Name: microinjection of crispr/cas9 protein into channel catfish, ictalurus punctatus, embryos for gene editing
Uploaded: 2018-01-20T13:00:00.000+00:00
Duration: 16 min 8 s
Description: Watch this Scientific Journal Video about Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing at JoVE.com

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><tbody><tr><td>Reproboost implant</td><td>Center of Marine Biotechnology</td><td></td><td>Luteinizing hormone releasing hormone analog (LHRHa) for induction of ovulation in channel catfish females</td></tr><tr><td>TRICAINE-S</td><td>Western Chemical. Inc.</td><td></td><td>For sedation of brood stock fish during hormone injection and egg stripping.&amp;nbsp;</td></tr><tr><td>Phenol red</td><td>Sigma-Aldrich</td><td>P0290</td><td>0.5%, sterile filtered</td></tr><tr><td>Stereo microscope</td><td>Olympus</td><td>213709</td><td>For visualizing the eggs during microinjection</td></tr><tr><td>Microinjector</td><td>ASI-Applied Scientific Instrumentation&amp;nbsp;</td><td>Model MPPI-3</td><td>For the delivery of the injection material into the embryos</td></tr><tr><td>Micromanipulator</td><td>ASI-Applied Scientific Instrumentation&amp;nbsp;</td><td>Model MM33&amp;nbsp;</td><td>For holding and controlling the movement of the injection needle.&amp;nbsp;</td></tr><tr><td>Eppendorf Microloader</td><td>Eppendorf</td><td>5242956.003</td><td>For loading injection solution into microinjection needles.</td></tr><tr><td>Vertical needle puller</td><td>David Kopf Instruments</td><td>Model 720</td><td>For pulling microinjection needles</td></tr><tr><td>Cas9 protein</td><td>PNA Bio Inc.&amp;nbsp;</td><td>CP01</td><td>Recombinant Cas9 protein from Streptococcus pyrogenes.</td></tr><tr><td>Expand High FidelityPLUS PCR System</td><td>&amp;nbsp;Roche Diagnostics, USA</td><td></td><td>For PCR and amplification of DNA templates to be used in gRNA preparation</td></tr><tr><td>Borosilicate glass capillaries</td><td>Fisher Scientific</td><td></td><td>1 mm outer diameter (OD), for making microinjection needles.</td></tr><tr><td>Petri dish</td><td>VWR</td><td>25384-302</td><td>For holding the embryos during the microinjection.</td></tr><tr><td>Crisco</td><td>The J.M. Smucker Company</td><td></td><td>Vegetable shortening for coating spawning pans and petri dishes.&amp;nbsp;</td></tr><tr><td>Holtfreter`s solution</td><td>Home Made</td><td></td><td>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.&amp;nbsp;</td></tr><tr><td>Doxycycline hyclate USP (monohydrate)</td><td>Letco Medical</td><td>690904</td><td>Antibiotic added to Holtfreter&#039;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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14.9K 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

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 ...

科研

遗传学

Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and behavioral phenotypes. The remarkably diverse phenotypes that have evolved as marine populations adapt to countless freshwater environments, combined with the ability to cross marine and freshwater forms, provide a rare vertebrate system in which genetics can be used to map genomic regions controlling evolved traits. Excellent genomic resources are now available, facilitating molecular genetic dissection of evolved changes. While mapping experiments generate lists of interesting candidate genes, functional genetic manipulations are required to test the roles of these genes. Gene regulation can be studied with transgenic reporter plasmids and BACs integrated into the genome using the Tol2 transposase system. Functions of specific candidate genes and cis-regulatory elements can be assessed by inducing targeted mutations with TALEN and CRISPR/Cas9 genome editing reagents. All methods require introducing nucleic acids into fertilized one-cell stickleback embryos, a task made challenging by the thick chorion of stickleback embryos and the relatively small and thin blastomere. Here, a detailed protocol for microinjection of nucleic acids into stickleback embryos is described for transgenic and genome editing applications to study gene expression and function, as well as techniques to assess the success of transgenesis and recover stable lines.

Transgenic manipulations and genome editing are critical for functionally testing the roles of genes and cis-regulatory elements. Here a detailed microinjection protocol for the generation of genomic modifications (including Tol2-mediated fluorescent reporter transgene constructs, TALENs, and CRISPRs) is presented for the emergent model fish, the threespine stickleback.

显微注射转基因的基因组，并在编辑Threespine刺鱼

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and ...

发育生物学

Manipulation of Gene Function in Mexican Cavefish

Cave animals provide a compelling system for investigating the evolutionary mechanisms and genetic bases underlying changes in numerous complex traits, including eye degeneration, albinism, sleep loss, hyperphagia, and sensory processing. Species of cavefish from around the world display a convergent evolution of morphological and behavioral traits due to shared environmental pressures between different cave systems. Diverse cave species have been studied in the laboratory setting. The Mexican tetra, Astyanax mexicanus, with sighted and blind forms, has provided unique insights into biological and molecular processes underlying the evolution of complex traits and is well-poised as an emerging model system. While candidate genes regulating the evolution of diverse biological processes have been identified in A. mexicanus, the ability to validate a role for individual genes has been limited. The application of transgenesis and gene-editing technology has the potential to overcome this significant impediment and to investigate the mechanisms underlying the evolution of complex traits. Here, we describe a different methodology for manipulating gene expression in A. mexicanus. Approaches include the use of morpholinos, Tol2 transgenesis, and gene-editing systems, commonly used in zebrafish and other fish models, to manipulate gene function in A. mexicanus. These protocols include detailed descriptions of timed breeding procedures, the collection of fertilized eggs, injections, and the selection of genetically modified animals. These methodological approaches will allow for the investigation of the genetic and neural mechanisms underlying the evolution of diverse traits in A. mexicanus.&#160;

We describe approaches for the manipulation of genes in the evolutionary model system Astyanax mexicanus. Three different techniques are described: Tol2-mediated transgenesis, targeted manipulation of the genome using CRISPR/Cas9, and knockdown of expression using morpholinos. These tools should facilitate the direct investigation of genes underlying the variation between surface- and cave-dwelling forms.

墨西哥鱼基因功能的操纵

Cave animals provide a compelling system for investigating the evolutionary mechanisms and genetic bases underlying changes in numerous complex traits, ...

Use of Freeze-thawed Embryos for High-efficiency Production of Genetically Modified Mice

The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. The CRISPR/Cas9 system allows researchers to modify the genome with unprecedented efficiency, fidelity, and simplicity. Harnessing this technology, researchers are seeking a rapid, efficient, and easy protocol for generating GM mice. Here we introduce an improved method for cryopreservation of one-cell embryos that leads to a higher developmental rate of the freeze-thawed embryos. By combining it with optimized electroporation conditions, this protocol allows for the generation of knockout and knock-in mice with high efficiency and low mosaic rates within a short time. Furthermore, we show a step-by-step explanation of our optimized protocol, covering CRISPR reagent preparation, in vitro fertilization, cryopreservation and thawing of one-cell embryos, electroporation of CRISPR reagents, mouse generation, and genotyping of the founders. Using this protocol, researchers should be able to prepare GM mice with unparalleled ease, speed, and efficiency.

Here, we present a modified method for cryopreservation of one-cell embryos as well as a protocol that couples the use of freeze-thawed embryos and electroporation for the efficient generation of genetically modified mice.

使用冷冻胚胎进行转基因小鼠高效生产

The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. ...

Determining the Role of Maternally-Expressed Genes in Early Development with Maternal Crispants

Early development depends on a pool of maternal factors incorporated into the mature oocyte during oogenesis that perform all cellular functions necessary for development until zygotic genome activation. Typically, genetic targeting of these maternal factors requires an additional generation to identify maternal-effect phenotypes, hindering the ability to determine the role of maternally-expressed genes during development. The discovery of the biallelic editing capabilities of CRISPR-Cas9 has allowed screening of embryonic phenotypes in somatic tissues of injected embryos or "crispants," augmenting the understanding of the role zygotically-expressed genes play in developmental programs. This article describes a protocol that is an extension of the crispant method. In this method, the biallelic editing of germ cells allows for the isolation of a maternal-effect phenotype in a single generation, or "maternal crispants." Multiplexing guide RNAs to a single target promotes the efficient production of maternal crispants, while sequence analysis of maternal crispant haploids provides a simple method to corroborate genetic lesions that produce a maternal-effect phenotype. The use of maternal crispants supports the rapid identification of essential maternally-expressed genes, thus facilitating the understanding of early development.

Early development is dependent on maternally-inherited products, and the role of many of these products is currently unknown. Herein, we described a protocol that uses CRISPR-Cas9 to identify maternal-effect phenotypes in a single generation.

确定母系表达基因在母体脆皮早期发育中的作用

Early development depends on a pool of maternal factors incorporated into the mature oocyte during oogenesis that perform all cellular functions necessary ...

CRISPR-Cas9 Genome Editing of Rat Embryos using Adeno-Associated Virus (AAV) and 2-Cell Embryo Electroporation

Genome editing technology is widely used to produce genetically modified animals, including rats. Cytoplasmic or pronuclear injection of DNA repair templates and CRISPR-Cas reagents is the most common delivery method into embryos. However, this type of micromanipulation necessitates access to specialized equipment, is laborious, and requires a certain level of technical skill. Moreover, microinjection techniques often result in lower embryo survival due to the mechanical stress on the embryo. In this protocol, we developed an optimized method to deliver large DNA repair templates to work in conjunction with CRISPR-Cas9 genome editing without the need for microinjection. This protocol combines AAV-mediated DNA delivery of single-stranded DNA donor templates along with the delivery of CRISPR-Cas9 ribonucleoprotein (RNP) by electroporation to modify 2-cell embryos. Using this novel strategy, we have successfully produced targeted knock-in rat models carrying insertion of DNA sequences from 1.2 to 3.0 kb in size with efficiencies between 42% and 90%.

CRISPR-Cas9 Genome Editing of Rat Embryos using Adeno-Associated Virus AAV and 2-Cell Embryo Electroporation

This protocol presents an optimized approach for producing genetically modified rat models. Adeno-associated virus (AAV) is used to deliver a DNA repair template, and electroporation is used to deliver CRISPR-Cas9 reagents to complete the genome editing process in 2-cell embryo.

使用腺相关病毒 （AAV） 和 2 细胞胚胎电穿孔对大鼠胚胎进行 CRISPR-Cas9 基因组编辑

Genome editing technology is widely used to produce genetically modified animals, including rats. Cytoplasmic or pronuclear injection of DNA repair ...

Zygote Microinjection for Creating Gene Cassette Knock-in and Flox Alleles in Mice

CRISPR-Cas technology has enabled the rapid and effortless generation of genetically modified mice. Specifically, mice and point mutant mice are readily produced by electroporation of CRISPR factors (and single-stranded oligo DNA donors) into the zygote. In contrast, gene cassette (&#62;1 kb) knock-in and floxed mice are mainly generated by microinjection of CRISPR factors and double-stranded DNA donors into zygotes. Genome editing technologies have also increased the flexibility of genetically modified mice production. It is now possible to introduce the intended mutations in the target genomic regions in a number of beneficial inbred mouse strains. Our team has produced over 200 gene cassette knock-in mouse lines, and over 110 floxed mouse lines by zygote microinjection of CRISPR-Cas9 following requests from several countries, including Japan. Some of these genome editing used BALB/c, C3H/HeJ, and C57BL/6N inbred strains, however most used C57BL/6J. Unlike the electroporation method, genome editing by zygote microinjection in various inbred strains of mice is not that easy. However, gene cassette knock-in and floxed mice on single inbred genetic backgrounds are as critical as genetic humanized, fluorescent reporter, and conditional knockout mouse models. Therefore, this article presents the protocol for the zygote microinjection of CRISPR factors and double-stranded DNA donors in C57BL/6J mice for generating gene cassette knock-in and floxed mice. This article exclusively focuses on nuclear injection rather than cytoplasmic injection. In addition to zygote microinjection, we outline the timeline for the production process and peripheral techniques such as induction of superovulation and embryo transfer.

The present protocol describes zygote microinjection of CRISPR-Cas9 and donor DNA to efficiently produce gene cassette knock-in and floxed mice.

受精卵显微注射用于在小鼠中产生基因盒敲入和絮状等位基因

CRISPR-Cas technology has enabled the rapid and effortless generation of genetically modified mice. Specifically, mice and point mutant mice are readily ...

Efficient Genome Editing of Mice by CRISPR Electroporation of Zygotes

With exceptional efficiency, accuracy, and ease, the CRISPR/Cas9 system has significantly improved genome editing in cell culture and lab animal experiments. When generating animal models, the electroporation of zygotes offers higher efficiency, simplicity, cost, and throughput as an alternative to the gold standard method of microinjection. Electroporation is also gentler, with higher viability, and reliably delivers Cas9/single-guide RNA (sgRNA) ribonucleoproteins (RNPs) into the zygotes of common laboratory mouse strains (e.g., C57BL/6J and C57BL/6N) that approaches 100% delivery efficiency. This technique enables insertion/deletion (indels) mutations, point mutations, the deletion of whole genes or exons, and small insertions in the range of 100-200 bp to insert LoxP or short tags like FLAG, HA, or V5. While constantly being improved, here we present the current state of CRISPR-EZ in a protocol that includes sgRNA production through in vitro transcription, embryo processing, RNP assembly, electroporation, and the genotyping of preimplantation embryos. A graduate-level researcher with minimal experience manipulating embryos can obtain genetically edited embryos in less than 1 week using this protocol. Here, we offer a straightforward, low-cost, efficient, high-capacity method that could be used with mouse embryos.

Here, we describe a simple technique intended for the efficient generation of genetically modified mice called CRISPR RNP Electroporation of Zygotes (CRISPR-EZ). This method delivers editing reagents by electroporation into embryos at an efficiency approaching 100%. This protocol is effective for point mutations, small genomic insertions, and deletions in mammalian embryos.

通过受精卵的CRISPR电穿孔对小鼠进行高效基因组编辑

With exceptional efficiency, accuracy, and ease, the CRISPR/Cas9 system has significantly improved genome editing in cell culture and lab animal ...

Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these independently derived phenotypes, which share a common genetic architecture. This is perhaps best exemplified by the numerous convergent features of gymnotiforms and mormyrids, two species-rich teleost clades that produce and detect weak electric fields and are called weakly electric fish. In the 50 years since the discovery that weakly electric fish use electricity to sense their surroundings and communicate, a growing community of scientists has gained tremendous insights into evolution of development, systems and circuits neuroscience, cellular physiology, ecology, evolutionary biology, and behavior. More recently, there has been a proliferation of genomic resources for electric fish. Use of these resources has already facilitated important insights with regards to the connection between genotype and phenotype in these species. A major obstacle to integrating genomics data with phenotypic data of weakly electric fish is a present lack of functional genomics tools. We report here a full protocol for performing CRISPR/Cas9 mutagenesis that utilizes endogenous DNA repair mechanisms in weakly electric fish. We demonstrate that this protocol is equally effective in both the mormyrid species Brienomyrus brachyistius and the gymnotiform Brachyhypopomus gauderio by using CRISPR/Cas9 to target indels and point mutations in the first exon of the sodium channel gene scn4aa. Using this protocol, embryos from both species were obtained and genotyped to confirm that the predicted mutations in the first exon of the sodium channel scn4aa were present. The knock-out success phenotype was confirmed with recordings showing reduced electric organ discharge amplitudes when compared to uninjected size-matched controls.

Here, a protocol is presented to produce and rear CRISPR/Cas9 genome knockout electric fish. Outlined in detail are the required molecular biology, breeding, and husbandry requirements for both a gymnotiform and a mormyrid, and injection techniques to produce Cas9-induced indel F0 larvae.

沉默的火花:CRISPR/Cas9基因组编辑在弱电鱼

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these ...

生物学

CRISPR-Cas9-Mediated Precise Knock-In Edits in Zebrafish Hearts

Clustered regularly interspaced short palindromic repeats (CRISPR) in animal models enable precise genetic manipulation for the study of physiological phenomena. Zebrafish have been used as an effective genetic model to study numerous questions related to heritable disease, development, and toxicology at the whole-organ and -organism level. Due to the well-annotated and mapped zebrafish genome, numerous tools for gene editing have been developed. However, the efficacy of generating and ease of detecting precise knock-in edits using CRISPR is a limiting factor. Described here is a CRISPR-Cas9-based knock-in approach with the simple detection of precise edits in a gene responsible for cardiac repolarization and associated with the electrical disorder, Long QT Syndrome (LQTS). This two-single-guide RNA (sgRNA) approach excises and replaces the target sequence and links a genetically encoded reporter gene. The utility of this approach is demonstrated by describing non-invasive phenotypic measurements of cardiac electrical function in wild-type and gene-edited zebrafish larvae. This approach enables the efficient study of disease-associated variants in a whole organism. Furthermore, this strategy offers possibilities for the insertion of exogenous sequences of choice, such as reporter genes, orthologs, or gene editors.

This protocol describes an approach to facilitate precise knock-in edits in zebrafish embryos using CRISPR-Cas9 technology. A phenotyping pipeline is presented to demonstrate the applicability of these techniques to model a Long QT Syndrome-associated gene variant.

CRISPR-Cas9介导的斑马鱼心脏中的精确敲入编辑

Clustered regularly interspaced short palindromic repeats (CRISPR) in animal models enable precise genetic manipulation for the study of physiological ...

CRISPR/Cas9 蛋白注射到沟鲶、鮰松毛虫、基因编辑的胚胎

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

显微注射转基因的基因组，并在编辑Threespine刺鱼

墨西哥鱼基因功能的操纵

使用冷冻胚胎进行转基因小鼠高效生产

确定母系表达基因在母体脆皮早期发育中的作用

使用腺相关病毒（AAV）和 2 细胞胚胎电穿孔对大鼠胚胎进行 CRISPR-Cas9 基因组编辑

受精卵显微注射用于在小鼠中产生基因盒敲入和絮状等位基因

通过受精卵的CRISPR电穿孔对小鼠进行高效基因组编辑

沉默的火花:CRISPR/Cas9基因组编辑在弱电鱼

CRISPR-Cas9介导的斑马鱼心脏中的精确敲入编辑

CRISPR/Cas9 蛋白注射到沟鲶、鮰松毛虫、基因编辑的胚胎

摘要

探索更多视频

此视频中的章节

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

显微注射转基因的基因组，并在编辑Threespine刺鱼

墨西哥鱼基因功能的操纵

使用冷冻胚胎进行转基因小鼠高效生产

确定母系表达基因在母体脆皮早期发育中的作用

使用腺相关病毒 （AAV） 和 2 细胞胚胎电穿孔对大鼠胚胎进行 CRISPR-Cas9 基因组编辑

受精卵显微注射用于在小鼠中产生基因盒敲入和絮状等位基因

通过受精卵的CRISPR电穿孔对小鼠进行高效基因组编辑

沉默的火花:CRISPR/Cas9基因组编辑在弱电鱼

CRISPR-Cas9介导的斑马鱼心脏中的精确敲入编辑

使用腺相关病毒（AAV）和 2 细胞胚胎电穿孔对大鼠胚胎进行 CRISPR-Cas9 基因组编辑