Name: introducing a gene knockout directly into the amastigote stage of trypanosoma cruzi using the crispr/cas9 system
Uploaded: 2019-07-31T15:00:00
Description: Watch this Scientific Journal Video about Introducing a Gene Knockout Directly Into the Amastigote Stage of Trypanosoma cruzi Using the CRISPR/Cas9 System at JoVE.com

这项工作部分得到了JSPS KAKENHI授予编号18K15141到Y.T.的部分支持。

注:图 1描述了整个实验流程的概述。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59962/59962fig01.jpg"> 图 1:使用 EA 的挖空实验概述。组织培养衍生的胰腺炎被收获并分化成EA.gRNA通过电穿孔转染成Cas9表达的乳腺,并且通过轴仿或通过宿主细胞入侵后的细胞内复制。<a href="https://www.jove.com/files/ftp_upload/59962/599...

<ol>
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我们证明,在CRISPR/Cas9介导的基因敲除中,通过将gRNA直接电化到Cas9表达的EA中,可以利用T.克鲁兹的乳腺的轴培养。这样,在乳腺发育阶段,可以评估靶基因的本质,而无需经历其他发育阶段。
乳腺转染的另一个有益方面是对大量靶基因的检测方便。一旦Cas9表达T.cruzi和宿主哺乳动物细胞的共同培养建立,它只需要几天时间将组织衍生的胰腺炎转化为EA和转染gRNA,以获得敲?...

锥虫病是恰加斯病的致病剂,主要在拉丁美洲1流行。T. cruzi具有独特的生命周期阶段,因为它在昆虫载体和哺乳动物宿主2之间传播。T. cruzi在吸血三胺虫的中肠道中复制为表皮球菌,在被沉积在人类或动物宿主之前,在后肠中分化成传染性代谢性胰小体球菌。一旦锥虫通过叮咬部位或通过粘膜进入宿主体,寄生虫就会侵入宿主细胞,并转化为称为乳腺的无旗体圆形。乳腺在宿主细胞内复制,并最终分化成锥虫,从宿主细胞中爆发并进入血流,感染另一个宿主细胞。
由于目前可用的化疗药物,苯甲酰氨基苯甲酸和尼富蒂莫克斯,引起不良副作用,并在疾病的慢性阶段无效3,这是极大的兴趣,确定针对T.cruzi的新药物靶点。近年来,CRISPR/Cas9系统已成为一个强大的工具,有效地执行基因敲除在T.cruzi,无论是通过转染单独或单质粒含有gRNA和Cas9 4,通过稳定表达Cas9和随后的引入gRNA 5,6,7或转录模板的gRNA8,或通过电穿孔预成型的gRNA/Cas9 RNP复合物7,9。这一技术进步被高度期待加速恰加斯病的药物靶点研究。
为了继续药物开发,验证靶基因或药物候选化合物在T.cruzi的药效中的重要性至关重要,因为它是寄生虫在哺乳动物宿主中的复制阶段。然而,这是一项具有挑战性的任务,因为由于存在阻塞宿主细胞,因此不能直接操纵乳腺。在利什曼尼亚,一种与T.cruzi密切相关的原生动物寄生虫,开发了一种斧头阿马斯蒂戈特培养方法,并已用于药物筛选测定方法10,11,12, 13.虽然轴突和细胞内乳腺14在化合物的易感性上有一些差异,但维持轴培养的能力为研究利什曼病临床相关阶段的基本生物学15、16。在T.cruzi的情况下,关于自然发生的细胞外乳腺(EA)17和EA 17,18,19的体外生产文献可追溯到几十年前。此外,EA已知具有传染性能力20,虽然小于锥形雄蒂戈特,和马斯蒂戈特宿主入侵的机制已阐明近年来(审查由Bonfim-Melo等人21)。然而,与利什曼尼亚不同,EA在T.cruzi中没有被用作实验工具,主要是因为EA被认为是细胞内寄生虫,因此在实际操作中并未被视为"复制形式"感。
最近,我们小组提议利用T.克鲁兹的EA作为时间轴文化22。T. 克鲁西图拉胡恩菌株的Amastigotes可在37°C下在LIT培养基中无宿主细胞复制长达10天,而不会严重恶化或丧失类似亲子的特性。在无宿主生长期,EA通过常规电穿孔、药物滴定测定与胰蛋白酶化合物和CRISPR/Cas9介导敲除,然后进行生长表型监测,成功地用于外源性基因表达。在本报告中,我们描述了在敲除实验中产生体外衍生EA和利用轴突腺体的详细方案。

<table>
	<tbody>
		<tr>
			<td>20% formalin solution</td>
			<td>FUJIFILM Wako Pure Chemical</td>
			<td>068-03863</td>
			<td>fixing cells</td>
		</tr>
		<tr>
			<td>25 cm2 double seal cap culture flask</td>
			<td>AGC Techno Glass</td>
			<td>3100-025</td>
			<td></td>
		</tr>
		<tr>
			<td>75 cm2 double seal cap culture flask</td>
			<td>AGC Techno Glass</td>
			<td>3110-075</td>
			<td></td>
		</tr>
		<tr>
			<td>All-in One Fluorescence Microscope</td>
			<td>Keyence</td>
			<td>BZ-X710</td>
			<td></td>
		</tr>
		<tr>
			<td>Alt-R CRISPR-Cas9 crRNA (for Control)</td>
			<td>IDT</td>
			<td>custom made</td>
			<td>target sequence = GGACGGCACCTTCATCTACAAGG</td>
		</tr>
		<tr>
			<td>Alt-R CRISPR-Cas9 crRNA (for TcCGM1)</td>
			<td>IDT</td>
			<td>custom made</td>
			<td>target sequence = TAGCCGCGATGGAGAGTTTATGG</td>
		</tr>
		<tr>
			<td>Alt-R CRISPR-Cas9 crRNA (for TcPAR1)</td>
			<td>IDT</td>
			<td>custom made</td>
			<td>target sequence = CGTGGAGAACGCCATTGCCACGG</td>
		</tr>
		<tr>
			<td>Alt-R CRISPR-Cas9 tracrRNA</td>
			<td>IDT</td>
			<td>1072532</td>
			<td>to anneal with crRNA</td>
		</tr>
		<tr>
			<td>Amaxa Nucleofector device</td>
			<td>LONZA</td>
			<td>AAN-1001</td>
			<td>electroporation</td>
		</tr>
		<tr>
			<td>Basic Parasite Nucleofector Kit 2</td>
			<td>LONZA</td>
			<td>VMI-1021</td>
			<td>electroporation</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich</td>
			<td>A3294</td>
			<td>component of the medium for in-vitro amastigogenesis</td>
		</tr>
		<tr>
			<td>Burker-Turk disposable hemocytometer</td>
			<td>Watson</td>
			<td>177-212C</td>
			<td>cell counting</td>
		</tr>
		<tr>
			<td>Coster 12-well Clear TC-Treated Multiple Well Plates</td>
			<td>Corning</td>
			<td>3513</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>FUJIFILM Wako Pure Chemical</td>
			<td>044-29765</td>
			<td>culture medium</td>
		</tr>
		<tr>
			<td>Fetal bovine serum, Defined</td>
			<td>Hyclone</td>
			<td>SH30070.03</td>
			<td>heat-inactivate before use</td>
		</tr>
		<tr>
			<td>G-418 Sulfate Solution</td>
			<td>FUJIFILM Wako Pure Chemical</td>
			<td>077-06433</td>
			<td>selection of transformant</td>
		</tr>
		<tr>
			<td>Hemin chloride</td>
			<td>Sigma-Aldrich</td>
			<td>H-5533</td>
			<td>component of LIT medium</td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Thermo Fisher Scientific</td>
			<td>H3570</td>
			<td>staining of nuclei</td>
		</tr>
		<tr>
			<td>Liver infusion broth, Difco</td>
			<td>Becton Dickinson</td>
			<td>226920</td>
			<td>component of LIT medium</td>
		</tr>
		<tr>
			<td>MES</td>
			<td>FUJIFILM Wako Pure Chemical</td>
			<td>349-01623</td>
			<td>component of the medium for in-vitro amastigogenesis</td>
		</tr>
		<tr>
			<td>PBS (&#8211;)</td>
			<td>FUJIFILM Wako Pure Chemical</td>
			<td>166-23555</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>Sigma-Aldrich</td>
			<td>P4864-10ML</td>
			<td>staining of dead cells</td>
		</tr>
		<tr>
			<td>RPMI 1646</td>
			<td>Sigma-Aldrich</td>
			<td>R8758</td>
			<td>medium for metacyclogenesis</td>
		</tr>
	</tbody>
</table>

introducing a gene knockout directly into the amastigote stage of trypanosoma cruzi using the crispr/cas9 system

锥虫病是一种致病原生动物寄生虫,主要在拉丁美洲引起恰加斯病。为了识别针对T.cruzi的新型药物靶点,验证靶向基因在寄生虫的哺乳动物阶段(乳腺)中的重要性是很重要的。T. 克鲁西的阿马斯蒂在宿主细胞内复制;因此,如果不经过其他发展阶段,就很难进行淘汰实验。最近,我们小组报告了一种生长状况,其中乳腺可异种复制长达10天,而不会失去其类似乳腺的特性。通过使用这种时间轴质乳腺培养,我们成功地将gRNA直接引入Cas9表达的乳腺,导致基因敲除,并仅在乳腺阶段分析其表型。在本报告中,我们描述了一个详细的方案,用于产生体外衍生的细胞外乳腺,并在CRISPR/Cas9介导的敲除实验中利用轴培养。敲除的乳腺的生长表型可以通过轴培养的细胞计数或宿主细胞入侵后细胞内乳腺的复制来评估。该方法绕过寄生虫阶段分化通常涉及生产转基因或敲除的乳腺。利用时间轴突培养具有扩大T.cruzi特定阶段研究的实验自由的潜力。

通过游出程序隔离胰岛
为了通过游出程序从污染旧的EA中收获新鲜的胰小球,细胞颗粒至少需要孵育1小时。 孵育颗粒超过2小时不会显著增加在溶液中游泳的胰小球的数量(图 2B.在这个特殊的实验中,在初始混合物中,胰小体测定的百分比为38%,在任意给定时间点,游出后百分比超过98%。从两个T-75瓶的汇合培养,我们经常获得3-4 x 107细胞的?...

Watch this Scientific Journal Video about Introducing a Gene Knockout Directly Into the Amastigote Stage of Trypanosoma cruzi Using the CRISPR/Cas9 System at JoVE.com

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

使用CRISPR/Cas9系统将基因挖空直接引入锥虫瘤的肌瘤阶段

在这里,我们描述了一个协议,使用CRISPR/Cas9系统将基因敲除引入锥虫的细胞外肌瘤。生长表型可以通过细胞计数来追踪轴酸乳腺培养物,或者通过宿主细胞入侵后细胞内乳腺增殖。

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

在实验中很难使用T.cruzi的宿主感染阶段，因为阿玛斯蒂戈特是细胞内寄生虫的强制。我们的培养技术允许阿玛蒂戈特在宿主细胞外暂时复制，因此我们可以直接在寄生虫的这个临床相关阶段进行实验。演示该程序将是 Yukie Akutsu， 我们研究所的技术人员。根据手稿指示保持宿主寄生虫共培养。当 Cas9 表达寄生虫准备好分化时，将上流液收集到圆锥管中，并在显微镜下检查样品质量。如果存在大量细胞外海子，则执行游出程序以隔离试管。将试药和海糖的混合物旋转15分钟，作为2，100倍G.然后丢弃大部分上流液，在管中留下0.5至1毫升的介质。松散地盖住管子，在37摄氏度下孵育颗粒一到两个小时，这将使活性的试药从颗粒中游出。孵育后，将上经剂与试管转移到1.5毫升微离心管中。向下旋转，用 5 毫升 DMEM 重新暂停颗粒。将寄生虫转移到T-25培养瓶中，在加湿的培养箱中孵化37摄氏度，在5%的二氧化碳下孵化。大约95%的寄生虫在24小时后会分化成食与动物。将细胞外海子的培养物在2，100倍G下离心15分钟，并丢弃上流液。将颗粒与含有电穿孔缓冲液的颗粒重新填充，最终细胞密度为每毫升10倍至第8个细胞。阿里配额100微升的重新暂停的寄生虫到1.5升微离心管。然后加入5至10微克的引导RNA，然后通过移液轻轻混合。将混合物转移到两毫米间隙电穿孔盒中，并应用电穿孔装置的脉冲。然后，将蛋瓶内装物转移到含有五毫升预热的 LIT 介质的 T-25 烧瓶中，使瓶盖松动，并在 37 摄氏度下孵育 5%的二氧化碳。监测细胞生长，要么通过继续轴培养，或在宿主细胞感染后作为细胞内母体。如果监测细胞生长为斧头胺类动物，轻轻摇动烧瓶，将细胞外阿米托酸盐重新放入溶液中，并在微离心管中将培养物的20微升与一微升的碘化氢溶液混合。重要的是，不要将培养瓶留在孵化器之外超过必要的时间，因为温度是导致阿玛底特的斧头增殖的因素之一。将样品应用到血细胞计上，并在荧光显微镜下观察。然后计算未被碘化丙二烯染色的可行阿米斯特里特的数量。为了监测敲除细胞的生长，作为细胞内海膜，种子宿主3T3细胞在12孔板与DMEM。电穿孔后一天通过离心收集敲除剂，丢弃上经剂，用两毫升DMEM重新暂停寄生虫。从宿主单元培养中删除介质，并添加重新暂停的主机。在37摄氏度下孵育200万分之二，两天，这将使阿米斯蒂戈特人建立感染。两天后，用DMEM清洗宿主细胞外的细胞外友爱，并加入新鲜的DMEM。继续孵育两天，然后可视化宿主细胞和细胞内阿米斯特的核。已经证明，与指南RNA对基本基因TcCGM1的T.Cruzi阿米斯特里特与对照组相比，显示出显著的生长缺陷。当监测细胞内机制的生长时，与T.Cruzi核相关的宿主核在TCCGM1敲解组中比对照组明显较低。相反，Guide RNA对一种副旗杆蛋白TcPAR1的转染在四天的斧头培养后不会显著影响细胞生长，或抑制细胞内寄生。然而，感染后五天，与控制共培养相比，在TcPAR1敲除共培养中，从宿主细胞中产生的试虫数量明显减少。此外，对照组中宿主单元内的分化试波马提格似乎比淘汰组中的多样试波面罩显得更活跃。此方法还可用于研究外源基因对阿玛斯蒂戈特阶段的影响，而不是将指南RNA转染成Cas9表达的亚马提格，您可以将质粒转染成野生型的阿玛底特，以表达外源基因。

Research

JoVE Journal

Genetics

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

Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format

用荧光PCR毛细管电泳技术在基因型CRISPR / Cas9介导的敲除突变体的高通量形式

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play ...

科研

遗传学

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

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 ...

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

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

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 ...

A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells

整合酶缺陷慢病毒载体载体的制备及其在细胞 CRISPR/Cas9-mediated 基因敲除中的表达

Lentiviral vectors are an ideal choice for delivering gene-editing components to cells due to their capacity for stably transducing a broad range of cells ...

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

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

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 ...

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

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. ...

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

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

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

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 ...

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

CRISPR/Cas9核糖核酸蛋白在急性骨髓性白血病细胞中的RUNX1电子消光器的转录作用研究

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding ...