Name: embryo microinjection and knockout mutant identification of crispr/cas9 genome-edited helicoverpa armigera (h&#252;bner)
Uploaded: 2021-07-01T13:00:00
Description: Watch this Scientific Journal Video about Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera HÃƒÂ¼bner at JoVE.com

这项工作得到了国家自然科学基金（31725023，31861133019 GW，31171912 CY）的支持。

1. 基因特异性引物的设计及sgRNA的制备
<ol><li>通过PCR扩增和测序分析验证目标基因中的保守基因组区域。从 臂状 芽胞杆菌的基因组DNA中扩增靶基因，并区分外显子和内含子。 注意：引导位点的序列特异性对于避免脱靶基因编辑是必要的。在外显子中搜索可能的引导位点接近基因的5'UTR。然后，重要的是要确保基因完全无功能。用于制备sgRNA的流路摘要如图 1所示。...

<ol>
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CRISPR/Cas9系统的应用为分析基因功能和基因间相互作用提供了有力的技术支持。我们在这里介绍的详细方案证明了通过CRISPR / Cas9基因组编辑在 H. armigera 中产生纯合子突变体。这种可靠的程序为 臂状芽孢杆菌的定向基因诱变提供了一种直接的方法。
CRISPR靶点的选择可能会影响诱变效率37.在该协议中，我们比较并分析了来自在线网站CRISOR的多个结...

基因组编辑技术的应用为实现不同物种的靶基因突变体提供了有效的工具。簇状规则间隔短回文重复序列（CRISPR）/相关蛋白9（Cas9）系统的出现提供了一种操纵基因组的新方法1。CRISPR/Cas9系统由引导RNA（gRNA）和Cas9内切酶2，3组成，而gRNA可以进一步分为两部分，靶向互补CRISPR RNA（crRNA）和反式激活crRNA（tracrRNA）。gRNA与Cas9内切酶整合并形成核糖核蛋白（RNP）。使用gRNA，Cas9内切酶可以通过碱基互补被引导到基因组的特定位点。Cas9的RuvC和HNH结构域在原始间隔体相邻基序（PAM）序列之前切割基因组的三个碱基的靶位点，并产生双链断裂（DSB）。然后，DNA切割可以通过两种机制进行修复，即非同源末端连接（NHEJ）或同源定向修复（HDR）4。DSB的修复引入了插入或缺失作为灭活靶向基因的方法，可能导致基因功能完全丧失。因此，CRISPR/Cas9系统的可异性和特异性使其成为表征体内基因功能和分析基因相互作用的可靠方法5。
CRISPR/Cas9系统具有诸多优点，已应用于生物医学6、7、基因治疗8、9、农业等各个领域10、11、12，并已用于各种生物系统，包括微生物13、植物14、15、线虫16 和哺乳动物17.在无脊椎动物中，许多昆虫物种都经过CRISPR/Cas9基因组编辑，如果蝇 果蝇黑腹果 蝇等18，19，20，21，22。
Helicoverpa armigera是全球最具破坏性的害虫之一23，并损害了许多作物，包括棉花，大豆和高粱24，25。随着测序技术的发展，阿米格拉氏菌的基因组以及一系列鳞翅目昆虫物种的基因组已经完全测序26，27，28，29。近年来，从这些昆虫中鉴定和表征了大量的抗性和嗅觉受体基因19，27，28，29。在臂状螺旋体中已经发现了一些与抗性相关的基因，例如编码钙粘蛋白30，ATP结合盒转运蛋白31，32以及HaTSPAN133的基因。使用CRISPR / Cas9技术敲除这些基因可导致对易感菌株中苏云金芽孢杆菌（BT）毒素的高度抗性。此外，Chang等人（2017）敲除了一种信息素受体，这验证了其在交配时间调节方面的重要功能19。这些报告表明，CRISPR/Cas9可以作为研究昆虫系统中体内基因功能的有效工具。然而，CRISPR/Cas9修饰在昆虫系统中的详细程序仍不完整，这限制了其在昆虫功能基因组学中的应用范围。
在这里，我们提出了一种使用CRISPR / Cas9系统敲除 臂状 芽蒿蒿中功能基因的方案。提供了详细的分步方案，包括设计和制备用于gRNA生产，胚胎收集，显微注射，昆虫饲养和突变体鉴定的基因特异性引物。该协议可作为操纵 臂状蛊 虫中任何功能基因的宝贵参考，并且可以扩展到其他鳞翅目物种。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2kb DNA ladder</td>
			<td>TransGen Biotech</td>
			<td>BM101</td>
			<td></td>
		</tr>
		<tr>
			<td>Capillary Glass</td>
			<td>World Precision Instrucments</td>
			<td>504949</td>
			<td>referred to as &#34;capillary glass&#34; in the protocol</td>
		</tr>
		<tr>
			<td>Double Sided Tape</td>
			<td>Minnesota Mining and Manufacturing Corporation</td>
			<td>665</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf FemtoJet 4i Microinjector</td>
			<td>Eppendorf Corporate</td>
			<td>E5252000021</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf InjectMan 4 micromanipulator</td>
			<td>Eppendorf Corporate</td>
			<td>5192000051</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Microloader Pipette Tips</td>
			<td>Eppendorf Corporate</td>
			<td>G2835241</td>
			<td></td>
		</tr>
		<tr>
			<td>GeneArt Precision gRNA Synthesis Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>A29377</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Slide</td>
			<td>Sail Brand</td>
			<td>7105</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus Microscope</td>
			<td>Olympus Corporation</td>
			<td>SZX16</td>
			<td></td>
		</tr>
		<tr>
			<td>PrimeSTAR HS (Premix)</td>
			<td>Takara Biomedical Technology</td>
			<td>R040</td>
			<td>used for mutant detection</td>
		</tr>
		<tr>
			<td>Sutter Micropipette Puller</td>
			<td>Sutter Instrument Company</td>
			<td>P-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>TIANamp Genomic DNA Kit</td>
			<td>TIANGEN Corporate</td>
			<td>DP304-03</td>
			<td></td>
		</tr>
		<tr>
			<td>TrueCut Cas9 Protein v2</td>
			<td>Thermo Fisher Scientific</td>
			<td>A36499</td>
			<td></td>
		</tr>
	</tbody>
</table>

embryo microinjection and knockout mutant identification of crispr/cas9 genome-edited helicoverpa armigera (h&#252;bner)

棉铃虫， Helicoverpa armigera，是世界上最具破坏性的害虫之一。分子遗传学，生理学，功能基因组学和行为研究的结合使 H. armigera 成为鳞翅目夜蛾科的模型物种。为了研究不同基因的体内功能和相互作用，簇状规则间隔的短回文重复序列（CRISPR）/相关蛋白9（Cas9）基因组编辑技术是一种用于进行功能基因组研究的方便有效的方法。在这项研究中，我们提供了一种循序渐进的系统方法，使用CRISPR / Cas9系统完成 臂状芽胞 杆菌中的基因敲除。详细描述了引导RNA（gRNA）的设计和合成。然后，总结了由引导RNA（gRNA）创建，胚胎收集，显微注射，昆虫饲养和突变体检测的基因特异性引物设计组成的后续步骤。最后，提供故障排除建议和注释，以提高基因编辑的效率。我们的方法将作为CRISPR / Cas9基因组编辑在 臂状芽孢 杆菌以及其他鳞翅目蛾中的应用的参考。

该协议提供了使用CRISPR / Cas9技术获得 臂状芽蒿 的基因敲除系的详细步骤。总结了通过该协议获得的代表性结果，用于gDNA选择，胚胎收集和注射，昆虫饲养和突变体检测。
在这项研究中，我们感兴趣的基因的靶位点位于其第二个外显子中（图2A）。该位点高度保守，使用琼脂糖凝胶电泳确认合成sg...

Watch this Scientific Journal Video about Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera HÃƒÂ¼bner at JoVE.com

Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera HÃƒÆ’Ã‚Â¼bner

这里展示的是 Helicoverpa armigera （Hübner）胚胎显微注射和通过CRISPR / Cas9基因组编辑创建的敲除突变体鉴定的方案。突变昆虫能够进一步研究体内不同基因的功能和相互作用。

CRISPR/Cas9基因组编辑的半球 藻（ Hübner）的胚胎显微注射和敲除突变体鉴定

The cotton bollworm, Helicoverpa armigera, is one of the most destructive pests in the world. A combination of molecular genetics, physiology, functional ...

棉铃虫是全球最具破坏性的害虫之一。使用CRISPR / Cas9技术的基因组编辑可以进一步研究该生物体中不同基因之间的基因功能和相互作用。该技术的两个主要优点是它具有高特异性并且可随地进行。为了选择sgRNA靶标，请访问CRISPR在线网站，搜索可能的引导位点和靠近基因五个主要未翻译区域的外显子。将外显子序列输入文本框，然后选择黑猩猩的靶基因组。根据网站的用户手册，为20 BPNGG选择PAM选项，并将其他设置保留在默认参数上。比较软件中预测的引导序列，并在五个主要未翻译区域附近选择包含一个或两个胍的20个碱基对的引导序列，这些导引序列具有最高的预测效率和最少的不匹配，以提高编辑效率并减少脱靶编辑。对于胚胎制备，从三天大的雄性和两天大的雌性中选择50个健康个体，并将它们混合在干净的网笼中。将一块含有10%糖溶液的湿棉放入笼子中。用纱布盖住网架，并用橡皮筋固定纱布。将水喷洒在纱布上，使其保持湿润。将一块黑布切成合适的尺寸，并用这块黑布代替湿润的纱布，使游离的卵子位置持续30分钟。将双面胶带粘贴到显微镜载玻片上。使用镊子，将带有卵的贴片连续粘贴在双面胶带的表面上。按每个贴片的边缘，确保它们牢固地粘在胶带上。每台显微镜载玻片收集50至100个卵。在显微注射之前，将显微镜载玻片保持在冰上以延缓胚胎的发育。如文本手稿中所述，使用微型移液器拉动毛细管玻璃来准备针头。使用微型研磨机将针尖研磨成锋利的尖端。通过将两微升商业化的Cas9蛋白和sgRNA加入PCR管中的无RNace水中来制备注射溶液，以获得10微升体积的混合物。通过移液混合，然后将其放在冰上。设置电子微注射器的参数。使用微量上样器移液器吸头将两微升的混合物装载到针头中，尽可能多地排出针尖中的残余空气。将注射针连接到显微操作器，并确保两个部件之间的紧密连接。将载玻片放入培养皿中，然后将其放在显微镜的载物台上。小心地将针尖以45度角插入胚胎的上半球。踩踏板将混合物输送到胚胎中，导致胚胎略有膨胀。立即从胚胎中取出针头，并用一只手移动培养皿，直到下一个胚胎靠近针头。使用相同的程序注射至少300个胚胎，以确保足够的孵化量。注射后盖上培养皿的盖子。当胚胎的表面颜色变暗时，将人造饮食放在显微镜载玻片周围的培养皿中。准备24孔培养板，并用人工饮食将每个孔填充到其容积容量的1/3。使用小画笔挑选出孵化的幼虫，并将它们转移到24孔培养板中，使得一个孔有一个幼虫。当幼虫长到第三龄阶段时，将它们转移到新的玻璃延展性乙型。将新封闭的G-zero雄性成虫和野生型雌性成虫以相等的比例转移到新鲜的网笼中。为他们提供10%的糖溶液滴在棉球中。后部昆虫使用常规方法，直到G-one化蛹。使用镊子小心地取出后腿，并将每条腿放入裂解矩阵管中。使用组织均质器使后腿均质化。根据制造商的说明，使用商用gDNA提取试剂盒提取均质化样品的基因组DNA。使用基因分型引物和文本手稿中的PCR反应条件扩增基因片段。通过基因测序服务确认基因型。将相同基因型的G-one个体放入一个网笼中，自我杂交G-one后代并继续使用相同的方法进行筛选。目的基因的目标位点位于其第二个外显子中。该位点高度保守，使用琼脂糖凝胶电泳确认合成sgRNA的目标条带片段。通过对来自G-one亲本单元的PCR产物进行测序来执行单个sgRNA靶标的突变检测。在不同的外显子上使用非重叠的sgRNA对的有效性得到了证明，因为突变体的大量缺失很容易与野生型平底锅区分开来。在获得一定数量的突变个体后，可以进行电生理或行为实验，以阐明基因功能和不同基因之间的相互作用。

Research

JoVE Journal

Behavior

Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera (H&#252;bner)

The Successive Alleys Test of Anxiety in Mice and Rats

在小鼠和大鼠连续的小巷测试焦虑

The plus-maze was derived from the early work of Montgomery. He observed that rats tended to avoid the open arms of a maze, preferring the enclosed ones.  ...

科研

行为学

Stereotaxic Microinjection of Viral Vectors Expressing Cre Recombinase to Study the Role of Target Genes in Cocaine Conditioned Place Preference

立体定向显微注射病毒载体表达Cre重组酶研究的靶基因的作用在可卡因条件性位置偏爱

Microinjecting recombinant adenoassociated viral (rAAV) vectors expressing Cre recombinase into distinct mouse brain regions to selectively knockout genes ...

Rodent Brain Microinjection to Study Molecular Substrates of Motivated Behavior

啮齿动物脑显微注射学习动机行为的分子基板

Brain microinjection can aid elucidation of the molecular substrates of complex behaviors, such as motivation. For this purpose rodents can serve as ...

Holistic Facial Composite Creation and Subsequent Video Line-up Eyewitness Identification Paradigm

整体面部复合创作以后的视频阵容目击者识别范式

The paradigm detailed in this manuscript describes an applied experimental method based on real police investigations during which an eyewitness or victim ...

Implantation and Recording of Wireless Electroretinogram and Visual Evoked Potential in Conscious Rats

植入和无线电图记录和视觉诱发电位清醒大鼠

The full-field electroretinogram (ERG) and visual evoked potential (VEP) are useful tools to assess retinal and visual pathway integrity in both ...

Manipulation of Epileptiform Electrocorticograms (ECoGs) and Sleep in Rats and Mice by Acupuncture

Manipulation of Epileptiform Electrocorticograms ECoGs and Sleep in Rats and Mice by Acupuncture

癫痫Electrocorticograms（ECoGs）的操作，并睡在针刺大鼠和小鼠

Ancient Chinese literature has documented that acupuncture possesses efficient therapeutic effects on epilepsy and insomnia. There is, however, little ...

Task Interruption and Resumption Paradigm for Testing the Activation and Pursuit of an Abstract Thinking Goal

任务中断和抽象思维的目标测试的激活和恢复追求范式

This protocol is based on the task interruption and resumption paradigm, the premise of which is that active goals lead to persistent behavior and thus a ...

Methods of Pairing and Pair Maintenance of New Zealand White Rabbits (Oryctolagus Cuniculus) Via Behavioral Ethogram, Monitoring, and Interventions

Methods of Pairing and Pair Maintenance of New Zealand White Rabbits Oryctolagus Cuniculus Via Behavioral Ethogram, Monitoring, and Interventions

新西兰白兔的配对和配对维护方法 (穴兔家兔) 通过行为 Ethogram、监视和干预

New Zealand White (NZW) laboratory rabbits (Oryctolagus cuniculus), as well as their ancestors the European Rabbit, are a social species that exhibit ...

Behavioral Tracking and Neuromast Imaging of Mexican Cavefish

墨西哥辣鱼的行为跟踪与神经吻合

Cave-dwelling animals have evolved a series of morphological and behavioral traits to adapt to their perpetually dark and food-sparse environments. Among ...

Transcranial Direct Current Stimulation (tDCS) of Wernicke's and Broca's Areas in Studies of Language Learning and Word Acquisition

Transcranial Direct Current Stimulation tDCS of Wernicke's and Broca's Areas in Studies of Language Learning and Word Acquisition

韦尼克和布罗卡地区语言学习和文字习得研究中的颅内直接电流刺激(tDCS)

Language is a highly important yet poorly understood function of the human brain. While studies of brain activation patterns during language comprehension ...

CRISPR/Cas9基因组编辑的半球 藻（ Hübner）的胚胎显微注射和敲除突变体鉴定

CRISPR/Cas9基因组编辑的半球藻（ Hübner）的胚胎显微注射和敲除突变体鉴定