作者感谢瓦妮莎·梅尔德纳-哈雷尔对手稿的批判性解读。

严格按照《国家卫生研究院实验室动物护理和使用指南》的建议，使用小鼠作为沙蝇喂养的血液来源。该议定书得到了国家艾滋病规划署动物护理和使用委员会（协议编号LPD 68E）的批准。无脊椎动物不在国家卫生研究院指南的涵盖范围之内。
1. 针头制备 （图1）
<ol><li>拉针和斜面，如在梅蒂和哈雷尔19描述。简言之，使用薄氧酸玻璃毛细管在双级玻璃微皮拉器上拉针。</li>
	<li>用一块额外的细石，在微尖斜面上用湿斜面切针。</li>
</ol>2. 胚胎收集和微操纵 （图2）
<ol><li>注射前五天，血液喂养沙蝇雌性，定期进行菌落维护。有关沙蝇群落饲养程序和所需材料的详细信息，请参阅律师等人1 在整个开发过程中，在 70% 湿度和 26 °C 的孵化器中保持昆虫。<ol>
<li>有一天，在喂血后，用石膏锅和侧端口的100-150组女性捕捉喂血的女性。通过用糖溶液（30%蔗糖-足够的体积浸泡一个小棉球）喂养雌性，这是通常的殖民地维护。</li>
	</ol>
</li>
	<li>在第2-3天献血后，不要滋润石膏锅。将雌性放在干燥的基材上可以防止过早产卵，因为雌性更喜欢在潮湿的环境中产卵。</li>
	<li>第5天进行血液喂养后，进行胚胎采集、微操纵和微注射。将湿润滤纸引入产卵室。新鲜铺设的胚胎没有完全发育的胆汁，呈白色。在手术过程中使用黑色滤纸，以促进白色胚胎的可视化。</li>
	<li>使用口腔吸气器将一小群女性从石膏锅转移到产卵室，通过侧端口。湿润基板（滤纸）的存在诱使女性渗出。</li>
	<li>30-60分钟后，小心地从腔室中取出新铺设的胚胎的滤纸。将滤纸和胚胎放在培养皿中长达3小时。在此期间，定期评估滤纸的水分水平，以确保其保持湿润。在此期间，重复步骤2.4至2.6与新的女性群体，并收集尽可能多的胚胎。</li>
	<li>准备显微镜幻灯片进行注射:用非常精细的画笔手动收集鸡蛋，并小心地将它们转移到另一张湿润的黑色滤纸上，放在显微镜滑梯上，上面有玻璃盖片。<ol>
<li>将水加入滤纸中，足以保持胚胎湿润，但不会使胚胎从盖片上浮出或被吸到盖片下。将胚胎与盖片对接。盖片起到后挡作用，防止卵子在注射过程中滚走。</li>
	</ol>
</li>
</ol>3. 胚胎注射 （图3）
<ol><li>鸡蛋收集开始后 2.5 至 3 小时开始注射。在室温和环境实验室湿度下进行注射。</li>
	<li>确保对齐的胚胎有足够的水，使其保持湿润。 注:在注射过程中，胚胎必须保持湿润。如果注射过程中没有湿润，注射变得困难，因为针头难以刺穿胚胎。正确的水量是胚胎周围有半月板水的点，但盖片不会漂浮在水面上，导致胚胎在盖片下推。在注射过程中监测胚胎周围的水量至关重要。根据需要加水以保持胚胎湿润。</li>
	<li>用水润湿刷子并将水转移到滤纸的后端，小心地加水。以这种方式添加水可以以缓慢和可控的方式添加水，以便添加足够的水来保持胚胎湿润。过多的水会导致胚胎浮出对齐，使胚胎难以注入。</li>
	<li>背负荷注射混合到微注射针。使用手绘的玻色酸针填充剂在针头中加入约 0.5 至 1μL 的注射混合物，还可用于凝胶加载管道尖端。注射组合由一个或几个导数RNA（每个80 ng/μL）组成，旨在针对与商业重组的Cas9蛋白（300 ng/μL）混合的特定轨迹。</li>
	<li>从 30 psi 的注射压力开始，按下注射触发器，将空气从针尖排出。这将迫使注射混合到针尖，使注射混合流动。此时，慢慢增加保持/恒定压力，直到注射材料开始流动，然后后退保持/恒定压力，使其略低于从针头流出的注射混合点。门控设置应提供足够的注射材料，以便可以看到少量的材料进入胚胎。 注:在卤烃油下注射沙蝇胚胎，类似于用于 果蝇 和蚊子的其他协议，初步测试，但这些注射的存活率非常低。通过在潮湿的滤纸上切换到注射，如上所述，生存得到了改善。</li>
	<li>将针头插入胚胎侧面。使用胚胎后面的盖片作为后盾，帮助针刺穿胚胎。在轻轻取出针头之前，将少量注射混合物放入胚胎中。</li>
	<li>取出针头后，立即按喷油器从针尖上取出任何回填材料。这将有助于防止针头堵塞。</li>
	<li>进入下一个胚胎。每次注射之间，确保滤纸湿润，针头不堵塞。</li>
	<li>一旦所有胚胎都被注射，计算注射的胚胎数量，以保持运行计数。</li>
	<li>通过在滤纸中加入水来去除盖片，使盖片浮动。此时，使用探针（用粘在尖端的昆虫别针粘住的木材施用器）将滤纸固定到位，同时用手指将盖片从注射的胚胎上拉开。</li>
	<li>一旦盖片被移除，就会从滤纸中清除多余的水。</li>
</ol>4. 注射后饲养 （G0） （图 4）
<ol><li>将滤纸与胚胎一起转移到放置在培养皿的几层湿润滤纸上，以保持湿度。在注射其他胚胎所需的时间将胚胎保留在这里。胚胎最多可保持3小时。注射后，它们会慢慢变成棕色。</li>
	<li>一旦所有胚胎都被注射，手动将它们转移到以前滋润的小尺寸石膏盆上。不要把太多的胚胎放在每个锅里，保持它们之间的距离，以避免个体之间可能的真菌污染。用屏幕覆盖锅，并存储它们，通常会做沙蝇殖民地鸡蛋，保持在孵化器在70%的湿度和26°C，在其整个发展。</li>
	<li>在注射后的第1天，用画笔取出所有受损和死亡的胚胎。在含有注射胚胎的石膏锅中加入 100 μL 的 0.5% 丙酸，一滴一滴，以限制真菌污染。细胞质从注射过程中受损的胚胎中释放，尤其有利于真菌生长。</li>
	<li>每天检查石膏盆，并去除任何坏或死胚胎。如果存在真菌，取出所有受感染的胚胎，并加入几滴0.5%的丙酸。</li>
	<li>第8天和第12天注射后，幼虫孵化。每天两次，将新出现的幼虫转移到新的石膏盆中，最多组5-10个。加入少量食物，每周检查食物2-3次。<ol>
<li>仔细监测幼虫食物的数量，因为过多会大大增加真菌污染的风险。沙蝇幼虫在群体中存活得更好，但是如果没有足够的食物，它们可能是食人族。</li>
	</ol>
</li>
	<li>当幼虫幼崽时，按性别分离它们，以保持雌性为处女。如果有很多幸存者，抛弃雄性，只保留女性。 注:男性和女性都可以携带和传递突变。然而，由于雌性在被穿越之前必须保持处女，因此更容易只使用G0 注射的女性，以避免不得不按性别分离昆虫。G0 男性可以保留作为备份， 如果成年幸存者的数量是低的， 在这种情况下， 将与排序的 wt 处女女性配合。</li>
</ol>5. 突变等位基因的选择和筛选 （图5）
<ol><li>大规模穿越G0 女性与wt男性在笼子里。血液喂养他们完全，将做正常的殖民地维护。</li>
	<li>一天后，用口腔吸气器将每名G0 女性与2-3 wt雄性转移到单独的石膏管中。每天在一个小棉球上用糖喂苍蝇，并使用水注射器，以确保石膏水分适合产卵。</li>
	<li>在G0 雌性产卵（对应于G1）后，将她们的身体收集在单独的、有注释的埃彭多夫管中，供以后的基因化。保持胚胎，通常做wt殖民地。</li>
	<li>通过选择的方法从G0 女性中提取DNA。</li>
	<li>筛选DNA是否存在突变，例如，使用围绕预期CRISPR切割区域的引物设计PCR检测。</li>
	<li>只保留 G1 女性铺设的 G1 胚胎， 以显示转化的证据。当这些G1 胚胎发育成幼崽时，按性别分离它们，只保留雌性。将 G1 女性从同一个 G0 女叉到 WT 男性，用血喂养它们，然后将 2 - 3 名女性和 4 - 5 名男性分成小尺寸容器。 注意:所有G1 个体都有机会携带一个独特的突变，所以如果幸存个体的数量不是太高，那么最好是单独交叉G1 女性与wt男性。几个女性携带不同的突变存在于同一管子的情况下是罕见的，但可能发生。如果在基因化后观察到这种情况，那么G2 后代雌性应该单独与wt雄性配对。这种交配方案避免了后代发生多重突变的可能性。</li>
	<li>产卵后，收集G1 女性身体，并筛选它们的突变。只保留至少有一名女性有突变证据的管子。</li>
	<li>为后代做单对交叉。产卵后，筛选父母存在突变。在每一代人中重复，直到父母双方都是同源的相同突变。一旦确定，它们将成为同源突变种群的创始人。</li>
</ol>

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我们在这里介绍了最近开发的胚胎微注射方法，用于在木薯沙蝇的CRISPR/Cas9靶向诱变。昆虫基因改造的胚胎微注射是在20世纪80年代中期在德罗索菲拉开发的，现在经常用于各种昆虫。其他基因改造材料的交付方法已经开发用于昆虫，如REMOT 20，21，22，23和电波<sup class="xr...

病媒传播疾病是持续演变过程中的主要公共卫生威胁。据世界卫生组织统计，数以百计的病媒物种分布在非常独特的植物家族（如蚊子、虱子、跳蚤）中，导致大量微生物病原体的传播，每年导致70多万人死亡。在向量昆虫中，植物素沙蝇（滴虫、心理虫）构成一个庞大的群体，80种经过验证的向量物种表现出不同的表型特征和向量能力，在不同地理区域发现。它们是 列什马尼亚属原生寄生虫的载体，每年造成约130万例新的莱什马尼亚病例和2万至3万例死亡。Leishmanias 的临床结果多种多样，症状从自我限制的皮性病变到内脏传播，在缺乏治疗的情况下是致命的。
沙蝇是严格的陆地昆虫。与其他 Diptera 相比，它们的生命周期相对较长，根据温度、湿度和营养等不同参数，寿命长达三个月。它包括一个胚胎阶段（6至11天），四个幼虫阶段（总共持续23至25天）和一个幼虫阶段（9至10天），然后是变质，然后是成年。沙蝇需要潮湿和温暖的环境来饲养。雄性和雌性都以从花蜜在野外获得的糖为食。只有女性是血液喂养者，因为他们需要从血液中获取蛋白质来生产卵子1。
研究的一个重要重点是确定导致可传播感染发展的病媒/寄生虫相互作用的性质。与其他向量昆虫一样，沙蝇固有的参数已被证明会影响它们的病媒能力，即它们携带病原体和将病原体传染给宿主的能力。例如，木瓜沙飞中古特细胞作为识别寄生虫表面成分的受体表达，可以直接影响其对莱什马尼亚主要2、3的载体能力。昆虫免疫反应途径，免疫缺陷（IMD），也是至关重要的木瓜沙蝇向量能力莱什马尼亚主要4。在伊蚊5、6、7、采采蝇格洛西纳摩尔西坦8和阿诺菲莱斯甘比亚蚊子9、10中，对病媒昆虫免疫反应途径在控制传染性病原体传播方面具有重要作用。
沙蝇/莱什马尼亚相互作用的研究因缺乏适应这些昆虫使用的基因表达修饰方法而受限。直到最近，只有通过小干扰RNA（siRNA）进行的基因下调节进行了11，12，13，14。该技术受成年女性微注射相关死亡率的限制，只导致部分功能丧失，无法代代相传。
CRISPR/Cas9技术彻底改变了沙蝇等非模型生物体的功能基因组研究。CRISPR Cas9系统从原核生物的适应性免疫系统中进行了改造，用于防御噬菌体15、16，并迅速被改造为用于包括昆虫在内的高级真核生物的基因组编辑工具。CRISPR/Cas9 目标基因组编辑的原则基于单一指南 RNA （sgRNA） 与特定基因组的互补性。Cas9核酶与sgRNA结合，在基因组DNA中产生双链DNA（dsDNA）断裂，sgRNA与其互补序列关联。Cas9-sgRNA 复合物由 sgRNA 中的 17 到 20 个互补基座引导到目标序列，然后可以通过两个独立的路径修复 dsDNA 中断:非声端连接 （NHEJ） 或同源定向修复 （HDR）17。NHEJ 修复涉及简单的中断关闭，但经常导致小的插入/删除事件。通过 HDR 修复的 DNA 使用与目标 DNA 共享同源的供体 DNA 分子作为修复模板。昆虫拥有两种机器。
CRISPR/Cas9 技术可以通过 NHEJ 修复途径在选定的轨迹中产生突变;或更复杂的基因组编辑策略，如敲击或表达记者，通过HDR通路与适当的捐赠者模板。在沙蝇中，免疫反应因子的 无 突变等位基因通过NHEJ介导的CRISPR在 木薯4中产生。在另一项研究中还注入了沙蝇胚胎，并结合了针对编码黄色的基因的CRISPR/Cas9组合。尽管如此，没有携带突变的成年人产生18。我们在这里描述了由NHEJ调解的CRISPR/Cas9的沙蝇靶向诱变的详细方法，特别侧重于胚胎微注射，这是协议的关键步骤。

<table>
	<tbody>
		<tr>
			<td>Black Filter Paper 4.25CM PK100</td>
			<td>VWR</td>
			<td>28342-012</td>
			<td>Cut into rectangles that are approximately 46 X 22mm. These are placed between the slide and the coverslip and act as a moist base layer for the embryos during injection.</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Fisher Scientific</td>
			<td>12-543A</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Microscope</td>
			<td>Any brand</td>
			<td></td>
			<td>For aligning embryos</td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-A3</td>
			<td>Base layer of the microinjection set up Figure 2A</td>
		</tr>
		<tr>
			<td>Insect cage</td>
			<td>custom made or several commercial options</td>
			<td></td>
			<td>polycarbonate cage for adults holding and mating Lawyer, Phillip, Mireille Killick-Kendrick, Tobin Rowland, Edgar Rowton, and Petr Volf. &#8220;Laboratory Colonization and Mass Rearing of Phlebotomine Sand Flies (Diptera, Psychodidae).&#8221; Parasite 24. Accessed August 6, 2020. https://doi.org/10.1051/parasite/2017041.</td>
		</tr>
		<tr>
			<td>Larval food</td>
			<td>custom made</td>
			<td></td>
			<td>a mix of rabbit chow and rabbit feces Lawyer, Phillip, Mireille Killick-Kendrick, Tobin Rowland, Edgar Rowton, and Petr Volf. &#8220;Laboratory Colonization and Mass Rearing of Phlebotomine Sand Flies (Diptera, Psychodidae).&#8221; Parasite 24. https://doi.org/10.1051/parasite/2017041.</td>
		</tr>
		<tr>
			<td>Microcaps 100 ml</td>
			<td>Drummond</td>
			<td>1-000-1000</td>
			<td>Used to back fill microinjection needles</td>
		</tr>
		<tr>
			<td>Mouth aspirator</td>
			<td>John W. Hock Company</td>
			<td>Model 612</td>
			<td>mouth aspirator with HEPA filter</td>
		</tr>
		<tr>
			<td>Olympus SZX12</td>
			<td>Olympus Life Sciences</td>
			<td></td>
			<td>Microinjection microscope</td>
		</tr>
		<tr>
			<td>Ovipots</td>
			<td>Nalge company</td>
			<td></td>
			<td>ovipots are made from 125-ml or 500-ml straigh-sided plolypropylene jars modified by drilling 2.5cm holes in the bottom and filled with 1cm of plaster of Paris. Lawyer, Phillip, Mireille Killick-Kendrick, Tobin Rowland, Edgar Rowton, and Petr Volf. &#8220;Laboratory Colonization and Mass Rearing of Phlebotomine Sand Flies (Diptera, Psychodidae).&#8221; Parasite 24. Accessed August 6, 2020. https://doi.org/10.1051/parasite/2017041.</td>
		</tr>
		<tr>
			<td>Paint Brush 6-0</td>
			<td>Any Art Supply Company</td>
			<td>n/a</td>
			<td>Used for aligning embryos</td>
		</tr>
		<tr>
			<td>Propionic acid</td>
			<td>Sigma-Aldrich</td>
			<td>402907</td>
			<td>antifungal agent</td>
		</tr>
		<tr>
			<td>Standard Glass Capillaries</td>
			<td>World Precision Instruments</td>
			<td>1B100-3</td>
			<td>Used for making microinjection needles</td>
		</tr>
		<tr>
			<td>Trio-MPC100 Controller and MP845 Manipulator</td>
			<td>Sutter Instruments</td>
			<td></td>
			<td>Microinjection Controller and Micromanipulator</td>
		</tr>
	</tbody>
</table>

sand fly (phlebotomus papatasi) embryo microinjection for crispr/cas9 mutagenesis

沙蝇是 莱什马尼亚 物种的自然载体，原生动物寄生虫产生从皮性病变到内脏病理学等多种症状。破译向量/寄生虫相互作用的性质对于更好地了解 莱什马尼亚 传染给宿主至关重要。在控制沙蝇向量能力的参数（即它们携带和传播病原体的能力）中，这些昆虫固有的参数被证明发挥着关键作用。例如，昆虫免疫反应会影响沙蝇向量对 莱什马尼亚的能力。由于缺乏适合用于这些非模型生物体的基因表达修饰方法，对此类参数的研究受到限制。通过小干扰RNA（siRNA）来降低基因调控是可能的，但除了在技术上具有挑战性之外，沉默只导致部分功能丧失，无法代代相传。CRISPR/Cas9技术的靶向突变最近被改编成 木薯沙 蝇。这项技术导致在一个专门选择的轨迹中产生可传播的突变，从而能够研究感兴趣的基因。CRISPR/Cas9 系统依赖于定向双链脱氧核糖核酸断裂的诱导，后来通过非同源端连接 （NHEJ） 或同源驱动修复 （HDR） 进行修复。NHEJ 包括一个简单的休息关闭，并经常导致小插入/删除事件。相比之下，HDR 使用与目标 DNA 共享同源的供体 DNA 分子的存在作为修复模板。在这里，我们提出了一种沙蝇胚胎微注射方法，用于CRISPR/Cas9使用NHEJ靶向突变，这是迄今为止唯一适应沙蝇载体的基因组修饰技术。

CRISPR/Cas9微注射协议在这里描述产生沙蝇突变体是在以前的出版物4中建立的。这种方法产生了高效的诱变，因为540个人中有11人幸存下来，其中9人是突变者。在为CRISPR/Cas9突变设计指南时，关键的第一步是对周围区域进行排序，以便成为目标。测序模板应该来自将用作注射胚胎来源的菌株。仅仅依靠已公布的基因组序列来设计指南是有风险的。在已发布的基因组和指南前设计序?...

Watch this Scientific Journal Video about Sand Fly Phlebotomus papatasi Embryo Microinjection for CRISPR/Cas9 Mutagenesis at JoVE.com

Sand Fly Phlebotomus papatasi Embryo Microinjection for CRISPR/Cas9 Mutagenesis

该协议详细介绍了CRISPR/Cas9在沙蝇中靶向诱变的步骤:胚胎采集、注射、昆虫饲养和识别，以及兴趣突变的选择。

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 ...

sand-fly-phlebotomus-papatasi-embryo-microinjection-for-crisprcas9

Research

JoVE Journal

Genetics

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

7.0K 次观看.  National Institutes of Health. 该协议详细介绍了CRISPR/Cas9在沙蝇中靶向诱变的步骤:胚胎采集、注射、昆虫饲养和识别，以及兴趣突变的选择。

视频: Sand Fly Phlebotomus papatasi Embryo Microinjection for CRISPR/Cas9 Mutagenesis

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the African clawed frog, Xenopus, a longstanding model organism for biomedical research. Traditional breeding schemes for creating homozygous mutant lines with CRISPR/Cas9-targeted mutagenesis have several time-consuming and laborious steps. To facilitate the creation of mutant embryos, particularly to overcome the obstacles associated with knocking out genes that are essential for embryogenesis, a new method called leapfrogging was developed. This technique leverages the robustness of Xenopus embryos to &#34;cut and paste&#34; embryological methods. Leapfrogging utilizes the transfer of primordial germ cells (PGCs) from efficiently-mutagenized donor embryos into PGC-ablated wildtype siblings. This method allows for the efficient mutation of essential genes by creating chimeric animals with wildtype somatic cells that carry a mutant germline. When two F0 animals carrying &#34;leapfrog transplants&#34; (i.e., mutant germ cells) are intercrossed, they produce homozygous, or compound heterozygous, null F1 embryos, thus saving a full generation time to obtain phenotypic data. Leapfrogging also provides a new approach for analyzing maternal effect genes, which are refractory to F0 phenotypic analysis following CRISPR/Cas9 mutagenesis. This manuscript details the method of leapfrogging, with special emphasis on how to successfully perform PGC transplantation.

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

Genes essential for survival pose technical hurdles for creating mutant lines. Leapfrogging circumvents lethality by combining genome editing with primordial germ cell transplantation to create wild-type animals carrying germline mutations. Leapfrogging also permits the efficient generation of homozygous null mutants in the F1 generation. Here, the transplantation step is demonstrated.

CRISPR/Cas9-based 跳跃的原始生殖细胞移植在爪

The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the ...

科研

遗传学

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

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene function. Gene knockout has been used to study these gene functions in vivo. The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system is a powerful tool used to edit genomic DNA sequences to alter gene function. While the traditional approach has been to introduce CRISPR/Cas9 mRNA into the single cell embryos through microinjection, this can be a slow and inefficient process in catfish. Here, a detailed protocol for microinjection of channel catfish embryos with CRISPR/Cas9 protein is described. Briefly, eggs and sperm were collected and then artificial fertilization performed. Fertilized eggs were transferred to a Petri dish containing Holtfreter's solution. Injection volume was calibrated and then guide RNAs/Cas9 targeting the toll/interleukin 1 receptor domain-containing adapter molecule (TICAM 1) gene and rhamnose binding lectin (RBL) gene were microinjected into the yolk of one-cell embryos. The gene knockout was successful as indels were confirmed by DNA sequencing. The predicted protein sequence alterations due to these mutations included frameshift and truncated protein due to premature stop codons.

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

A simple and efficient microinjection protocol for gene editing in channel catfish embryos using the CRISPR/Cas9 system is presented. In this protocol, guide RNAs and Cas9 protein were microinjected into the yolk of one-cell embryos. This protocol has been validated by knocking out two channel catfish immune-related genes.

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 ...

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

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

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

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

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

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

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex determination, venom synthesis, and host-symbiont interactions, among others. A major limitation of working with this organism is the lack of effective protocols to perform directed genome modifications. An important part of genome modification is delivery of editing reagents, including CRISPR/Cas9 molecules, into embryos through microinjection. While microinjection is well established in many model organisms, this technique is particularly challenging to perform in N. vitripennis primarily due to its small embryo size, and the fact that embryonic development occurs entirely within a parasitized blowfly pupa. The following procedure overcomes these significant challenges while demonstrating a streamlined, visual procedure for effectively removing wasp embryos from parasitized host pupae, microinjecting them, and carefully transplanting them back into the host for continuation and completion of development. This protocol will strongly enhance the capability of research groups to perform advanced genome modifications in this organism.

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

Microinjection of Nasonia vitripennis embryos is an essential method for generating heritable genome modifications. Described here is a detailed procedure for microinjection and transplantation of Nasonia vitripennis embryos, which will greatly facilitate future genome manipulation in this organism.

金小蜂金小蜂基因组操作的胚胎显微注射和移植技术

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex ...

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 ...

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise heterozygous genetic modifications is challenging due to CRISPR-CAS9 mediated indel formation in the second allele. Here, we demonstrate a protocol to help overcome this difficulty by using two repair templates in which only one expresses the desired sequence change, while both templates contain silent mutations to prevent re-cutting and indel formation. This methodology is most advantageous for gene editing coding regions of DNA to generate isogenic control and mutant human stem cell lines for studying human disease and biology. In addition, optimization of transfection and screening methodologies have been performed to reduce labor and cost of a gene editing experiment. Overall, this protocol is widely applicable to many genome editing projects utilizing the human pluripotent stem cell model.

This protocol provides a method to facilitate the generation of defined heterozygous or homozygous nucleotide changes using CRISPR-CAS9 in human pluripotent stem cells.

在人类多能干细胞中使用CRISPR-CAS9生成定义的基因组修饰

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise ...

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 ...

Embryo Microinjection Techniques for Efficient Site-Specific Mutagenesis in Culex quinquefasciatus

Culex quinquefasciatus is a vector of a diverse range of vector-borne diseases such as avian malaria, West Nile virus (WNV), Japanese encephalitis, Eastern equine encephalitis, lymphatic filariasis, and Saint Louis encephalitis. Notably, avian malaria has played a major role in the extinction of numerous endemic island bird species, while WNV has become an important vector-borne disease in the United States. To gain further insight into C. quinquefasciatus biology and expand their genetic control toolbox, we need to develop more efficient and affordable methods for genome engineering in this species. However, some biological traits unique to Culex mosquitoes, particularly their egg rafts, have made it difficult to perform microinjection procedures required for genome engineering. To address these challenges, we have developed an optimized embryo microinjection protocol that focuses on mitigating the technical obstacles associated with the unique characteristics of Culex mosquitoes. These procedures demonstrate optimized methods for egg collection, egg raft separation and other handling procedures essential for successful microinjection in C. quinquefasciatus. When coupled with the CRISPR/Cas9 genome editing technology, these procedures allow us to achieve site-specific, efficient and heritable germline mutations, which are required to perform advanced genome engineering and develop genetic control technologies in this important, but currently understudied, disease vector.

Embryo Microinjection Techniques for Efficient Site-Specific Mutagenesis in Culex quinquefasciatus

This protocol describes microinjection procedures for Culex quinquefasciatus embryos that are optimized to work with CRISPR/Cas9 gene editing tools. This technique can efficiently generate site-specific, heritable, germline mutations that can be used for building genetic technologies in this understudied disease vector.

胚胎微注射技术，用于在库莱克斯五角星高效位点特异性突变体

Culex quinquefasciatus is a vector of a diverse range of vector-borne diseases such as avian malaria, West Nile virus (WNV), Japanese encephalitis, ...

Embryo Injections for CRISPR-Mediated Mutagenesis in the Ant Harpegnathos saltator

The unique traits of eusocial insects, such as social behavior and reproductive division of labor, are controlled by their genetic system. To address how genes regulate social traits, we have developed mutant ants via delivery of CRISPR complex into young embryos during their syncytial stage. Here, we provide a protocol of CRISPR-mediated mutagenesis in Harpegnathos saltator, a ponerine ant species that displays striking phenotypic plasticity. H. saltator ants are readily reared in a laboratory setting. Embryos are collected for microinjection with Cas9 proteins and in vitro synthesized small guide RNAs (sgRNAs) using home-made quartz needles. Post-injection embryos are reared outside the colony. Following emergence of the first larva, all embryos and larvae are transported to a nest box with a few nursing workers for further development. This protocol is suitable for inducing mutagenesis for analysis of caste-specific physiology and social behavior in ants, but may also be applied to a broader spectrum of hymenopterans and other insects.

Embryo Injections for CRISPR-Mediated Mutagenesis in the Ant Harpegnathos saltator

Many characteristics of insect eusociality rely on within-colony communication and division of labor. Genetic manipulation of key regulatory genes in ant embryos via microinjection and CRISPR-mediated mutagenesis provides insights into the nature of altruistic behavior in eusocial insects.

胚胎注射用于CRISPR介导的诱变在蚂蚁Harpegnathos盐化剂中

The unique traits of eusocial insects, such as social behavior and reproductive division of labor, are controlled by their genetic system. To address how ...