Laboratório de Genômica Funcional de Parasitos (GFP), Universidade Federal de Paraná, Paraná, Brazil; Facultad de Ciencias Básicas Biomédicas, Universidad Simón Bolívar, Barranquilla, Colombia.
Laboratório de Genômica Funcional de Parasitos (GFP), Universidade Federal de Paraná, Paraná, Brazil.
Int J Parasitol. 2020 Nov;50(13):1067-1077. doi: 10.1016/j.ijpara.2020.06.010. Epub 2020 Aug 25.
The genetic manipulation of Trypanosoma cruzi continues to be a challenge, mainly due to the lack of available and efficient molecular tools. The CRE-lox recombination system is a site-specific recombinase technology, widely used method of achieving conditional targeted deletions, inversions, insertions, gene activation, translocation, and other modifications in chromosomal or episomal DNA. In the present study, the CRE-lox system was adapted to expand the current genetic toolbox for this hard-to-manipulate parasite. For this, evaluations of whether direct protein delivery of CRE recombinase through electroporation could improve CRE-mediated recombination in T. cruzi were performed. CRE recombinase was fused to the C-terminus of T. cruzi histone H2B, which carries the nuclear localization signal and is expressed in the prokaryotic system. The fusion protein was affinity purified and directly introduced into epimastigotes and tissue culture-derived trypomastigotes. This enabled the control of gene expression as demonstrated by turning on a tandem dimer fluorescent protein reporter gene that had been previously transfected into parasites, achieving CRE-mediated recombination in up to 85% of parasites. This system was further tested for its ability to turn off gene expression, remove selectable markers integrated into the genome, and conditionally knock down the nitroreductase gene, which is involved in drug resistance. Additionally, CREditing also enabled the control of gene expression in tissue culture trypomastigotes, which are more difficult to transfect than epimastigotes. The considerable advances in genomic manipulation of T. cruzi shown in this study can be used by others to aid in the greater understanding of this parasite through gain- or loss-of-function approaches.
克氏锥虫的基因操作仍然是一个挑战，主要是由于缺乏可用的和有效的分子工具。CRE-lox 重组系统是一种位点特异性重组酶技术，广泛用于实现条件性靶向缺失、反转、插入、基因激活、易位和染色体或附加体 DNA 中的其他修饰。在本研究中，适应性地调整了 CRE-lox 系统，以扩展该难以操作的寄生虫的现有遗传工具包。为此，评估了通过电穿孔直接递送 CRE 重组酶是否可以提高 T. cruzi 中的 CRE 介导的重组。将 CRE 重组酶融合到 T. cruzi 组蛋白 H2B 的 C 末端，该末端带有核定位信号，并在原核系统中表达。融合蛋白通过亲和纯化并直接引入原生质体和组织培养衍生的锥虫体。这使得能够控制基因表达，如先前转染到寄生虫中的串联二聚体荧光蛋白报告基因的开启所示，实现高达 85%的寄生虫中的 CRE 介导的重组。该系统进一步测试了其关闭基因表达、去除整合到基因组中的选择性标记以及条件性敲低参与药物抗性的硝基还原酶基因的能力。此外，CREditing 还能够控制组织培养锥虫体中的基因表达，与原生质体相比，组织培养锥虫体更难转染。本研究中显示的克氏锥虫基因组操作的重大进展可被他人用于通过获得或丧失功能方法来帮助更好地理解这种寄生虫。
