CRISPR-Cas9-based genome-wide screening of Toxoplasma gondii.
CRISPR-Cas9-based genome-wide screening of Toxoplasma gondii.
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DOI：
10.1038/nprot.2017.131
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发表时间：
2018-01
期刊：
影响因子：
14.8
通讯作者：
Lourido S
中科院分区：
文献类型：
--
作者：
Sidik SM;Huet D;Lourido S
顶复门寄生虫会导致人类和牲畜显着发病和死亡，这凸显了对其分子生物学有更深入了解的必要性。虽然用于产生靶向基因破坏的技术早已可用于顶端复合物，但此类方法不容易扩展到整个基因组。我们最近使用 CRISPR/Cas9 以混合形式破坏弓形虫中的所有核蛋白编码基因。该方法依赖于将指导 RNA 文库转染到组成型表达 Cas9 的寄生虫中。在这里，我们介绍了这种筛选的完整工作流程，包括指导RNA文库的制备、受体菌株的生长和测试、突变体群体的产生、筛选的培养条件、基因组DNA文库的制备、指导RNA位点的下一代测序以及检测赋予适应性的基因的分析。该方法可用于研究培养条件如何影响寄生虫的遗传需求，这将使研究其代谢需求、宿主特异性和耐药机制成为可能。此外，通过操纵进行筛选的背景，研究人员将能够研究遗传相互作用，这可能会发现寄生虫基因组中的冗余或上位性。使用这种方法，经过大约一个月的准备后，全基因组筛选及其分析可以在三周内完成，使其成为揭示顶复门寄生虫中功能重要基因的强大工具。
Apicomplexan parasites cause significant morbidity and mortality in humans and livestock, highlighting the need for a deeper understanding of their molecular biology. While techniques for generating targeted gene disruptions have long been available for apicomplexans, such methods are not readily scalable to the entire genome. We recently used CRISPR/Cas9 to disrupt all nuclear protein-coding genes in Toxoplasma gondii using a pooled format. The method relies on transfecting a guide RNA library into parasites constitutively expressing Cas9. Here, we present the complete workflow of such a screen, including preparation of the guide RNA library, growth and testing of the recipient strain, generation of the mutant population, culture conditions for the screen, preparation of genomic DNA libraries, next-generation sequencing of the guide RNA loci, and analysis to detect fitness-conferring genes. This method can be deployed to study how culture conditions affect the parasite’s genetic requirements, which will enable studies of their metabolic needs, host-specificity, and drug-resistance mechanisms. Additionally, by manipulating the background in which the screen is performed, researchers will be able to investigate genetic interactions, which may uncover redundancy or epistasis in the parasite genome. Using this method, a genome-wide screen and its analysis can be completed in three weeks after approximately one month of preparation, making it a powerful tool to uncover functionally important genes in apicomplexan parasites.
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DOI：
10.1126/science.1165771
发表时间：
2008-12-19
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Marraffini LA;Sontheimer EJ
通讯作者：
Sontheimer EJ
影响因子：
46.9
作者：
Doench JG;Fusi N;Sullender M;Hegde M;Vaimberg EW;Donovan KF;Smith I;Tothova Z;Wilen C;Orchard R;Virgin HW;Listgarten J;Root DE
通讯作者：
Root DE
影响因子：
64.5
作者：
Hsu PD;Lander ES;Zhang F
通讯作者：
Zhang F
影响因子：
48
作者：
Gibson, Daniel G.;Young, Lei;Smith, Hamilton O.
通讯作者：
Smith, Hamilton O.
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle