Apicomplexan parasites, such as Toxoplasma gondii, cause extensive morbidity and mortality in humans and livestock, highlighting the need for a deeper understanding of their molecular biology. Although techniques for the generation of 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 T. gondii using a pooled format. The method relies on transfection of 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 repertoire of genes needed by parasites, which will enable studies of their metabolic needs, host specificity, and drug-resistance mechanisms. In addition, by manipulating the background in which the screen is performed, researchers will be able to investigate genetic interactions, which may help uncover redundancy or epistasis in the parasite genome. Using this method, a genome-wide screen and its analysis can be completed in 3 weeks, after ∼1 month of preparation to generate the library and grow the cells needed, making it a powerful tool for uncovering functionally important genes in apicomplexan parasites.

中文翻译：

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基于 CRISPR-Cas9 的弓形虫全基因组筛选。


弓形虫等顶复门寄生虫会导致人类和牲畜广泛发病和死亡，这凸显了对其分子生物学有更深入了解的必要性。尽管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。该方法依赖于将引导 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、下一代测序指导RNA位点的分析，以及检测适应性赋予基因的分析。这种方法可用于研究培养条件如何影响寄生虫所需的基因库，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，这可能有助于发现寄生虫基因组中的冗余或上位性。使用这种方法，经过约 1 个月的准备以生成文库并培养所需细胞后，可在 3 周内完成全基因组筛选及其分析，使其成为发现顶复门寄生虫中功能重要基因的强大工具。