Winiger Rahel R, Hehl Adrian B
Laboratory of Molecular Parasitology, Institute of Parasitology, University of Zurich, Switzerland.
J Biol Methods. 2020 Dec 19;7(4):e140. doi: 10.14440/jbm.2020.343. eCollection 2020.
() and () are closely related coccidian parasites belonging to the phylum Apicomplexa, which comprises many other important pathogens of humans and livestock. is considered a model organism for studying the cell biology of Apicomplexa mainly due to the ease of propagation in diverse host cells and the availability of a wide range of genetic tools. Conversely, culture systems currently exist only for the acute phase of infection, and genetic manipulation has proven much more challenging. In recent years, the targeted editing of chromosomal DNA by the programmable CRISPR-associated (Cas)9 enzyme has greatly improved the scope and accuracy of genetic manipulation in and related parasites but is still lagging in . The CRISPR/Cas9 technology enables the introduction of single point and insertion/deletion mutations, precise integration of in-frame epitope tags, and deletions of genes at reduced time and cost compared to previous methods. Current protocols for CRISPR-mediated genome editing in rely on either constitutive or transient expression of Cas9 as well as target specific sgRNAs encoded separately or together on transfected plasmid vectors. Constitutively expressed Cas9 carries the risk of toxicity, whilst the transient approach is laborious and error-prone. Here we present a protocol for plasmid vector-independent genome-editing using chemically synthesized and modified sgRNAs. This protocol allows for rapid and cost-effective generation of mutant cell lines of and B. .
（）和（）是密切相关的球虫寄生虫，属于顶复门，该门包含许多其他重要的人类和牲畜病原体。（）被认为是研究顶复门细胞生物学的模式生物，主要是因为它易于在多种宿主细胞中繁殖，并且有广泛的遗传工具可用。相反，（）的培养系统目前仅存在于感染的急性期，并且基因操作已被证明更具挑战性。近年来，可编程的CRISPR相关（Cas）9酶对染色体DNA的靶向编辑极大地提高了（）和相关寄生虫中基因操作的范围和准确性，但在（）中仍然滞后。与以前的方法相比，CRISPR/Cas9技术能够引入单点和插入/缺失突变、框内表位标签的精确整合以及基因缺失，且时间和成本更低。目前在（）中CRISPR介导的基因组编辑方案依赖于Cas9的组成型或瞬时表达以及在转染质粒载体上单独或一起编码的靶标特异性sgRNA。组成型表达的Cas9具有毒性风险，而瞬时方法既费力又容易出错。在这里，我们提出了一种使用化学合成和修饰的sgRNA进行不依赖质粒载体的基因组编辑的方案。该方案允许快速且经济高效地生成（）和（）的突变细胞系。
