Table_1_Effective Genome Editing in Leishmania (Viannia) braziliensis Stably Expressing Cas9 and T7 RNA Polymerase.xlsx

Until 2015, loss-of-function studies to elucidate protein function in Leishmania relied on gene disruption through homologous recombination. Then, the CRISPR/Cas9 revolution reached these protozoan parasites allowing efficient genome editing with one round of transfection. In addition, the development of LeishGEdit, a PCR-based toolkit for generating knockouts and tagged lines using CRISPR/Cas9, allowed a more straightforward and effective genome editing. In this system, the plasmid pTB007 is delivered to Leishmania for episomal expression or integration in the β-tubulin locus and for the stable expression of T7 RNA polymerase and Cas9. In South America, and especially in Brazil, Leishmania (Viannia) braziliensis is the most frequent etiological agent of tegumentary leishmaniasis. The L. braziliensis β-tubulin locus presents significant sequence divergence in comparison with Leishmania major, which precludes the efficient integration of pTB007 and the stable expression of Cas9. To overcome this limitation, the L. major β-tubulin sequences, present in the pTB007, were replaced by a Leishmania (Viannia) β-tubulin conserved sequence generating the pTB007_Viannia plasmid. This modification allowed the successful integration of the pTB007_Viannia cassette in the L. braziliensis M2903 genome, and in silico predictions suggest that this can also be achieved in other Viannia species. The activity of Cas9 was evaluated by knocking out the flagellar protein PF16, which caused a phenotype of immobility in these transfectants. Endogenous PF16 was also successfully tagged with mNeonGreen, and an in-locus complementation strategy was employed to return a C-terminally tagged copy of the PF16 gene to the original locus, which resulted in the recovery of swimming capacity. The modified plasmid pTB007_Viannia allowed the integration and stable expression of both T7 RNA polymerase and Cas9 in L. braziliensis and provided an important tool for the study of the biology of this parasite.

直至2015年，阐明利什曼原虫（Leishmania）蛋白质功能的功能丧失研究，均依赖于通过同源重组实现的基因敲除。随后，CRISPR/Cas9技术革新惠及这类原生动物寄生虫，仅需一轮转染即可实现高效基因组编辑。此外，基于聚合酶链式反应（PCR）的工具包LeishGEdit的开发，可利用CRISPR/Cas9构建基因敲除株与标记细胞系，使基因组编辑流程更为简便高效。在该系统中，质粒pTB007被导入利什曼原虫，用于实现游离表达或整合至β-微管蛋白基因座，同时实现T7 RNA聚合酶与Cas9的稳定表达。
在南美洲，尤其是巴西，巴西利什曼原虫（Leishmania (Viannia) braziliensis）是皮肤利什曼病最常见的致病病原体。与硕大利什曼原虫（Leishmania major）相比，巴西利什曼原虫的β-微管蛋白基因座存在显著序列差异，这阻碍了pTB007的高效整合以及Cas9的稳定表达。为克服这一局限，研究人员将pTB007中携带的硕大利什曼原虫β-微管蛋白序列，替换为维安尼亚亚属利什曼原虫的β-微管蛋白保守序列，由此构建得到pTB007_Viannia质粒。这一修饰使得pTB007_Viannia基因盒成功整合至巴西利什曼原虫M2903的基因组中，且计算机预测显示，该方法同样可应用于其他维安尼亚亚属利什曼原虫物种。
研究通过敲除鞭毛蛋白PF16评估了Cas9的活性，该操作使这些转染株产生了运动能力丧失的表型。内源PF16也成功被mNeonGreen荧光蛋白标记；同时研究采用基因座内互补策略，将携带C端标签的PF16基因拷贝放回其原始基因座，使转染株恢复了运动能力。改造后的pTB007_Viannia质粒可实现T7 RNA聚合酶与Cas9在巴西利什曼原虫中的整合与稳定表达，为该寄生虫的生物学研究提供了重要工具。