。<ol>
<li>针对沟道鲶鱼基因组序列, 并选择无潜在靶点的目标。</li>
		<li>加入酚红, 以使 gRNA/Cas9 蛋白混合到总体积的三分之一。</li>
		<li>调整最终浓度的 gRNA/Cas9 蛋白混合与核酸游离水, 使每个胚胎注射 50 nL 含有约 2.5 ng gRNA 和 7.5 ng Cas9 蛋白。使用前在冰上孵育10分钟的混合物。</li>
	</ol>
</li>
	<li>用 microloader, 负载5-10 µL 的 gRNA/Cas9 混合物进入注射针通过插入 microloader 到针阀杆, 并慢慢地排出混合物, 而撤回 microloader 提示 (图 3)。避免在里面捕捉气泡。</li>
	<li>将针固定在微上, 确保连接紧密, 然后将支架固定在微上。确保自由稳定的运动。通过打开氮气缸和调整压力调节器来施加注射压力。 注: 注射量可受压力、针孔直径及注射时间的影响。要确定注射量, 注入一滴矿物油放在例上。如果需要, 压力, 注射时间, 针直径可以调整, 以增加或减少注射量。在矿物油中注入多次, 以确保体积一致。</li>
</ol>5. 鲶鱼胚胎的显微注射
<ol><li>应用一个非常薄的一层蔬菜缩短到100毫米清洁培养皿。</li>
	<li>使用油脂塑料植物标签, 转移50-100 鸡蛋从受精盘到培养皿和覆盖他们与 Holtfreter 的解决方案 (材料表)。在一个单独的层中对齐鸡蛋。在注射过程中, 这种排列应该能保持卵的位置。</li>
	<li>将培养皿与卵放在显微镜的舞台上, 用一只手握住。用另一只手 (图 3) 降低针头, 直到它将绒毛膜和蛋黄穿透到一个平滑的运动中。针头的尖端应尽可能靠近 blastodisc, 然后再送上注射材料。<ol>
<li>按住踏板将注射材料送进蛋黄。如果 blastodisc 不清楚地被看见, 则射入材料可以被传播入不同的位置在蛋黄。为了做到这一点 , 针入到蛋黄的最末端 , 然后压抑踏板和撤回针同时进行 , 通过不同地区的蛋黄传播的注射材料。多达 50 nanoliters 可以注射到沟鲶胚胎的卵黄, 但是, gRNA/Cas9 蛋白的数量需要优化每个基因, 以达到最佳的结果突变率和胚胎生存32。</li>
	</ol>
</li>
	<li>将针头平滑地缩回, 以免损坏鸡蛋。移动培养皿以相同的程序注入另一个卵子。避免在注射过程中培养皿的运动, 因为这可能会损害卵子或折断针头。除去因注射而破裂或损坏的卵。注射可以开始在 15-20 min 在受精以后和继续, 只要胚胎仍然在一个细胞阶段 (直到大约 60-90 min 在受精以后)。</li>
	<li>将注入的胚胎放回 Holtfreter 的溶液中, 用 10 ppm 的强力霉素和连续温和曝气6-7 天, 直到孵化40。每天更换解决方案并清除死胚胎。</li>
</ol>6. 突变检测
<ol><li>随机收集几个注射胚胎在72小时后受精, 去除蛋黄和提取基因组 DNA。包括3-5 非注射胚胎作为对照。 注: 用蛋白酶 K 消化和乙醇沉淀去除卵壳和卵黄物质后, 可以从胚胎中提取基因组 DNA32。</li>
	<li>使用高保真度 Taq 聚合酶32, 在每个基因的 gRNA 上放大一个 DNA 片段, 以此来预测突变的发生。对于 TICAM 1, 引物 5 "GCTGCTGAATGTCTGATTATG 3" (正向) 和 5 "GTCCTCCACACTCCTGAAG 3" (反向) 用于放大 750 bp, 而对于 RBL, 引物 5 "TCATTATTTCCTGGTACTCAGTA 3" (向前) 和 5 "AGAGATTAGTCACACATCATTATT 3" (反向) 用于从 RBL 基因中放大 375 bp 段。<ol>
<li>使用以下 pcr 反应: 高达20µL pcr 级水;1x 高保真 Taq 酶反应缓冲区与氯化镁2, 0.2 毫米 dNTP 混合, 0.4 µM 为每一个前向和反向底漆相同的集合, 100-300 ng 基因组 DNA 和1.25 单位高保真 Taq 酶混合。执行 PCR 循环条件: 初始变性在94° c 为3分钟;35循环变性在94° c 为三十年代, 退火在60° c 为五十五年代为 TICAM, 并且四十年代为 RBL, 引伸在72° c 为1极小/kb;并且最后的引伸在72° c 为 10 min。</li>
	</ol>
</li>
	<li>利用突变检测方法检测突变胚, 如纯化 PCR 产物序列、测量突变检测法、heteroduplex 导纳法、限制酶识别点的损失或任何其他的突变检测方法适合目标。在本协议中, 检测突变胚胎使用的测量突变检测的 TICAM 1 和限制酶SapI切割现场, 以及序列的纯化 PCR 产品的 RBL 基因。</li>
	<li>鉴定突变等位基因, 克隆 PCR 产物和单个菌落的序列向量。在图 4和图 6中出现的突变等位基因来自注入的胚胎。PCR 产物从六微量和3控制胚胎为每个基因被汇集了在两个单独管, 一个为微量胚胎和另一个为控制胚胎, 快速地克隆和86传染媒介为 TICAM 1 和48传染媒介为 RBL 基因测序, 包括8序列反应从3控制非注射胚胎为每个基因。</li>
	<li>为了识别不同的变异等位基因, 使用任何 DNA 序列对准工具 (如爆炸或 T 咖啡) 将突变胚胎的序列与野生类型序列进行比较。</li>
</ol>

作者声明他们没有利益冲突。

本文介绍了一种用于实现基因敲除的沟鲶胚显微注射的详细方案。CRISPR/Cas9 蛋白在蛋黄中的注射更简单, 节省时间, 不需要广泛的训练时, microinjecting blastodisc 的一细胞胚胎, 这是最常见的技术, 大多数基因转移和基因编辑与鱼30,42. 目前的协议证明是成功的诱导 indels 沟鲶 TICAM 1 和 RBL 基因。这些可靠和有效的程序为今后在沟鲶中产生基因敲除奠定了基础。...

微注射是一种常用的实验室技术, 通过玻璃毛细管1将少量的物质 (如 DNA、RNA、蛋白质和其他大分子) 输送到细胞或胚胎中。显微注射是使用特殊的设备设置, 包括微量, 微, 和显微镜2。这项技术已经被研究者用来通过转基因、基因敲除和基因治疗来改变许多生物体, 目的是了解细胞内组件的动力学3,4,5。
沟鲶 (鮰松毛虫) 是美国水产养殖和休闲捕鱼活动中最流行的鲶鱼种类。越来越需要研究沟鲶的功能基因组学, 而沟鲶完整基因组测序提高了基因组编辑工具的应用价值6,7。了解基因功能不仅可以丰富对鲶鱼进行的研究, 而且还能导致更有效的基因改良计划, 以提高鲶鱼产业。一旦确定了某一特定性状的关键基因, 它们就可以通过基因组编辑来改进鲶鱼的生产, 从而产生有益的等位基因, 选择这些等位基因, 抑制有害等位基因, 转移通过转基因的有益等位基因, 或这些选项的一些组合。将不同品种的不同商业利益性状的最佳基因结合在一起, 将大大提高鱼类生产作业的生产率和利润率8。
基因敲除是一种直接的方法来研究基因功能在体内。DNA 水平的突变将被后代继承, 这将有助于研究它们在不同世代的影响。不同的基因组编辑工具已经开发, 包括锌指核酸 (ZFNs), 转录激活剂样效应核酸 (TALENs), 和聚集定期 interspaced 短复发重复/CRISPR 相关蛋白 9 (CRISPR/Cas9) 系统9,10,11,12。
CRISPR/Cas9 系统是一个强大的, 有效的工具, 已被用来编辑基因组 dna 序列包括在鱼通过 RNA 引导的站点特定的 dna 劈裂5,13。该系统由一个引导 RNA (gRNA) 组成, 它决定了基因组中的目标序列和 DNA 切酶, Cas9。CRISPR/Cas9 系统可以被设计成针对基因组中的任何序列, 具有优于 ZFNs 和 TALENs 的几个优点: (1) 更低的成本 (2) 更容易的工程 (3) 更具体的指南 RNA 绑定到目标序列, 并减少离靶突变 (4)多个序列可以同时针对不同的 gRNA (5) 基因的高诱变率, 不能由 TALENs 突变, 和 (6) 改善系传输率的突变高达6倍, 相比 ZFNs 和 TALENs14, 15、16、17、18。
微注射的主要替代方法是电穿孔, 其中电动脉冲应用于胚胎或细胞, 以提高膜通透性和生物分子的摄取量19,20。一些转基因鱼的产生使用电穿孔, 如鳉 (Oryzias latipes), 斑马鱼 (斑马鱼), 鳞鲑 (虹 tshawytscha), 沟鲶, 海鲷 (鲷 sarba), 并鲤鱼 (鲤鱼)21,22,23,24,25,26。
电穿孔已被用来提供质粒 DNA, RNA, 和 Cas9 蛋白的基因敲除。在哺乳动物细胞中, Cas9/gRNA 质粒 DNA、Cas9 mRNA/gRNA 和 Cas9 蛋白/gRNA 配合物通过电穿孔传递, 而诱变率最高的是使用 Cas9 蛋白/gRNA 配合物来测试大多数电穿孔条件下的27。在海脊索动物 (Ciona 性) 中, TALENs 表达的构造被 electroporated 成受精卵, 以诱导多种基因的敲除28。ZFNs 表达质粒结构是 electroporated 在沟鲶中敲出黄体生成素的途径29。然而, 一旦引入细胞, 质粒结构将需要转录到 rna 和翻译成功能蛋白之前, 他们可以针对预期的 DNA 序列, 这将可能延迟的时间, 与微注射 rna/蛋白.随着胚胎的发育, 延迟诱变增加了注射嵌的形成。此外, 转基因表达不太可能通过微注射来达到表达水平, 降低诱变效率。
作为一种将基因组编辑工具引入细胞的方法, 微注射在电穿孔方面有几个优点。基因组编辑分子可以通过显微注射30可靠地引入细胞或胚胎。需要较少的注塑材料。很容易确定注入的材料的数量。更高的变异率与低嵌在创建者鱼可以达到, 将改进系传输变异到 F1。由于突变率较高, 需要分析生产第一代鱼的创始人较少。在电穿孔, 更多的创始人鱼需要分析, 这将增加成本。然而, 沟鲶微量胚的生存率低于 electroporated, 但可以通过注入更多的胚胎31,32来克服。在沟鲶中, 卵黄微量胚的存活范围从16到55% 不等, 这取决于所针对的基因和 gRNA/Cas9 蛋白的用量32。
定期注射鲶鱼胚胎在技术上要求和耗时, 然而, 一个快速和有效的 CRISPR/Cas9 蛋白显微注射协议提出的沟鲶胚胎。这一协议需要更少的时间和专业知识, 因为 CRISPR/Cas9 蛋白溶液被注射到一个细胞胚胎的蛋黄。数以百计的受精卵可以在1小时内注射 (大约是第一次发生细胞分裂所需的时间)。为了验证该协议, 在沟鲶、toll/白细胞介素1受体域内含有的适配器分子 (TICAM1) 基因和李结合凝集蛋白 (RBL) 基因中, 两种病易感性相关基因被淘汰。TICAM 1 参与了由 toll 样受体 (TLR) 3 启动的信号通路。在沟鲶, TICAM 1 是显着上调以下细菌的挑战与爱德华 ictaluri, 而它是 downregulated 在蓝鲶, 一个物种耐E. ictaluri33。RBL 在早期感染中起重要作用,杆菌柱, 杆菌病的致病剂, 在上调敏感的通道鲶鱼菌株中记录了急性和强健的杆菌, 与杆菌电阻应变34。

<table border="0" cellpadding="0" cellspacing="0" width="520">
	<tbody>
		<tr height="100">
			<td height="100" style="height:100px;width:136px;">Reproboost implant</td>
			<td style="width:115px;">Center of Marine Biotechnology</td>
			<td style="width:99px;"></td>
			<td style="width:171px;">Luteinizing hormone releasing hormone analog (LHRHa) for induction of ovulation in channel catfish females</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:136px;">TRICAINE-S</td>
			<td style="width:115px;">Western Chemical. Inc.</td>
			<td style="width:99px;"></td>
			<td style="width:171px;">For sedation of brood stock fish during hormone injection and egg stripping.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:136px;">Phenol red</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:99px;">P0290</td>
			<td style="width:171px;">0.5%, sterile filtered</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:136px;">Stereo microscope</td>
			<td style="width:115px;">Olympus</td>
			<td style="width:99px;">213709</td>
			<td style="width:171px;">For visualizing the eggs during microinjection</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:136px;">Microinjector</td>
			<td style="width:115px;">ASI-Applied Scientific Instrumentation&#160;</td>
			<td style="width:99px;">Model MPPI-3</td>
			<td style="width:171px;">For the delivery of the injection material into the embryos</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:136px;">Micromanipulator</td>
			<td style="width:115px;">ASI-Applied Scientific Instrumentation&#160;</td>
			<td style="width:99px;">Model MM33&#160;</td>
			<td style="width:171px;">For holding and controlling the movement of the injection needle.&#160;</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:136px;">Eppendorf Microloader</td>
			<td style="width:115px;">Eppendorf</td>
			<td style="width:99px;">5242956.003</td>
			<td style="width:171px;">For loading injection solution into microinjection needles.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:136px;">Vertical needle puller</td>
			<td style="width:115px;">David Kopf Instruments</td>
			<td style="width:99px;">Model 720</td>
			<td style="width:171px;">For pulling microinjection needles</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:136px;">Cas9 protein</td>
			<td style="width:115px;">PNA Bio Inc.&#160;</td>
			<td style="width:99px;">CP01</td>
			<td style="width:171px;">Recombinant Cas9 protein from Streptococcus pyrogenes.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:136px;">Expand High FidelityPLUS PCR System</td>
			<td style="width:115px;">&#160;Roche Diagnostics, USA</td>
			<td style="width:99px;"></td>
			<td style="width:171px;">For PCR and amplification of DNA templates to be used in gRNA preparation</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:136px;">Borosilicate glass capillaries</td>
			<td style="width:115px;">Fisher Scientific</td>
			<td style="width:99px;"></td>
			<td style="width:171px;">1 mm outer diameter (OD), for making microinjection needles.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:136px;">Petri dish</td>
			<td style="width:115px;">VWR</td>
			<td style="width:99px;">25384-302</td>
			<td style="width:171px;">For holding the embryos during the microinjection.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:136px;">Crisco</td>
			<td style="width:115px;">The J.M. Smucker Company</td>
			<td style="width:99px;"></td>
			<td style="width:171px;">Vegetable shortening for coating spawning pans and petri dishes.&#160;</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:136px;">Holtfreter`s solution</td>
			<td style="width:115px;">Home Made</td>
			<td style="width:99px;"></td>
			<td style="width:171px;">59 mM NaCl, 0.67 mM KCl, 2.4 mM NaHCO3, 0.76 mM CaCl2, 1.67 mM MgSO4 to incubate the microinjected embryos till hatch.&#160;</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:136px;">Doxycycline hyclate USP (monohydrate)</td>
			<td style="width:115px;">Letco Medical</td>
			<td style="width:99px;">690904</td>
			<td style="width:171px;">Antibiotic added to Holtfreter&#39;s solution to 10 ppm to prevent bacterial infections.</td>
		</tr>
	</tbody>
</table>

microinjection of crispr/cas9 protein into channel catfish, ictalurus punctatus, embryos for gene editing

整个基因组的渠道鲶鱼,鮰松毛虫, 已排序, 导致更多的机会研究渠道鲶鱼基因功能。基因敲除已被用来研究这些基因功能在体内。聚类定期 interspaced 短复发重复/CRISPR 相关蛋白 9 (CRISPR/Cas9) 系统是一个强大的工具, 用于编辑基因组 DNA 序列, 以改变其功能。传统的方法是通过显微注射将 CRISPR/Cas9 mRNA 引入单细胞胚胎, 这在鲶鱼中可能是一个缓慢而低效的过程。本文介绍了 CRISPR/Cas9 蛋白微注射鲶鱼胚胎的详细方案。简单地, 收集卵子和精子, 然后进行人工受精。受精卵被转移到含有 Holtfreter 溶液的培养皿中。注射量被校准, 然后引导 RNAs/Cas9 靶向的收费/白细胞介素1受体域包含的适配器分子 (TICAM 1) 基因和李结合凝集蛋白 (RBL) 基因微量到卵黄的一细胞胚胎。基因剔除是成功的, indels 通过 DNA 测序得到证实。由于这些突变的预测蛋白质序列的变化包括 frameshift 和截断蛋白质由于过早停止密码。

为了证明微注射协议的有效性, gRNAs 设计的靶向沟鲶费/白细胞介素1受体域包含的适配器分子 (TICAM1) 基因和李结合凝集蛋白 (RBL) 基因微量。
TICAM 1 DNA 测序的载体从单个殖民地, 克隆 PCR 产品揭示了 indel 突变诱导的 TICAM 1 基因。突变率为 79%, 在24人分析。图 4和图 5说明?...

Watch this Scientific Journal Video about Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing at JoVE.com

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

本文介绍了一种简单高效的 CRISPR/Cas9 系统在沟鲶胚胎基因编辑中的微注射协议。在这个协议中, 引导 rna 和 Cas9 蛋白被微量到一细胞胚胎的卵黄中。通过敲出两条沟鲶免疫相关基因来验证这个协议。

CRISPR/Cas9 蛋白注射到沟鲶、鮰松毛虫、基因编辑的胚胎

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene ...

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Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

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14.7K 次观看.  Auburn University. 本文介绍了一种简单高效的 CRISPR/Cas9 系统在沟鲶胚胎基因编辑中的微注射协议。在这个协议中, 引导 rna 和 Cas9 蛋白被微量到一细胞胚胎的卵黄中。通过敲出两条沟鲶免疫相关基因来验证这个协议。

视频: Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the identification and verification of a drug resistance gene in the Apicomplexan parasite Toxoplasma gondii, the method of Quantitative Trait Locus (QTL) mapping using a Whole Genome Sequence (WGS) -based genetic map and the method of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 -based gene editing. The approach of QTL mapping allows one to test if there is a correlation between a genomic region(s) and a phenotype. Two datasets are required to run a QTL scan, a genetic map based on the progeny of a recombinant cross and a quantifiable phenotype assessed in each of the progeny of that cross. These datasets are then formatted to be compatible with R/qtl software that generates a QTL scan to identify significant loci correlated with the phenotype. Although this can greatly narrow the search window of possible candidates, QTLs span regions containing a number of genes from which the causal gene needs to be identified. Having WGS of the progeny was critical to identify the causal drug resistance mutation at the gene level. Once identified, the candidate mutation can be verified by genetic manipulation of drug sensitive parasites. The most facile and efficient method to genetically modify T. gondii is the CRISPR/Cas9 system. This system comprised of just 2 components both encoded on a single plasmid, a single guide RNA (gRNA) containing a 20 bp sequence complementary to the genomic target and the Cas9 endonuclease that generates a double-strand DNA break (DSB) at the target, repair of which allows for insertion or deletion of sequences around the break site. This article provides detailed protocols to use CRISPR/Cas9 based genome editing tools to verify the gene responsible for sinefungin resistance and to construct transgenic parasites.

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

Details are presented on how QTL mapping with a whole genome sequence based genetic map can be used to identify a drug resistance gene in Toxoplasma gondii and how this can be verified with the CRISPR/Cas9 system that efficiently edits a genomic target, in this case the drug resistance gene.

QTL定位和CRISPR / Cas9编辑识别抗药性基因<em&gt;弓形虫</em

Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the ...

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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 ...

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 ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. Complete gene ablation in HSCs required the generation of knockout mice from which HSCs could be isolated, and gene ablation in primary human HSCs was not possible. Viral transduction could be used for knock-down approaches, but these suffered from variable efficacy. In general, genetic manipulation of human and mouse hematopoietic cells was hampered by low efficiencies and extensive time and cost commitments. Recently, CRISPR/Cas9 has dramatically expanded the ability to engineer the DNA of mammalian cells. However, the application of CRISPR/Cas9 to hematopoietic cells has been challenging, mainly due to their low transfection efficiencies, the toxicity of plasmid-based approaches and the slow turnaround time of virus-based protocols.
A rapid method to perform CRISPR/Cas9-mediated gene editing in murine and human hematopoietic stem and progenitor cells with knockout efficiencies of up to 90% is provided in this article. This approach utilizes a ribonucleoprotein (RNP) delivery strategy with a streamlined three-day workflow. The use of Cas9-sgRNA RNP allows for a hit-and-run approach, introducing no exogenous DNA sequences in the genome of edited cells and reducing off-target effects. The RNP-based method is fast and straightforward: it does not require cloning of sgRNAs, virus preparation or specific sgRNA chemical modification. With this protocol, scientists should be able to successfully generate knockouts of a gene of interest in primary hematopoietic cells within a week, including downtimes for oligonucleotide synthesis. This approach will allow a much broader group of users to adapt this protocol for their needs.

A protocol for fast CRISPR/Cas9-mediated gene disruption in mouse and human primary hematopoietic cells is described in this article. Cas9-sgRNA ribonucleoproteins are introduced via electroporation with sgRNAs generated through in vitro transcription and commercial Cas9. High editing efficiencies are achieved with limited time and financial cost.

CRISPR/Cas9 对小鼠和人造血祖细胞的高效基因破坏

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical regions, is caused by infection with Plasmodium parasites. The development of more effective drugs to combat malaria can be accelerated by improving our understanding of the biology of this complex parasite. Genetic manipulation of these parasites is key to understanding their biology; however, historically the genome of P. falciparum has been difficult to manipulate. Recently, CRISPR/Cas9 genome editing has been utilized in malaria parasites, allowing for easier protein tagging, generation of conditional protein knockdowns, and deletion of genes. CRISPR/Cas9 genome editing has proven to be a powerful tool for advancing the field of malaria research. Here, we describe a CRISPR/Cas9 method for generating glmS-based conditional knockdown mutants in P. falciparum. This method is highly adaptable to other types of genetic manipulations, including protein tagging and gene knockouts.

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

We describe a method for generating glmS-based conditional knockdown mutants in Plasmodium falciparum using CRISPR/Cas9 genome editing.

CRISPR/Cas9 基因编辑制作人类疟疾寄生虫的条件突变体

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical ...

Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or C-terminal fluorescent tags. The prokaryotic CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) may be used to introduce large exogenous sequences into genomic loci via homology directed repair (HDR). To achieve the desired knock-in, this protocol employs the ribonucleoprotein (RNP)-based approach where wild type Streptococcus pyogenes Cas9 protein, synthetic 2-part guide RNA (gRNA), and a donor template plasmid are delivered to the cells via electroporation. Putatively edited cells expressing the fluorescently tagged proteins are enriched by fluorescence activated cell sorting (FACS). Clonal lines are then generated and can be analyzed for precise editing outcomes. By introducing the fluorescent tag at the genomic locus of the gene of interest, the resulting subcellular localization and dynamics of the fusion protein can be studied under endogenous regulatory control, a key improvement over conventional overexpression systems. The use of hiPSCs as a model system for gene tagging provides the opportunity to study the tagged proteins in diploid, nontransformed cells. Since hiPSCs can be differentiated into multiple cell types, this approach provides the opportunity to create and study tagged proteins in a variety of isogenic cellular contexts.

Described here is a protocol for tagging endogenously expressed proteins with fluorescent tags in human induced pluripotent stem cells using CRISPR/Cas9. Putatively edited cells are enriched by fluorescence activated cell sorting and clonal cell lines are generated.

CRISPR/Cas9 诱导人多潜能干细胞内源蛋白标记的应用

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or ...

CRISPR/Cas9 Ribonucleoprotein-mediated Precise Gene Editing by Tube Electroporation

Gene editing nucleases, represented by CRISPR-associated protein 9 (Cas9), are becoming mainstream tools in biomedical research. Successful delivery of CRISPR/Cas9 elements into the target cells by transfection is a prerequisite for efficient gene editing. This protocol demonstrates that tube electroporation (TE) machine-mediated delivery of CRISPR/Cas9 ribonucleoprotein (RNP), along with single-stranded oligodeoxynucleotide (ssODN) donor templates to different types of mammalian cells, leads to robust precise gene editing events. First, TE was applied to deliver CRISPR/Cas9 RNP and ssODNs to induce disease-causing mutations in the interleukin 2 receptor subunit gamma (IL2RG) gene and sepiapterin reductase (SPR) gene in rabbit fibroblast cells. Precise mutation rates of 3.57%-20% were achieved as determined by bacterial TA cloning sequencing. The same strategy was then used in human iPSCs on several clinically relevant genes including epidermal growth factor receptor (EGFR), myosin binding protein C, cardiac (Mybpc3), and hemoglobin subunit beta (HBB). Consistently, highly precise mutation rates were achieved (11.65%-37.92%) as determined by deep sequencing (DeepSeq). The present work demonstrates that tube electroporation of CRISPR/Cas9 RNP represents an efficient transfection protocol for gene editing in mammalian cells.

Presented here is a protocol for efficient CRISPR/Cas9 ribonucleoprotein-mediated gene editing in mammalian cells using tube electroporation.

CRISPR/Cas9 核糖核酸蛋白介导的精确基因编辑通过管电穿孔

Gene editing nucleases, represented by CRISPR-associated protein 9 (Cas9), are becoming mainstream tools in biomedical research. Successful delivery of ...

Introducing a Gene Knockout Directly Into the Amastigote Stage of Trypanosoma cruzi Using the CRISPR/Cas9 System

Trypanosoma cruzi is a pathogenic protozoan parasite that causes Chagas&#8217; disease mainly in Latin America. In order to identify a novel drug target against T. cruzi, it is important to validate the essentiality of the target gene in the mammalian stage of the parasite, the amastigote. Amastigotes of T. cruzi replicate inside the host cell; thus, it is difficult to conduct a knockout experiment without going through other developmental stages. Recently, our group reported a growth condition in which the amastigote can replicate axenically for up to 10 days without losing its amastigote-like properties. By using this temporal axenic amastigote culture, we successfully introduced gRNAs directly into the Cas9-expressing amastigote to cause gene knockouts and analyzed their phenotypes exclusively in the amastigote stage. In this report, we describe a detailed protocol to produce in vitro derived extracellular amastigotes, and to utilize the axenic culture in a CRISPR/Cas9-mediated knockout experiment. The growth phenotype of knockout amastigotes can be evaluated either by cell counts of the axenic culture, or by replication of intracellular amastigote after host cell invasion. This method bypasses the parasite stage differentiation normally involved in producing a transgenic or a knockout amastigote. Utilization of the temporal axenic amastigote culture has the potential to expand the experimental freedom of stage-specific studies in T. cruzi.

Introducing a Gene Knockout Directly Into the Amastigote Stage of Trypanosoma cruzi Using the CRISPR/Cas9 System

Here, we describe a protocol to introduce a gene knockout into the extracellular amastigote of Trypanosoma cruzi, using the CRISPR/Cas9 system. The growth phenotype can be followed up either by cell counting of axenic amastigote culture or by proliferation of intracellular amastigotes after host cell invasion.

使用CRISPR/Cas9系统将基因挖空直接引入锥虫瘤的肌瘤阶段

Trypanosoma cruzi is a pathogenic protozoan parasite that causes Chagas&#8217; disease mainly in Latin America. In order to identify a novel drug target ...

CRISPR-Cas9-Mediated Genome Editing in the Filamentous Ascomycete Huntiella omanensis

The CRISPR-Cas9 genome editing system is a molecular tool that can be used to introduce precise changes into the genomes of model and non-model species alike. This technology can be used for a variety of genome editing approaches, from gene knockouts and knockins to more specific changes like the introduction of a few nucleotides at a targeted location. Genome editing can be used for a multitude of applications, including the partial functional characterization of genes, the production of transgenic organisms and the development of diagnostic tools. Compared to previously available gene editing strategies, the CRISPR-Cas9 system has been shown to be easy to establish in new species and boasts high efficiency and specificity. The primary reason for this is that the editing tool uses an RNA molecule to target the gene or sequence of interest, making target molecule design straightforward, given that standard base pairing rules can be exploited. Similar to other genome editing systems, CRISPR-Cas9-based methods also require efficient and effective transformation protocols as well as access to good quality sequence data for the design of the targeting RNA and DNA molecules. Since the introduction of this system in 2013, it has been used to genetically engineer a variety of model species, including Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster and Mus musculus. Subsequently, researchers working on non-model species have taken advantage of the system and used it for the study of genes involved in processes as diverse as secondary metabolism in fungi, nematode growth and disease resistance in plants, among many others. This protocol detailed below describes the use of the CRISPR-Cas9 genome editing protocol for the truncation of a gene involved in the sexual cycle of Huntiella omanensis, a filamentous ascomycete fungus belonging to the Ceratocystidaceae family.

CRISPR-Cas9-Mediated Genome Editing in the Filamentous Ascomycete Huntiella omanensis

The CRISPR-Cas9 genome editing system is an easy-to-use genome editor that has been used in model and non-model species. Here we present a protein-based version of this system that was used to introduce a premature stop codon into a mating gene of a non-model filamentous ascomycete fungus.

CRISPR-Cas9-中切基因组编辑在纤维阿莫西特 亨蒂埃拉阿曼尼西斯

The CRISPR-Cas9 genome editing system is a molecular tool that can be used to introduce precise changes into the genomes of model and non-model species ...

Sand Fly (Phlebotomus papatasi) Embryo Microinjection for CRISPR/Cas9 Mutagenesis

Sand flies are the natural vectors for Leishmania species, protozoan parasites producing a broad spectrum of symptoms ranging from cutaneous lesions to visceral pathology. Deciphering the nature of the vector/parasite interactions is of primary importance for better understanding of Leishmania transmission to their hosts. Among the parameters controlling the sand fly vector competence (i.e. their ability to carry and transmit pathogens), parameters intrinsic to these insects were shown to play a key role. Insect immune response, for example, impacts sand fly vector competence to Leishmania. The study of such parameters has been limited by the lack of methods of gene expression modification adapted for use in these non-model organisms. Gene downregulation by small interfering RNA (siRNA) is possible, but in addition to being technically challenging, the silencing leads to only a partial loss of function, which cannot be transmitted from generation to generation. Targeted mutagenesis by CRISPR/Cas9 technology was recently adapted to the Phlebotomus papatasi sand fly. This technique leads to the generation of transmissible mutations in a specifically chosen locus, allowing to study the genes of interest. The CRISPR/Cas9 system relies on the induction of targeted double-strand DNA breaks, later repaired by either Non-Homologous End Joining (NHEJ) or by Homology Driven Repair (HDR). NHEJ consists of a simple closure of the break and frequently leads to small insertion/deletion events. In contrast, HDR uses the presence of a donor DNA molecule sharing homology with the target DNA as a template for repair. Here, we present a sand fly embryo microinjection method for targeted mutagenesis by CRISPR/Cas9 using NHEJ, which is the only genome modification technique adapted to sand fly vectors to date.

Sand Fly Phlebotomus papatasi Embryo Microinjection for CRISPR/Cas9 Mutagenesis

This protocol details the steps of CRISPR/Cas9 targeted mutagenesis in sand flies: embryo collection, injection, insect rearing, and identification as well as selection of mutations of interest.

沙飞（木薯）胚胎微注射为CRISPR/Cas9穆塔赫内西

Sand flies are the natural vectors for Leishmania species, protozoan parasites producing a broad spectrum of symptoms ranging from cutaneous lesions to ...