我们要感谢L. David Sibley和Asis Khan对这些协议所基于的原始出版物的贡献。这项工作由美国国家卫生研究院拨款AI108721资助。

The authors declare that they have no competing financial interests.

这些方案提出了几种方法，当组合时，可以鉴定弓形虫中的耐药性基因。两个特别是项目的组成部分，相对经验丰富的QTL定位方法和最近开发的CRISPR / Cas9基因编辑方法。 Lander和Botstein在1989年发表了有影响力的论文，展示了将遗传基因座与表型36相关联的QTL定位。最近在2012年，Dounda和Charpentier描述了在化脓链球菌 22中的CRISPR / Cas9编辑系统，被迅速适应为许多不同模型的遗传工具，包括弓形虫13，17 。两种方法在这里是有用的，其中QTL定位定义了使用基于WGS的SNP检测最终鉴定的含有耐药性突变的基因座，CRISPR / Cas9编辑提供了我<html>确认SNR1是正弦蛋白药物抗性基因。 最初开发的ME49-FUDR r X VAND-SNF r cross 8用于询问毒力表型19 ，但是亲本菌株也发生了另外的表型差异，其中因果基因未知，VAND亲本中的正辛酸抗性。这突出了十字架的一个好处，因为当观察到父母的其他表型差异时，它们可以重新利用。另外一种类型为2×3型的弓形体十字架是这种情况，其中通过绘制多种表型37,38,39,40 ，发现了涉及毒力的几个基因。迄今为止，已经描述了四种不同的弓形虫十字交叉，用于描绘负责表型的基因ss ="xref"&gt; 8,37,41,42，所有这些都有可能被重新用于绘制遗传基础未知的新表型。按照这些方法，已经对代表已知全球遗传多样性的62个弓形虫菌株的基因组进行了测序。可以从这个池中获得新的杂交，以获得不同有趣表型的菌株。夸耀了QTL映射的优点，需要说的是产生十字架不是一个小事。还有其他方法可用于鉴定因果基因。一个强大的技术使用化学诱变来产生可以筛选表型的突变体。为了找到致病基因，突变体可以用粘粒文库44进行补充，也可以使用基因组重排序方法找到因果突变45 。对于莫在这里，参见Coleman 等人的两篇JoVE文章。和Walwyn 等人概述了这些方法46,47 。 导致鉴定因果SNF r突变的许多步骤（方案2和3）依赖于可免费获得学术用途的软件进行的计算方法。提供每个步骤的详细命令，当运行正确的文件将允许用户重新创建必要的数据集以找到因果SNF r SNP。请记住，命令中的一些语法指的是可以修改为用户首选项的文件名或目录结构（$ PATH）。尽管这里给出的命令肯定不会耗尽分析QTL分析和基于WGS的SNP识别的交叉方式，但它们足够全面，可以重复本文所述的实验，并且应允许用户变为m<html>熟悉如何以一步一步的方式利用这些方法。 虽然QTL作图和基于WGS序列的SNP检测足以鉴定候选SNF r基因，但需要额外的实验来证实其在耐药性中的作用。这可以通过基因破坏或敲除技术令人信服地显示，这两种技术可以使用CRISPR / Cas9基因编辑实现。给出了使用CRISPR / Cas9产生indel突变或将转基因构建体插入到弓形虫中的靶基因的详细方法。 CRISPR / Cas9提供的靶向特异性提高了基因编辑与传统方法的效率。此外，这有效地提高了使用非实验室适应的菌株进行以前难以修改的遗传研究的可能性13 。即使在起步阶段，CRISPR / Cas9已被用于使基因中断lass ="xref"&gt; 2，13，17，18，48，49，标签基因17 ，并使得弓形虫中的基因敲除16,19,50,51,52,53,54有望成为有用的工具对于未来的许多其他研究。 SNR1失活导致正辛宁抗性的发现使得SNR1基因座成为转基因插入或遗传互补的有希望的位点。为了最大限度地利用CRISPR / Cas9介导的基因靶向来指导转基因整合到SNR1基因座中的成功，应该考虑以下几个方面红色在实验设计。首先，推荐将选择标记包括在转基因构建体中以增加菌株构建的效率。如果包括选择标记，则可以使用阳性和阴性选择，导致几乎100％的双选择的寄生虫是在GI1整合在SNR1基因座处的GOI转基因的。相比之下，如果转基因构建体不含有其他可选性状并且依赖于在SNR1基因座处的阴性选择，则成功转基因的效率在很大程度上取决于CRISPR质粒和转基因构建体的共转染效率。 第二，虽然CRISPR / Cas9介导的非同源DNA片段的位点特异性整合经常用于互补和转基因，但是应该注意的是，在这种情况下，不能保证插入的方向是可能的。这可能会造成问题ms到某些应用程序。例如，为了补充具有不同基因等位基因的突变体，难以确保所有等位基因以相同方向插入，并且取向的差异可能导致表达差异。对于这种类型的应用，推荐具有与SNR1基因座同源的DNA构建体，这将驱动适当的整合方向（ 图6 ）。 第三，在转染过程中，CRISPR质粒与转基因DNA分子之间的比例对成功转基因株系的构建至关重要。这个比例需要根据选择策略进行调整。推荐以下指南:1）如果转基因构建体含有耐药标记，相应的药物是用于产生转基因寄生虫的唯一选择， 即不使用正辛宁素，则建议的转基因构建体与CRISPR等离子体的摩尔比id为1:5。在这种情况下使用更多的CRISPR质粒增加接受转基因构建体的寄生虫也将接受CRISPR质粒的可能性，因此耐药性寄生虫更可能将标记插入CRISPR靶向位点。如果比例相反，接受转基因构建体的大多数寄生虫将不会得到CRISPR质粒。因此，绝大多数耐药寄生虫通过与CRISPR / CAS9介导的位点特异性插入不相关的随机整合获得转基因构建体。 2）如果转基因构建体含有耐药性标记，并且相应的药物与sinefungin一起使用以选择转基因寄生虫，则建议的转基因构建体与CRISPR质粒之间的摩尔比为1:1。这种策略提供了转基因株系施工效率最高。 3）如果转基因结构不包含可选择的制造商，则依赖于否定选择通过sinefungin单独获得转基因寄生虫，转基因构建体和CRISPR质粒之间的建议摩尔比为5:1。此设计的理由与上述第一条指导相同。由于在方案5中使用了乙胺嘧啶和正辛宁作选择，所以将DHFR * mini基因与靶向CRISPR质粒的SNR1之比设定为1:1。 总而言之，这里概述的方法具有不可能在识别SNR1 2的原始出版物中传达的细节水平。这些协议;具体来说，命令行语法，所使用的程序的顺序布局以及CRISPR / Cas9的使用应有助于未来确定新基因的表型。

宿主范围决定了寄生虫的流行程度。一些寄生虫具有非常具体的宿主要求，限制了它们被发现的区域，另一些是通才。一种这样的通才是弓形虫（ 弓形虫） 。这种寄生虫在世界各地被发现，因为它可以感染所有哺乳动物和许多鸟类。人类也很易受感染，估计全球大约有1/3的人口已被感染。幸运的是，强大的免疫反应通常控制寄生虫的生长，但是在免疫系统受损的情况下，寄生虫可以不加控制地生长并引起疾病，通常是脑炎。此外，如果先前未感染的妇女在怀孕期间感染，这种寄生虫可能会导致先天性疾病，因为它们缺乏免疫记忆，以迅速限制寄生虫的传播。另外，眼睛弓形体病的负担可导致视力丧失1 。对于这些reasons 弓形虫已经成为研究的焦点，由于为研究开发了许多分子方法，Apicomplexan寄生虫的模型。这里将讨论的两种方法是定量性状位点（QTL）映射和聚类定期间隔短回归重复（CRISPR）/ Cas9基因编辑。 QTL定位和CRISPR / Cas9编辑分别是以前研究中使用的正向和反向遗传方法，用于鉴定和/或表征弓形虫毒力基因。在这里，组合这些方法以鉴定和确认正弦蛋白抗性（SNF r ）基因，TgME49_290860及其直系同源物（注释为SNR1 ） 2的功能 。 虽然弓形虫可以感染多种中间宿主，但它必须通过并感染小肠上皮细胞以完成生命周期。猫是寄生虫的最终寄主产生外部阶段，并通过减数分裂进行遗传重组。进行QTL研究必须创建一个遗传交叉，而在弓形虫的情况下，这就意味着通过猫的两种不同的寄生虫种类，通过猫来表现出不同的寄生虫种类，即亲本污渍，以产生重组子代3 。在喂养猫之前，使亲本菌株对分离的药物具有抗性，以通过后代4的双重药物选择来更有效地鉴定重组体。为此，在弓形虫中使用了三种药物;尿嘧啶磷酸核糖转移酶（ UPRT ）为抗性基因5的氟脱氧核糖（FUDR），腺苷激酶（ AK ）为抗性基因6的阿糖胞苷（ARA）和抗性基因未知的正辛宁（SNF） 7 。几个遗传十字架已经对于弓形虫而言 ，仅使用全基因组测序（WGS） 8对ME49-FUDR r X VAND-SNF r交叉的24个后代进行基因分型。这使得通过使用该交叉作图和鉴定SNF抗性基因的可能性，因为通过药物敏感性VAND（VAND-SNF s ）株的化学诱变使VAND亲本被制成正辛宁抗性，并且使用VAND- SNF s菌株因此允许鉴定后代WGS和VAND-SNF参考基因组之间的所有多态性，包括遗传的父母VAND-SNF r突变，其使一些后代正辛酸抗性。 为了识别SNF r后代中的因果单核苷酸多态性（SNP），可以使用几种基于计算的开源资源来分析数据。为了创建ME49-FUDR r X VAND-SNF r通过REDHORSE软件套件9开发，使用父母和子代的WGS比对来准确检测基因交换的基因组位置。然后可以将该映射信息与表型数据（后代中的SNF r ）组合以格式化与R统计编程软件中的"qtl"包10兼容的数据集，其中可以运行QTL扫描以显示与表型。为了鉴定位于QTL基因座内的因果SNP，使用Bowtie 2比对程序11可以使用VarScan mpileup2snp变体呼叫者程序12来调用SNP，从而使后代的WGS读数可以单独对准正链霉素敏感性VAND基因组。使用这些SNP，然后可以扫描QTL基因座，存在于SNF r中但不存在的多态性在SNF 的后代。利用在基因编码区域鉴定的因子SNP，可以在SNF s株中进行候选SNF r基因的遗传修饰，以验证耐药性。 CRISPR / Cas9基因组编辑系统最近在Toxoplasma 13中成立，其中增加了用于探索这种寄生虫的复杂生物学的重要工具，特别是用于非实验室适应菌株的遗传研究。由于WT 弓形体细胞中非活性的非同源末端连接（NHEJ）活性，靶基因组修饰难以实现，因为外源引入的DNA以极高的频率14随机整合到基因组中。为了增加基因座特异性修饰的成功率，已经采用不同的方法来提高同源重组的效率和/或降低the NHEJ活动15,16 。其中一种方法是CRISPR / Cas9系统。与其他方法相比，CRISPR / Cas9系统在引入特定于站点的修改方面效率高，易于设计13,17,18 。此外，它可以用于任何弓形体，不需要额外修改寄生虫13,19。 CRISPR / Cas9系统起源于化脓性链球菌的适应性免疫系统，其用于捍卫移植遗传因子（如噬菌体20,21,22）的入侵。该系统利用RNA引导的DNA核酸内切酶Cas9将双链DNA断裂（DSB）引入目标，随后repa由容易出错的NHEJ通过短的indel突变来灭活靶基因，或通过同源性定向重组来改变目标基因座，如同设计的23,24 。靶特异性由称为单导向RNA（gRNA）的小RNA分子确定，其包含与靶DNA22具有100％同源性的单独设计的20nt序列。 gRNA分子还含有Cas9识别的鉴定指导核酸酶到靶位点，其中包括一个特殊的Protospacer相邻基序（PAM，序列为&#39;NGG&#39;） 25,26 。因此，gRNA分子和PAM序列共同作用来确定基因组中的Cas9切割位点。人们可以很容易地将gRNA序列改变成不同的位点进行切割。 当CRISPR / Cas9系统首次在Toxop开发时使用表达Cas9核酸酶和gRNA分子的单个质粒的拉斯马引导DSB在靶向位点13,17。已经表明，CRISPR / Cas9系统不仅通过同源重组，而且外源DNA的非同源整合，大大增加了位点特异性基因组修饰的效率。它在含有NHEJ活性的野生型菌株中进行。因此，该系统可用于基本上任何弓形体菌株进行有效的基因组编辑。在典型的实验中，目标特异性CRISPR质粒和用于修饰靶标的DNA片段共转染入寄生虫。如果用于修饰靶标的DNA片段含有与靶基因座的同源序列，则可以使用同源重组来修复由CRISPR / Cas9引入的DSB，以允许目标物的精确修饰。另一方面，如果intDNA片段不含同源序列，仍可以整合入CRISPR / Cas9靶向位点。后者通常用于通过插入选择性标记来破坏基因，或者在允许阴性选择的位点上补体突变体13 。这里的SNR1基因座作为一个例子来说明CRISPR / Cas9如何用于基因破坏和转基因寄生虫的产生。

<table><tbody><tr><td>T25 flasks</td><td>Corning</td><td>430639</td><td></td></tr><tr><td>Human Foreskin Fibroblasts (HFF)</td><td>ATCC</td><td>SCRC-1041</td><td></td></tr><tr><td>&lt;em&gt;T. gondii&lt;/em&gt; ME49 strain</td><td>ATCC</td><td>50840</td><td></td></tr><tr><td>&lt;em&gt;T. gondii&lt;/em&gt;VAND strain</td><td>ATCC</td><td>PRA-344</td><td></td></tr><tr><td>DMEM (No Sodium Bicarbonate)</td><td>Life Sciences</td><td>12800017</td><td></td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma Aldrich</td><td>S5761</td><td></td></tr><tr><td>HEPES</td><td>Sigma Aldrich</td><td>H3375</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS) Premium Grade</td><td>VWR International</td><td>97068-085</td><td></td></tr><tr><td>L-Glutamine&amp;nbsp; 200 mM</td><td>Sigma Aldrich</td><td>G7513</td><td></td></tr><tr><td>Gentamicin (10 mg/mL)</td><td>Life Technologies&amp;nbsp;</td><td>15710064</td><td></td></tr><tr><td>Cell Scraper for Flasks</td><td>VWR International</td><td>10062-904</td><td></td></tr><tr><td>Syringe 10 mL</td><td>BD</td><td>309604</td><td></td></tr><tr><td>Blunt needles 22 G x 1&quot;</td><td>BRICO Products</td><td>BN2210</td><td></td></tr><tr><td>Nuclepore Filter 3.0 &amp;micro;m, 25 mm</td><td>GE Healthcare</td><td>110612</td><td></td></tr><tr><td>Swin-Lok Filter Holder 25 mm</td><td>GE Healthcare</td><td>420200</td><td></td></tr><tr><td>Hemacytometer</td><td>Propper MFG</td><td>90001</td><td></td></tr><tr><td>Sinefungin</td><td>Enzo Life Sciences</td><td>380-070-M001</td><td></td></tr><tr><td>R&amp;nbsp;</td><td>The R Foundation</td><td>https://www.r-project.org/</td><td></td></tr><tr><td>J/qtl</td><td>The Churchill Group</td><td>http://churchill.jax.org/software/jqtl.shtml</td><td></td></tr><tr><td>Bowtie2</td><td>John Hopkins University</td><td>http://bowtie-bio.sourceforge.net/bowtie2/index.shtml</td><td></td></tr><tr><td>NCBI SRA Toolkit</td><td>NCBI</td><td>http://www.ncbi.nlm.nih.gov/Traces/sra/?view=toolkit_doc</td><td></td></tr><tr><td>SAMtools</td><td>Wellcome Trust Sanger Institute</td><td>http://www.htslib.org/</td><td></td></tr><tr><td>VarScan</td><td>Washington University, St Louis</td><td>http://varscan.sourceforge.net/</td><td></td></tr><tr><td>MUMmer</td><td>JCVI &amp;amp; Univ Hamburg</td><td>http://mummer.sourceforge.net/</td><td></td></tr><tr><td>pSAG1::CAS9-U6::sgUPRT</td><td>Addgene</td><td>Plasmid #54467</td><td>&lt;em&gt;T. gondii&lt;/em&gt; CRISPR plasmid to cut the UPRT gene</td></tr><tr><td>pSAG1::CAS9-U6::sg290860-6</td><td>Addgene</td><td>Plasmid #59855</td><td>&lt;em&gt;T. gondii&lt;/em&gt; CRISPR plasmid to cut the TG*_290860 SNR1 gene</td></tr><tr><td>pUPRT::DHFR-D</td><td>Addgene</td><td>Plasmid #58528</td><td>Template for &lt;em&gt;DHFR*&lt;/em&gt; mini gene</td></tr><tr><td>Q5 Site-Directed Mutagenesis Kit&amp;nbsp;</td><td>New England Biolabs</td><td>E0552S</td><td></td></tr><tr><td>QIAprep Spin Miniprep Kit</td><td>QIAGEN</td><td>27106</td><td></td></tr><tr><td>NanoDrop 2000</td><td>Thermo Scientific</td><td>ND-2000</td><td></td></tr><tr><td>LB Broth</td><td>Fisher Scientific</td><td>DF0446-07-5</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma Aldrich</td><td>P9541</td><td></td></tr><tr><td>Calcium chloride</td><td>Sigma Aldrich</td><td>746495</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma Aldrich</td><td>P9791</td><td></td></tr><tr><td>Potassium phosphate dibasic</td><td>Sigma Aldrich</td><td>P5504</td><td></td></tr><tr><td>EDTA</td><td>Sigma Aldrich</td><td>E5134</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma Aldrich</td><td>M1028</td><td></td></tr><tr><td>Adenosine triposhpate (ATP)</td><td>Sigma Aldrich</td><td>A6419</td><td></td></tr><tr><td>L-glutathione (GSH)</td><td>Sigma Aldrich</td><td>G4251</td><td></td></tr><tr><td>ECM 830 Electroporation System</td><td>BTX</td><td>45-0002</td><td></td></tr><tr><td>Electroporation Cuvette&amp;nbsp; 4 mm</td><td>Harvard Apparatus</td><td>45-0126</td><td></td></tr><tr><td>Crytal Violet</td><td>Alfa Aesar</td><td>B21932-14</td><td></td></tr><tr><td>6-well TC plate</td><td>Corning</td><td>353046</td><td></td></tr><tr><td>24-well TC plate</td><td>Corning</td><td>353935</td><td></td></tr><tr><td>Cover glass 12 mm round</td><td>VWR International</td><td>89015-724</td><td></td></tr><tr><td>96-well TC plate</td><td>Corning</td><td>353075</td><td></td></tr><tr><td>Gibson Assembly Cloning Kit (Multi-fragment)</td><td>New England Biolabs</td><td>E5510S</td><td></td></tr><tr><td>Q5 High-Fidelity Polymerase&amp;nbsp;</td><td>New England Biolabs</td><td>M0491S</td><td></td></tr><tr><td>Pyrimethamine</td><td>TCI AMERICA</td><td>P2037-1G</td><td>Use cuation when using pyrimethamine resistant parasites - see Protocol</td></tr></tbody></table>

qtl mapping and crispr/cas9 editing to identify a drug resistance gene in toxoplasma gondii

科学知识与现有的技术和方法有着固有的联系。本文将介绍两种允许鉴别和鉴定弓形虫弓形虫弓形虫药物抗性基因的方法，该方法采用基于全基因组序列（WGS）的遗传图谱进行定量性状位点（QTL）定位及其方法的集群定期间隔短回归重复（CRISPR）/基于Cas9的基因编辑。 QTL定位的方法可以测试基因组区域和表型之间是否存在相关性。需要两个数据集来进行QTL扫描，这是基于在该交叉的每个后代中评估的重组交叉的后代和可量化表型的遗传图谱。然后将这些数据集格式化为与生成QTL扫描以识别与表型相关的显着位点的R / qtl软件兼容。虽然这可以大大缩小搜索范围可能的候选人的遗传，QTL跨越包含许多基因的区域，需要识别因果基因。确定后代的WGS对于鉴定基因水平的致病药物耐药突变是至关重要的。一旦鉴定，候选突变可以通过药物敏感寄生虫的遗传操作来验证。遗传修饰弓形虫最简单有效的方法是CRISPR / Cas9系统。该系统由仅在单个质粒上编码的两个组分组成，包含与基因组靶标互补的20bp序列的单个引导RNA（gRNA）和在靶标上产生双链DNA断裂（DSB）的Cas9内切核酸酶，其修复允许插入或删除断点位置周围的序列。本文提供详细的协议，使用基于CRISPR / Cas9的基因组编辑工具来验证负责丝氨酸蛋白酶抗性的基因并构建转基因寄生虫。

本文概述了可以连续使用以鉴定负责耐药性的基因的几种方法（ 图1 ）。评估ME49-FUDR r X VAND-SNF r交叉的24个后代，如方案1所述对药物sinefungin的抗性进行评估。使用遗传图谱和后代的SNF r表型，在R / qtl - 协议2（ 图2 ）。这导致在染色体IX上跨越约1Mbp的一个显着的峰。在这个地区，因果突变位于。 为了鉴定因果突变，使用Bowtie2 - Protocol 3.1将来自后代的WGS读数与VAND-SNF参考基因组进行比对，使用VarScan mpileup2snp - Protocol 3.2调用SNPs，并使用MUMmer鉴定了VAND基因组中的QTL位点 - Protoc醇3.3。提取QTL基因座内的后代SNP并扫描其中SNF r后代具有SNP并且SNF不存在的模式，这是可行的，因为通过与VAND-SNF参考基因组比较获得后代SNP（ 图3 ） 。只有一个SNP匹配这种模式，导致名为SNR1的推定的氨基酸转运蛋白基因中的早期终止密码子。 使用CRISPR / Cas9系统确认SNR1是SNF r抗性基因。制备了用于革兰氏阴性菌基因编辑的新的CRISPR / Cas9质粒，其含有靶向在后代 - 方案4中鉴定的SNF r突变附近的SNR1基因的gRNA（ 图4A ）。将针对CRISPR / Cas9质粒的SNR1电穿孔入SNF s WT寄生虫菌株，当培养时获得抗性突变体在0.3μM正弦蛋白中。当用针对CRISPR / Cas9质粒的UPRT电穿孔时，没有获得SNF r寄生虫。克隆了几种SNF r CRISPR突变体，并对SNR1 gRNA靶标周围的区域进行了测序。每个突变体具有破坏SNR1基因的编码序列的indel （ 图4B ）。该方法也可用于通过NHEJ（ 图5）或HR（ 图6 ） - 补充文件2将靶向构建体插入SNR1基因座。 <img alt="图1" src="/files/ftp_upload/55185/55185fig1.jpg" /> 图1:协议中概述的实验示意图工作流程。使用ME49-FUDR r X VAND-SNF r交叉鉴定和确认SNF r基因的主要步骤参照t中列出的适当方案他的文章。 <a href="http://ecsource.jove.com/files/ftp_upload/55185/55185fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" src="/files/ftp_upload/55185/55185fig2.jpg" /> 图2:R / qtl在SNF r表型上运行QTL扫描的命令。协议2中概述的命令使用qtl包在R中运行。显示代表性的命令和情节。 <a href="http://ecsource.jove.com/files/ftp_upload/55185/55185fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" src="/files/ftp_upload/55185/55185fig3.jpg" /> 图3:因果SNP的位置。使用Bowtie 2将后代WGS读数与VAND-SNF参考基因组进行比对，使用VarScan调用SNP，并对应<html>用MUMmer鉴定的VAND QTL位点。位于QTL基因座内的SNP被导入到电子表格中，并鉴定了因果SNP。 SNF r后代（黄色），SNF后代（绿色），SNF r SNP（红色）（参见数据文件3 ）。 <a href="http://ecsource.jove.com/files/ftp_upload/55185/55185fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图4" src="/files/ftp_upload/55185/55185fig4.jpg" /> 图4:使用CRISPR / Cas9将SNR1确认为SNF r基因。 （ A ）CRISPR / Cas9在SNR1基因座中诱导吲哚突变（绿色）。 （ B ）由两种不同的SNR1特异性CRISPR质粒（分别为C5和C6）引起的SNR1中的代表性插入片段。 RH是WT菌株，C5和C6是SNF r突变体。 （B取自参考文献s ="xref"&gt; 2）。 <a href="http://ecsource.jove.com/files/ftp_upload/55185/55185fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图5" src="/files/ftp_upload/55185/55185fig5.jpg" /> 图5. 使用CRISPR / Cas9的插入诱变。 （ A ）通过CRISPR / Cas9介导的位点特异性整合将补体或转基因基因插入到SNR1基因座中。插入的DHFR * mini基因和用于鉴定的引物，F1 / 2和R1 / 2的两个可能取向代表用于诊断PCR的寡核苷酸的引发位点。 （ B ）将一个snr1 :: DHFR *克隆，RH的诊断PCR用作WT对照。包括GRA1p的PCR作为对照，检查基因组DNA的质量作为模板。星号表示具有非特异性谱带的泳道（图5B取自参考文献ce 2 ）。 <a href="http://ecsource.jove.com/files/ftp_upload/55185/55185fig5large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图6" src="/files/ftp_upload/55185/55185fig6.jpg" /> 图6: 使用CRISPR / Cas9的基因敲除。 CRISPR / Cas9介导的弓形虫同源基因替代的经典设计。在这种情况下，插入的转基因的方向是固定的。 F1 / R1，F2 / R2和F3 / R3代表用于诊断PCR的寡核苷酸的引物位点。 <a href="http://ecsource.jove.com/files/ftp_upload/55185/55185fig6large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 数据文件1:ME49-FUDR r X VAND-SNF r 交叉文件f或用于R / qtl，"csv"格式。一个.csv文件，可以使用format ="csv"选项加载到R / qtl中。 <a href="http://ecsource.jove.com/files/ftp_upload/55185/Data_File_1.csv" target="_blank">请点击这里下载此文件。</a> 数据文件2:（chrid txt，基因型txt，markerpos.txt，mname.txt，phenonames.txt和phenos.txt）。 ME49-FUDR r X VAND-SNF r 用于R / qtl，"gary"格式的Cross文件。可以使用format ="gary"选项加载到R / qtl中的六个.txt文件。 <a href="http://ecsource.jove.com/files/ftp_upload/55185/Data_File_2.zip" target="_blank">请点击这里下载此文件。</a> 数据文件3.跨SNF的后代SNP的电子表格 r <strong&gt; QTL轨迹。包含一个与后代SNP的工作表，第二个工作表与父母的表型数据和十字架的后代。 <a href="http://ecsource.jove.com/files/ftp_upload/55185/Data_File_3.xlsx" target="_blank">请点击这里下载此文件。</a> 补充文件1:附加协议详细信息。 <a href="http://ecsource.jove.com/files/ftp_upload/55185/Supplementary_File_1_-_101916.docx" target="_blank">请点击这里下载此文件。</a> 补充文件2:方案5 - 利用 遗传互补或转基因菌株构建 的 SNR1位点 处的负选择 。 <a href="http://ecsource.jove.com/files/ftp_upload/55185/Supplementary_File_2_-_101916.docx" target="_blank">请点击这里下载此文件。</a></a>

Watch this Scientific Journal Video about QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii at JoVE.com

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

详细介绍了如何使用基于全基因组序列的遗传图谱进行QTL定位，以鉴定弓形虫中的药物抗性基因，以及如何使用有效编辑基因组靶标的CRISPR / Cas9系统进行验证，在这种情况下耐药基因。

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 ...

qtl-mapping-crisprcas9-editing-to-identify-drug-resistance-gene

Research

JoVE Journal

Genetics

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

16.2K 次观看.  Huazhong Agricultural University. 详细介绍了如何使用基于全基因组序列的遗传图谱进行QTL定位，以鉴定弓形虫中的药物抗性基因，以及如何使用有效编辑基因组靶标的CRISPR / Cas9系统进行验证，在这种情况下耐药基因。 

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

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

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide RNA (sgRNA) that confers specificity, the Cas9 protein cleaves both DNA strands at the targeted locus. The DNA break can trigger either non-homologous end joining (NHEJ) or homology directed repair (HDR). NHEJ can introduce small deletions or insertions which lead to frame-shift mutations, while HDR allows for larger and more precise perturbations. Here, we present protocols for generating knockout cell lines by coupling established CRISPR/Cas9 methods with two options for downstream selection/screening. The NHEJ approach uses a single sgRNA cut site and selection-independent screening, where protein production is assessed by dot immunoblot in a high-throughput manner. The HDR approach uses two sgRNA cut sites that span the gene of interest. Together with a provided HDR template, this method can achieve deletion of tens of kb, aided by the inserted selectable resistance marker. The appropriate applications and advantages of each method are discussed.

Recent advances in the ability to genetically manipulate somatic cell lines hold great potential for basic and applied research. Here, we present two approaches for CRISPR/Cas9 generated knockout production and screening in mammalian cell lines, with and without the use of selectable markers.

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 ...

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

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site&#8212;in essence, analyzing onsite mutagenesis.

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

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

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical regions, is caused by infection with Plasmodium parasites. The development of more effective drugs to combat malaria can be accelerated by improving our understanding of the biology of this complex parasite. Genetic manipulation of these parasites is key to understanding their biology; however, historically the genome of P. falciparum has been difficult to manipulate. Recently, CRISPR/Cas9 genome editing has been utilized in malaria parasites, allowing for easier protein tagging, generation of conditional protein knockdowns, and deletion of genes. CRISPR/Cas9 genome editing has proven to be a powerful tool for advancing the field of malaria research. Here, we describe a CRISPR/Cas9 method for generating glmS-based conditional knockdown mutants in P. falciparum. This method is highly adaptable to other types of genetic manipulations, including protein tagging and gene knockouts.

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

We describe a method for generating glmS-based conditional knockdown mutants in Plasmodium falciparum using CRISPR/Cas9 genome editing.

CRISPR/Cas9 基因编辑制作人类疟疾寄生虫的条件突变体

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

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth defects upon congenital infection. The lytic replication cycle is characterized by three stages: 1. active invasion of a nucleated host cell; 2. replication inside the host cell; 3. active egress from the host cell. The mechanism of egress is increasingly being appreciated as a unique, highly regulated process, which is still poorly understood at the molecular level. The signaling pathways underlying egress have been characterized through the use of pharmacological agents acting on different aspects of the pathways1-5. As such, several independent triggers of egress have been identified which all converge on the release of intracellular Ca2+, a signal that is also critical for host cell invasion6-8. This insight informed a candidate gene approach which led to the identification of plant like calcium dependent protein kinase (CDPK) involved in egress9. In addition, several recent breakthroughs in understanding egress have been made using (chemical) genetic approaches10-12. To combine the wealth of pharmacological information with the increasing genetic accessibility of Toxoplasma we recently established a screen permitting the enrichment for parasite mutants with a defect in host cell egress13. Although chemical mutagenesis using N-ethyl-N-nitrosourea (ENU) or ethyl methanesulfonate (EMS) has been used for decades in the study of Toxoplasma biology11,14,15, only recently has genetic mapping of mutations underlying the phenotypes become routine16-18. Furthermore, by generating temperature-sensitive mutants, essential processes can be dissected and the underlying genes directly identified. These mutants behave as wild-type under the permissive temperature (35 &deg;C), but fail to proliferate at the restrictive temperature (40 &deg;C) as a result of the mutation in question. Here we illustrate a new phenotypic screening method to isolate mutants with a temperature-sensitive egress phenotype13. The challenge for egress screens is to separate egressed from non-egressed parasites, which is complicated by fast re-invasion and general stickiness of the parasites to host cells. A previously established egress screen was based on a cumbersome series of biotinylation steps to separate intracellular from extracellular parasites11. This method also did not generate conditional mutants resulting in weak phenotypes. The method described here overcomes the strong attachment of egressing parasites by including a glycan competitor, dextran sulfate (DS), that prevents parasites from sticking to the host cell19. Moreover, extracellular parasites are specifically killed off by pyrrolidine dithiocarbamate (PDTC), which leaves intracellular parasites unharmed20. Therefore, with a new phenotypic screen to specifically isolate parasite mutants with defects in induced egress, the power of genetics can now be fully deployed to unravel the molecular mechanisms underlying host cell egress.

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

Forward genetics is a powerful method to unravel the molecular level of how Toxoplasma egresses from its host cell. Protocols are provided to chemically mutagenize parasites, enrich for mutants with defects in induced egress, and validate the phenotype of cloned mutants.

遗传筛选隔离弓形虫宿主细胞的出口突变

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth ...

免疫与感染

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

Trypanosoma cruzi is a pathogenic protozoan parasite that causes Chagas&#8217; disease mainly in Latin America. In order to identify a novel drug target against T. cruzi, it is important to validate the essentiality of the target gene in the mammalian stage of the parasite, the amastigote. Amastigotes of T. cruzi replicate inside the host cell; thus, it is difficult to conduct a knockout experiment without going through other developmental stages. Recently, our group reported a growth condition in which the amastigote can replicate axenically for up to 10 days without losing its amastigote-like properties. By using this temporal axenic amastigote culture, we successfully introduced gRNAs directly into the Cas9-expressing amastigote to cause gene knockouts and analyzed their phenotypes exclusively in the amastigote stage. In this report, we describe a detailed protocol to produce in vitro derived extracellular amastigotes, and to utilize the axenic culture in a CRISPR/Cas9-mediated knockout experiment. The growth phenotype of knockout amastigotes can be evaluated either by cell counts of the axenic culture, or by replication of intracellular amastigote after host cell invasion. This method bypasses the parasite stage differentiation normally involved in producing a transgenic or a knockout amastigote. Utilization of the temporal axenic amastigote culture has the potential to expand the experimental freedom of stage-specific studies in T. cruzi.

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

Here, we describe a protocol to introduce a gene knockout into the extracellular amastigote of Trypanosoma cruzi, using the CRISPR/Cas9 system. The growth phenotype can be followed up either by cell counting of axenic amastigote culture or by proliferation of intracellular amastigotes after host cell invasion.

使用CRISPR/Cas9系统将基因挖空直接引入锥虫瘤的肌瘤阶段

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

Genetic Manipulation in &Delta;ku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination.
Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii by deleting the gene encoding the KU80 protein1,2. The &Delta;ku80 strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro and in vivo and exhibit essentially a 100% frequency of homologous recombination. The &Delta;ku80 strains make functional genomic studies feasible on the single gene as well as on the genome scale1-4.
Here, we report methods for using type I and type II &Delta;ku80&Delta;hxgprt strains to advance gene targeting approaches in T. gondii. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) selectable marker. The described gene targeting protocol can be used in a variety of ways in &Delta;ku80 strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii and related significant human pathogens that cause malaria (Plasmodium sp.) and cryptosporidiosis (Cryptosporidium).

Here we report a method for using type I and type II &Delta;ku80 strains of Toxoplasma gondii to efficiently generate targeted gene deletions and gene replacements for functional genomic analysis.

遗传操纵<em&gt;Δku80</em&gt;株功能基因组分析<em&gt;弓形虫</em

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene ...

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-&#947;-Dependent Cell Autonomous Immunity

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within virtually any warm blooded vertebrate cell type. During parasite invasion of a host cell, the parasite creates a parasitophorous vacuole (PV) that originates from the host cell membrane independent of phagocytosis within which the parasite replicates. While IFN-dependent-innate and cell mediated immunity is important for eventual control of infection, innate immune cells, including neutrophils, monocytes and dendritic cells, can also serve as vehicles for systemic dissemination of the parasite early in infection. An approach is described that utilizes the host innate immune response, in this case macrophages, in a forward genetic screen to identify parasite mutants with a fitness defect in infected macrophages following activation but normal invasion and replication in na&#239;ve macrophages. Thus, the screen isolates parasite mutants that have a specific defect in their ability to resist the effects of macrophage activation. The paper describes two broad phenotypes of mutant parasites following activation of infected macrophages: parasite stasis versus parasite degradation, often in amorphous vacuoles. The parasite mutants are then analyzed to identify the responsible parasite genes specifically important for resistance to induced mediators of cell autonomous immunity. The paper presents a general approach for the forward genetics screen that, in theory, can be modified to target parasite genes important for resistance to specific antimicrobial mediators. It also describes an approach to evaluate the specific macrophage antimicrobial mediators to which the parasite mutant is susceptible. Activation of infected macrophages can also promote parasite differentiation from the tachyzoite to bradyzoite stage that maintains chronic infection. Therefore, methodology is presented to evaluate the importance of the identified parasite gene to establishment of chronic infection.

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-&#947;-Dependent Cell Autonomous Immunity

Forward genetics is a powerful approach to identify genes in intracellular pathogens important for resistance to cell autonomous immunity. The current approach uses innate immune cells, specifically macrophages, to identify novel Toxoplasma gondii genes important for immune evasion.

正向遗传学屏风使用巨噬细胞识别<em&gt;弓形虫</em&gt;基因的重要的抗IFN-γ依赖性细胞免疫自主

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within ...

Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay

Toxoplasma gondii is a protozoan pathogen that widely affects the human population. The current antibiotics used for treating clinical toxoplasmosis are limited. In addition, they exhibit adverse side effects in certain groups of people. Therefore, discovery of novel therapeutics for clinical toxoplasmosis is imperative. The first step of novel antibiotic development is to identify chemical compounds showing high efficacy in inhibition of parasite growth using a high throughput screening strategy. As an obligate intracellular pathogen, Toxoplasma can only replicate within host cells, which prohibits the use of optical absorbance measurements as a quick indicator of growth. Presented here is a detailed protocol for a luciferase-based growth assay. As an example, this method is used to calculate the doubling time of wild-type Toxoplasma parasites and measure the efficacy of morpholinurea-leucyl-homophenyl-vinyl sulfone phenyl (LHVS, a cysteine protease-targeting compound) regarding inhibition of parasite intracellular growth. Also described, is a CRISPR-Cas9-based gene deletion protocol in Toxoplasma using 50 bp homologous regions for homology-dependent recombination (HDR). By quantifying the inhibition efficacies of LHVS in wild-type and TgCPL (Toxoplasma cathepsin L-like protease)-deficient parasites, it is shown that LHVS inhibits wild-type parasite growth more efficiently than &#916;cpl growth, suggesting that TgCPL is a target that LHVS binds to in Toxoplasma. The high sensitivity and easy operation of this luciferase-based growth assay make it suitable for monitoring Toxoplasma proliferation and evaluating drug efficacy in a high throughput manner.

Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay

Presented here is a protocol to evaluate the inhibition efficacy of chemical compounds against in vitro intracellular growth of Toxoplasma gondii using a luciferase-based growth assay. The technique is used to confirm inhibition specificity by genetic deletion of the corresponding target gene. The inhibition of LHVS against TgCPL protease is evaluated as an example.

使用基于路西法抹去的生长测定，确定化学抑制剂对细胞内弓形体贡迪生长的抑制效率

Toxoplasma gondii is a protozoan pathogen that widely affects the human population. The current antibiotics used for treating clinical toxoplasmosis are ...

<meta charset="utf8"></head><body>鉴定疟疾寄生虫中的激酶重新布线:一种化学遗传学方法

Understanding the Development of Compensatory Pathways in a Mutant Malaria Parasite Harbouring Hypomorphic Allele of Plant-Like Kinases

One of the mechanisms for subverting the effect of drugs by&#160;the malaria parasite is through rewiring of its transcriptome. The effect is more pronounced for target genes belonging to the multigene family. Plasmodium falciparum protein kinases belonging to the CDPK family are essential for blood stage development. As such, CDPKs are considered good targets for the development of anti-malarial compounds. The chemical genetics approach has been historically used to elucidate the function of protein kinases in higher eukaryotes. It requires the substitution of gatekeeper residue for another amino acid with a different side chain through genetic manipulation. Amino acid substitution at the gatekeeper position modulates the activity of a protein kinase and changes its susceptibility to a specific class of compounds known as bumped kinase inhibitors (BKIs) that help in the functional identification of the target gene. Here, we have exploited the chemical genetics approach to understand compensatory mechanisms evolved by a mutant parasite harboring a hypomorphic allele of cdpk1. Overall, our approach helps in identifying compensatory pathways that may be simultaneously targeted to prevent the development of drug resistance against individual kinases.

<meta charset="utf8"></head><body>作者聚焦:鉴定含有必需蛋白激酶亚型等位基因的疟疾寄生虫中的代偿途径

Chemical genetics involves the substitution of a gatekeeper residue with an amino acid containing a different side chain at the target locus. Here, we have generated a mutant parasite containing a hypomorphic allele of cdpk1 and identified compensatory pathways adopted by the parasite in the mutant background.

QTL定位和CRISPR / Cas9编辑识别抗药性基因

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