State Key Laboratory for Animal Disease Control and Prevention, South China Agricultural University, Guangzhou, 510642, China.
Guangdong Laboratory for Lingnan Modern Agriculture, Center for Emerging and Zoonotic Diseases, College of Veterinary Medicine, South China Agricultural University, Guangzhou, 510642, China.
Parasit Vectors. 2024 Aug 21;17(1):352. doi: 10.1186/s13071-024-06431-1.
The dimerizable Cre recombinase system (DiCre) exhibits increased leaky activity in Cryptosporidium, leading to unintended gene editing in the absence of induction. Therefore, optimization of the current DiCre technique is necessary for functional studies of essential Cryptosporidium genes.
Based on the results of transcriptomic analysis of Cryptosporidium parvum stages, seven promoters with different transcriptional capabilities were screened to drive the expression of Cre fragments (FKBP-Cre59 and FRB-Cre60). Transient transfection was performed to assess the effect of promoter strength on leakage activity. In vitro and in vivo experiments were performed to evaluate the leaky activity and cleavage efficiency of the optimized DiCre system by polymerase chain reaction (PCR), nanoluciferase, and fluorescence analyses.
The use of promoters with lower transcriptional activity, such as pcgd6_4110 and pcgd3_260, as opposed to strong promoters such as pActin, pα-Tubulin, and pEnolase, reduced the leakage rate of the system from 35-75% to nearly undetectable levels, as verified by transient transfection. Subsequent in vitro and in vivo experiments using stable lines further demonstrated that the optimized DiCre system had no detectable leaky activity. The system achieved 71% cleavage efficiency in vitro. In mice, a single dose of the inducer resulted in a 10% conditional gene knockout and fluorescent protein expression in oocysts. These fluorescently tagged transgenic oocysts could be enriched by flow sorting for further infection studies.
A DiCre conditional gene knockout system for Cryptosporidium with good cleavage efficiency and reduced leaky activity has been successfully established.
二聚化 Cre 重组酶系统（DiCre）在隐孢子虫中表现出增加的漏活性，导致在没有诱导的情况下发生意外的基因编辑。因此，需要优化当前的 DiCre 技术，以进行必需的隐孢子虫基因的功能研究。
基于隐孢子虫各阶段转录组分析的结果，筛选了七个具有不同转录能力的启动子来驱动 Cre 片段（FKBP-Cre59 和 FRB-Cre60）的表达。进行瞬时转染以评估启动子强度对漏活性的影响。通过聚合酶链反应（PCR）、纳米荧光素酶和荧光分析进行体外和体内实验，评估优化的 DiCre 系统的漏活性和切割效率。
使用转录活性较低的启动子，如 pcgd6_4110 和 pcgd3_260，而不是强启动子，如 pActin、pα-Tubulin 和 pEnolase，可将系统的漏率从 35-75%降低到几乎无法检测的水平，这在瞬时转染中得到了验证。随后使用稳定系进行的体外和体内实验进一步表明，优化的 DiCre 系统没有检测到漏活性。该系统在体外实现了 71%的切割效率。在小鼠中，单次给予诱导剂可导致 10%的条件性基因敲除和卵囊中荧光蛋白的表达。这些荧光标记的转基因卵囊可通过流式细胞分选进行富集，用于进一步的感染研究。
成功建立了一种具有良好切割效率和降低漏活性的隐孢子虫 DiCre 条件性基因敲除系统。
