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，广泛用于在染色体或游离DNA中实现条件性靶向缺失、倒位、插入、基因激活、易位和其他修饰的方法。在本研究中，CRE- lox系统适用于扩展这种难以操纵的寄生虫的当前遗传工具箱。为此，评估 CRE 重组酶通过电穿孔的直接蛋白质传递是否可以改善T. cruzi 中CRE 介导的重组进行了。CRE 重组酶与T. cruzi的 C 端融合组蛋白 H2B，携带核定位信号并在原核系统中表达。融合蛋白经过亲和纯化后直接引入上鞭毛体和组织培养衍生的锥鞭毛体。这可以控制基因表达，正如通过打开先前已转染到寄生虫中的串联二聚体荧光蛋白报告基因所证明的那样，在高达 85% 的寄生虫中实现 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标记以及有条件地敲除与耐药性有关的硝基还原酶基因的能力。此外，CREditing 还可以控制组织培养锥鞭毛体中的基因表达，锥鞭毛体比上鞭毛体更难转染。本研究中显示的T. cruzi可以被其他人用来通过获得或丧失功能的方法来帮助更好地了解这种寄生虫。