Name: crispr/cas9 gene editing to make conditional mutants of human malaria parasite p. falciparum
Uploaded: 2018-09-18T12:30:00
Description: Watch this Scientific Journal Video about CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum at JoVE.com

我们感谢格鲁吉亚大学的 Muthugapatti Kandasamy (UGA) 生物医学显微镜核心技术援助和何塞-胡安洛佩兹-卢比奥分享 pUF1-Cas9 和 pL6 质粒。这项工作得到了弧形基金会奖项的支持, D.W.C. 和 H.M.K., UGA 启动资金 V.M., 赠款从3月的硬币基金会 (罗勒 O ' 康纳启动学者研究奖) V.M., 和美国国立卫生研究院 (R00AI099156 和R01AI130139) V.M. 和 (T32AI060546) H.M.K。

恶性疟原虫的持续培养需要使用人类的红细胞, 我们利用了商业上购买的血液, 这些单位被剥夺了所有的识别和匿名。格鲁吉亚大学的机构审查委员会和生物安全办公室审查了我们的议定书, 并批准了我们实验室使用的所有议定书。
1. 选择 gRNA 序列
<ol><li>转到 CHOPCHOP (http://chopchop.cbu.uib.no/) 并选择"Fasta 目标"。在"目标"下, 将200基对从基因的开放阅读框架 (ORF...

<ol>
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CRISPR/Cas9 在恶性疟原虫中的实施, 与以往的遗传操作方法相比, 提高了对寄生虫基因组进行修改所需的时间和效率。该综合协议概述了在恶性疟原虫中使用 CRISPR/Cas9 生成条件突变体所需的步骤。虽然这里的方法专门针对glmS突变体的生成, 但这种策略可以适应各种需求, 包括基因标记、基因挖空和点突变的引入。
此协议中的一个关键的早期步骤是选择一个 gRN...

疟疾是疟原虫属原生动物寄生虫造成的一种破坏性疾病。恶性疟原虫是人类最致命的疟疾寄生虫, 每年造成约44.5万人死亡, 其中大部分为五岁以下儿童.疟原虫有一个复杂的生命周期, 涉及蚊子载体和脊椎动物宿主。当被感染的蚊子吃了一顿血时, 人类首先会被感染。然后, 寄生虫侵入它生长、发育和分裂大约一周的肝脏。在这个过程之后, 寄生虫被释放入血液, 在那里他们接受无性复制在红细胞 (红细胞)。红细胞内寄生虫的生长直接负责与疟疾有关的临床症状2。
直到最近, 转基因的恶性疟原虫的生产是一个费力的过程, 涉及几轮的药物选择, 花了几个月, 并有很高的失败率。这一耗时的程序 relieson 在感兴趣的地区产生随机 DNA 断裂和寄生虫的内生能力, 修复其基因组, 虽然同源维修3,4,5,6.最近, 聚集定期 Interspaced 回文 Repeat/Cas9 (CRISPR/Cas9) 基因组编辑已成功地应用于恶性疟原虫7,8。在疟疾研究中引入这种新技术对于促进对这些致命疟原虫的生物学的了解至关重要。CRISPR/Cas9 允许特定的基因靶向, 通过引导 rna (gRNAs) 是同源的基因的兴趣。gRNA/Cas9 复合体通过 gRNA 识别该基因, Cas9 然后引入双链断裂, 强制启动修复机制在生物体9,10。由于恶性疟原虫缺乏通过非同源端连接修复 DNA 断裂的机制, 它利用同源重组机制, 整合转染的同源 dna 模板修复 Cas9/gRNA-induced双绞线断裂11,12。
在这里, 我们提出了一个协议, 以产生条件下的突变体恶性疟原虫使用 CRISPR/Cas9 基因组编辑。该协议表明使用glmS核酶的条件下击倒蛋白水平的 PfHsp70x (PF3D7_0831700), 一个陪同下, 由P. 恶性疟原虫出口到主机红细胞13,14。glmS核酶是通过治疗与氨基葡萄糖 (转化为 glucosamine-6-phosphate 细胞), 以切割其相关的 mRNA, 导致减少蛋白质14。该协议可以很容易地适应使用其他条件击倒工具, 如不稳定域或 RNA 适配子4,5,15。我们的协议详细描述了由血凝素 (HA) 标记和glmS核酶编码序列组成的修复质粒的生成, 这些顺序由与 PfHsp70x 开放阅读框架 (ORF) 和 3 '-UTR 相同源的序列所构成。我们还描述了第二种质粒的生成, 以驱动 gRNA 的表达。这两种质粒, 连同第三个驱动 Cas9 的表达, 被转染成红细胞, 用于改变恶性疟原虫的基因组。最后, 我们描述了一种聚合酶链反应 (PCR) 技术, 以验证标签和glmS核酶的整合。该协议对于任何恶性疟原虫基因的修改或完全挖空具有很强的适应性, 增强了我们对疟原虫生物学的新见解的能力。

<table>
	<tbody>
		<tr>
			<td>Gene Pulser Xcell Electroporator</td>
			<td>Bio-Rad</td>
			<td>1652660</td>
			<td></td>
		</tr>
		<tr>
			<td>Gene Pulser Xcell Electroporator</td>
			<td>Bio-Rad</td>
			<td>165-2086</td>
			<td>We buy the ones that are individually wrapped</td>
		</tr>
		<tr>
			<td>Sodium Acetate</td>
			<td>Sigma-Aldrich</td>
			<td>S2889-250g</td>
			<td></td>
		</tr>
		<tr>
			<td>DSM1</td>
			<td>Gift from Akhil Vaidya lab</td>
			<td>Ganesan et al. Mol. Biochem. Parasitol. 2011 177:29-34</td>
			<td></td>
		</tr>
		<tr>
			<td>TPP Tissue Culture 6 Well Plates</td>
			<td>MIDSCI</td>
			<td>TP92006</td>
			<td></td>
		</tr>
		<tr>
			<td>TPP 100mm Tissue Culture Dishes (12 mL Plate)</td>
			<td>MIDSCI</td>
			<td>TP93100</td>
			<td></td>
		</tr>
		<tr>
			<td>TPP Tissue Culture 96 Well Plates</td>
			<td>MIDSCI</td>
			<td>TP92096</td>
			<td></td>
		</tr>
		<tr>
			<td>TPP Tissue Culture 24 Well Plates</td>
			<td>MIDSCI</td>
			<td>TP92024</td>
			<td></td>
		</tr>
		<tr>
			<td>NEBuffer 2</td>
			<td>New England Biolabs</td>
			<td>#B7002S</td>
			<td></td>
		</tr>
		<tr>
			<td>NEBuffer 2.1</td>
			<td>New England Biolabs</td>
			<td>#B7202S</td>
			<td></td>
		</tr>
		<tr>
			<td>BtgZI</td>
			<td>New England Biolabs</td>
			<td>#R0703L</td>
			<td></td>
		</tr>
		<tr>
			<td>SacII</td>
			<td>New England Biolabs</td>
			<td>#R0157L</td>
			<td></td>
		</tr>
		<tr>
			<td>HindIII-HF</td>
			<td>New England Biolabs</td>
			<td>#R3104S</td>
			<td></td>
		</tr>
		<tr>
			<td>Afe1</td>
			<td>New England Biolabs</td>
			<td>#R0652S</td>
			<td></td>
		</tr>
		<tr>
			<td>Nhe1-HF</td>
			<td>New England Biolabs</td>
			<td>#R3131L</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Polymerase</td>
			<td>New England Biolabs</td>
			<td>#M0203S</td>
			<td></td>
		</tr>
		<tr>
			<td>500 mL Steritop bottle top filter unit</td>
			<td>Millipore</td>
			<td>SCGPU10RE</td>
			<td>You can use any size that fits your needs</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>E4378-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9333-500g</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C7902-500g</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>M8266-100g</td>
			<td></td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Fisher</td>
			<td>P288-500</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H4034-500g</td>
			<td></td>
		</tr>
		<tr>
			<td>pMK-U6</td>
			<td>Generated by the Muralidharan Lab</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>pHA-glmS</td>
			<td>Generated by the Muralidharan Lab</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>pUF1-Cas9</td>
			<td>Gift from the Jose-Juan Lopez-Rubio Lab</td>
			<td>Ghorbal et al. Nature Biotech 2014</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7021-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S5761-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td>P5280-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypoxanthine</td>
			<td>Sigma-Aldrich</td>
			<td>H9636-25g</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin Reagent</td>
			<td>Gibco</td>
			<td>15710-064</td>
			<td></td>
		</tr>
		<tr>
			<td>Thymidine</td>
			<td>Sigma-Aldrich</td>
			<td>T1895-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>PL6-eGFP BSD</td>
			<td>Generated by the Muralidharan Lab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Puf1-cas9 eGFP gRNA</td>
			<td>Generated by the Muralidharan Lab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NucleoSpin Gel and PCR Clean-up</td>
			<td>Macherey-Nagel</td>
			<td>740609.250</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumax I</td>
			<td>Life Technologies</td>
			<td>N/A</td>
			<td>You will want to try a few batches to find out what the parasites will grow in best</td>
		</tr>
		<tr>
			<td>Human Red Blood Cells</td>
			<td>Interstate Blood Bank, Inc</td>
			<td>Email or call them directly for ordering</td>
			<td>We typically use O+ blood</td>
		</tr>
		<tr>
			<td>3D7 parasite line</td>
			<td>Available upon request</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysogeny Broth (LB)</td>
			<td>Fisher</td>
			<td>BP1426-2</td>
			<td>You can make your own, it is not necessary to use exactly this</td>
		</tr>
		<tr>
			<td>Ampicilin</td>
			<td>Fisher</td>
			<td>BP1760-25</td>
			<td>We make a 1000X stock at 100mg/ml in water and store in the -20C</td>
		</tr>
		<tr>
			<td>Ampicilin</td>
			<td>Clonetech</td>
			<td>R050A</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-EF1alpha</td>
			<td>Dr. Daniel Goldberg&#39;s Lab</td>
			<td>Washington University in St. Louis</td>
			<td>You can use your preferred loading control for western blots. This is just the one we use in our laboratory</td>
		</tr>
		<tr>
			<td>Rat Anti-HA Clone 3F10, monoclonal</td>
			<td>Made by Roche, sold by Sigma</td>
			<td>11867423001</td>
			<td>You can use your preferred anti-HA antibody</td>
		</tr>
		<tr>
			<td>0.6 mL tubes</td>
			<td>Fisher</td>
			<td>AB0350</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisher HealthCare* PROTOCOL* Hema 3* Manual Staining System (Fixative+Solution I and II)</td>
			<td>Fisher</td>
			<td>22-122-911</td>
			<td>You can also use giemsa stain</td>
		</tr>
		<tr>
			<td>Fisherfines Premium Frosted Microscope Slides - Size: 3 x 1 in.</td>
			<td>Fisher</td>
			<td>12-544-3</td>
			<td></td>
		</tr>
	</tbody>
</table>

crispr/cas9 gene editing to make conditional mutants of human malaria parasite p. falciparum

疟疾是全世界发病率和死亡率的一个重要原因。这种疾病主要影响那些生活在热带和亚热带地区的人, 是由疟原虫感染引起的。通过提高我们对这种复杂寄生虫生物学的认识, 可以加速开发更有效的防治疟疾药物。这些寄生虫的基因操纵是了解他们的生物学的关键;然而, 在历史上,恶性疟原虫的基因组一直难以操作。最近, CRISPR/Cas9 基因组编辑已被用于疟疾寄生虫, 允许更容易的蛋白质标记, 产生条件蛋白 knockdowns, 并删除基因。CRISPR/Cas9 基因组编辑已被证明是促进疟疾研究领域的有力工具。在这里, 我们描述了一个 CRISPR/Cas9 的方法来产生glmS的条件下的突变体恶性疟原虫。这种方法非常适合其他类型的基因操作, 包括蛋白质标记和基因挖空。

图 1显示了这种方法中使用的质粒示意图以及盾构突变的例子。如图 2所示, 在转染后如何识别突变体寄生虫, PCRs 对 HA-glmS结构集成的检查结果。图 3显示了克隆板的一个代表性图像, 以证明在寄生虫存在时培养基的颜色变化。在图 4中显示了免疫荧光检测和西方印迹实验的结果,...

Watch this Scientific Journal Video about CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum at JoVE.com

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

CRISPR/Cas9 基因编辑制作人类疟疾寄生虫的条件突变体

我们描述了一种利用 CRISPR/Cas9 基因组编辑在恶性疟原虫中产生glmS的条件击倒突变体的方法。

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

这种方法可以帮助回答人类疟疾寄生虫P.Falciparum生物学中的关键问题，通过条件性敲除帮助发现蛋白质功能。与以前的方法相比，该技术的主要优点是，它允许相对容易地生成条件突变体。首先，转到"斩波"并选择 Fasta Target'下目标，粘贴来自基因开放读取帧三个黄金端的 200 个碱基对，以及从基因的三个主要 UTR 开始的 200 个碱基对粘贴。在"选择 P.Falciparum"和选择 CRISPR/Cas9'下一步下，单击"查找目标站点"从显示的选项中选择 gRNA 序列，优先选择最接近修改站点且离目标站点最近的最高效的 gRNA。购买 gRNA 序列及其反向补品作为聚丙烯酰胺凝胶电泳纯化寡糖。用于定位PfH-sp70x的gRNA序列可以在文本协议中找到。将 pMK-U6、pUF1-Cas9 和 pHA-glmS DNA 各加入 40 微克，加入无菌 1.5 毫米离心管。将水中三摩尔醋酸钠的DNA体积的十分之一加入到管子中，然后用涡流混合。然后，将100%乙醇的体积的2.5倍添加到管子中，使用涡流混合至少30秒。将管子放在冰上或在零下20摄氏度下放置30分钟。然后，在18，300次G下将管离心，在4摄氏度下30分钟。离心后，小心地从管中去除上经液，而不会干扰颗粒。将70%乙醇的体积加三倍到管子中，然后用涡流短暂混合。在 4 摄氏度下将管在 18，300 次 G 下离心 30 分钟。再次，小心地从管中去除上一液，而不干扰颗粒。使管保持打开状态，让颗粒风干 15 分钟。将沉淀的DNA储存在零下20摄氏度，直到转染需要。要转染 RBC，请准备 1X 细胞混合缓冲区以及文本协议中描述的不完整和完整的 RPMI。使用 0.22 微米过滤器对缓冲液进行灭菌。将 380 微升的 1X 细胞混合添加到 DNA 和涡流中溶解。让DNA溶解在1X细胞混合中10分钟，每三分钟旋转10秒。在无菌的15毫升锥形管中，将300微升的分离人类RBC与4毫升1X细胞混合相结合。将 RBC 以 870 倍 G 离心三分钟。然后从 RBC 颗粒中去除上因。用DNA-细胞混合混合物重新悬浮RBC颗粒，并转移到0.2厘米电穿孔的细胞中。使用文本协议中列出的条件对 RBC 进行波分。电穿孔后，将内维从圆锥体转移到含有5毫升完整RPMI的15毫升圆锥管中。在20摄氏度下以870倍G离心管，3分钟。然后，将上一点液去。现在，将颗粒重新悬浮在四毫升完整的RPMI中，并转移到六井组织培养板中的一个井中。在传输的 RBC 中加入 400 个高氧化氮培养物的微升，在 3% 的氧气、3% 的 CO2 和 94% 的氮气下保持所有培养物的温度。第二天，用四毫升完整的RPMI洗涤培养。将培养在 870 倍 G 下离心三分钟。吸上一道。在四毫升完整的 RPMI 中重新挂起培养。48 小时后，用四毫升完整的 RPMI 洗涤培养。然后，在包含一个微摩尔 DSM1 的完整 RPMI 中重新挂起培养基，以选择 Cas9 质粒。在此点之后，将培养基替换为新鲜完整的 RPMI，每 48 小时更换一个微摩尔 DSM1。每天继续用完整的RPMI清洗培养物，直到血液涂片不再可见寄生虫。为了进行血液涂片，移液器150微升培养成0.6毫升的离心管。在1，700次G下进行离心造粒后，吸走上经剂。使用移液器将颗粒细胞转移到玻璃滑梯上。使用第二个玻璃幻灯片以 45 度角保持到第一个幻灯片，涂抹血滴。根据制造商的协议，使用市售的染色套件对幻灯片进行染色。使用 100 次油浸目标查看寄生虫。从转染后五天开始，在培养媒介中重新悬浮 RBC 时，去除两毫升培养的培养。以2%的血红蛋白加入两毫升的新鲜介质和血液。以这种方式每周添加一次新鲜血液，直到寄生虫再次出现，由瘦血涂片决定。对寄生虫培养物进行连续稀释，以达到每200微升0.5个寄生虫的最终浓度。在 96 孔组织培养板的井中加入 200 微升稀释培养剂。保持克隆板，直到在井中检测到寄生虫。每48小时更换96井板中的介质，用新鲜介质。每周一次，从开始克隆板开始的五天后开始，从每一个井中去除100微升，并添加100微升的新鲜介质加血液。要识别任何含有寄生虫的井，请将 96 孔板以 45 度角放置约 20 分钟，使血液在板内以一定角度沉淀。然后，将 96 井板放在灯箱上。请注意，含有寄生虫的井中含有黄色的介质，与无寄生虫井的粉红色介质相比，由于寄生虫对介质的酸化。使用血清移液器，将含有寄生虫的井的内容移动到24孔组织培养板，以允许寄生虫的膨胀。使用 PCR 分析，检查这些克隆寄生虫线是否正确整合。此处显示了免疫荧光测定的结果，证明了PfH-sp70x的HA标记成功。PfH-sp70x glmS 寄生虫被固定并染色，并染色为核标记物，以及 HA 抗体。膜相关组蛋白丰富的蛋白质一，蛋白质出口到宿主RBC的标记。此处显示了使用葡萄糖胺治疗后 PfH-sp70x 蛋白质水平的西式 Blot 分析。如预期的那样，在治疗期间蛋白质水平会降低。这项技术为寄生虫生物学领域的研究人员研究寄生虫生物学中的基本问题铺平了道路。重要的是要记住，P.Falciparum是一种血液传播的病原体，在进行此程序时应佩戴适当的个人防护设备。

Research

JoVE Journal

Genetics

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

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

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

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 ...

科研

遗传学

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 ...

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

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

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 ...

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. ...

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

利用 CRISPR/Cas9 Ribonucleoproteins 快速简便地生成线虫基因组点突变体的管道

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been ...

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 ...

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

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

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 ...