CRISPR/Cas9 基因编辑制作人类疟疾寄生虫的条件突变体
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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 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…
Representative Results
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,…
Discussion
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…
Declarações
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Gene Pulser Xcell Electroporator | Bio-Rad | 1652660 | |
| Gene Pulser Xcell Electroporator | Bio-Rad | 165-2086 | We buy the ones that are individually wrapped |
| Sodium Acetate | Sigma-Aldrich | S2889-250g | |
| DSM1 | Gift from Akhil Vaidya lab | Ganesan et al. Mol. Biochem. Parasitol. 2011 177:29-34 | |
| TPP Tissue Culture 6 Well Plates | MIDSCI | TP92006 | |
| TPP 100mm Tissue Culture Dishes (12 mL Plate) | MIDSCI | TP93100 | |
| TPP Tissue Culture 96 Well Plates | MIDSCI | TP92096 | |
| TPP Tissue Culture 24 Well Plates | MIDSCI | TP92024 | |
| NEBuffer 2 | New England Biolabs | #B7002S | |
| NEBuffer 2.1 | New England Biolabs | #B7202S | |
| BtgZI | New England Biolabs | #R0703L | |
| SacII | New England Biolabs | #R0157L | |
| HindIII-HF | New England Biolabs | #R3104S | |
| Afe1 | New England Biolabs | #R0652S | |
| Nhe1-HF | New England Biolabs | #R3131L | |
| T4 DNA Polymerase | New England Biolabs | #M0203S | |
| 500 mL Steritop bottle top filter unit | Millipore | SCGPU10RE | You can use any size that fits your needs |
| EGTA | Sigma | E4378-100G | |
| KCl | Sigma-Aldrich | P9333-500g | |
| CaCl2 | Sigma-Aldrich | C7902-500g | |
| MgCl2 | Sigma-Aldrich | M8266-100g | |
| K2HPO4 | Fisher | P288-500 | |
| HEPES | Sigma-Aldrich | H4034-500g | |
| pMK-U6 | Generated by the Muralidharan Lab | n/a | |
| pHA-glmS | Generated by the Muralidharan Lab | n/a | |
| pUF1-Cas9 | Gift from the Jose-Juan Lopez-Rubio Lab | Ghorbal et al. Nature Biotech 2014 | |
| Glucose | Sigma-Aldrich | G7021-1KG | |
| Sodium bicarbonate | Sigma-Aldrich | S5761-500G | |
| Sodium pyruvate | Sigma-Aldrich | P5280-100G | |
| Hypoxanthine | Sigma-Aldrich | H9636-25g | |
| Gentamicin Reagent | Gibco | 15710-064 | |
| Thymidine | Sigma-Aldrich | T1895-1G | |
| PL6-eGFP BSD | Generated by the Muralidharan Lab | ||
| Puf1-cas9 eGFP gRNA | Generated by the Muralidharan Lab | ||
| NucleoSpin Gel and PCR Clean-up | Macherey-Nagel | 740609.250 | |
| Albumax I | Life Technologies | N/A | You will want to try a few batches to find out what the parasites will grow in best |
| Human Red Blood Cells | Interstate Blood Bank, Inc | Email or call them directly for ordering | We typically use O+ blood |
| 3D7 parasite line | Available upon request | N/A | |
| Lysogeny Broth (LB) | Fisher | BP1426-2 | You can make your own, it is not necessary to use exactly this |
| Ampicilin | Fisher | BP1760-25 | We make a 1000X stock at 100mg/ml in water and store in the -20C |
| Ampicilin | Clonetech | R050A | |
| Anti-EF1alpha | Dr. Daniel Goldberg's Lab | Washington University in St. Louis | You can use your preferred loading control for western blots. This is just the one we use in our laboratory |
| Rat Anti-HA Clone 3F10, monoclonal | Made by Roche, sold by Sigma | 11867423001 | You can use your preferred anti-HA antibody |
| 0.6 mL tubes | Fisher | AB0350 | |
| Fisher HealthCare* PROTOCOL* Hema 3* Manual Staining System (Fixative+Solution I and II) | Fisher | 22-122-911 | You can also use giemsa stain |
| Fisherfines Premium Frosted Microscope Slides – Size: 3 x 1 in. | Fisher | 12-544-3 |
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Citar este artigo
Kudyba, H. M., Cobb, D. W., Florentin, A., Krakowiak, M., Muralidharan, V. CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum. J. Vis. Exp. (139), e57747, doi:10.3791/57747 (2018).