State Key Laboratory of Genetic Engineering, Ministry of Education Key Laboratory of Contemporary Anthropology, Department of Microbiology and Microbial Engineering, School of Life Science, Fudan University, Shanghai, 200433, China.
Key Laboratory of Parasite and Vector Biology of MOH, WHO Cooperation Center for Tropical Diseases, National Institute of Parasitic Diseases, Chinese Center for Diseases Control and Prevention, Shanghai, 200025, China.
Parasit Vectors. 2017 Jun 5;10(1):281. doi: 10.1186/s13071-017-2207-4.
RNA polymerase III promoters have been widely used to express short hairpin-RNA (shRNA), microRNA (miRNA), and small guide RNA (sgRNA) in gene functional analysis in a variety of organisms including Schistosoma mansoni. However, no endogenous RNA polymerase III promoters have been identified in Schistosoma japonicum. The lack of appropriate promoters in S. japonicum has hindered its gene functional analysis. Identification of functional promoters in S. japonicum is therefore in urgent need.
Via sequence alignment, a 347 bp sequence upstream from the coding region of S. japonicum U6 small nuclear RNA (snRNA) was identified, cloned, and named as S. japonicum U6 (sjU6) promoter. A sgRNA sequence named as sgRNA970 was designed, and its Cas9 nuclease guiding activity was confirmed by in vitro cleavage assay. The sjU6 promoter was ligated with sgRNA970 coding sequence by overlap PCR to generate a sjU6-sgRNA970 expression cassette. The expression cassette was inserted into a lentiviral plasmid to construct the pHBLV-sgRNA970 plasmid. First, we tested the sjU6 promoter activity in HEK293 cells by transfecting HEK293 cells with the pHBLV-sgRNA970 plasmid. RT-PCR amplification of the total RNA from the transfected HEK293 cells confirmed the presence of sgRNA970 transcript and indicated sjU6 promoter was functional to initiate transcription in HEK293 cells. Then we transduced the lentivirus expressing Cas9-ZsGreen fusion protein into 14 dpi schistosomula to test whether lentivirus was capable to induce exogenous gene expression in S. japonicum. Fluorescence microscopy and western blot results confirmed the expression of Cas9-ZsGreen fusion protein in S. japonicum. Therefore, this lentiviral system was adapted to test promoter activity in S. japonicum. Finally, we transduced 14 dpi S. japonicum with lentivirus produced from the pHBLV-sgRNA970 plasmid. RT-PCR amplification of the total RNA from transduced schistosomula confirmed the presence of sgRNA970 transcript and therefore indicated sjU6 promoter was functional to initiate transcription in S. japonicum.
To our knowledge, sjU6 promoter would be the first identified and validated endogenous RNA polymerase III promoter in S. japonicum, which could be used for future CRISPR/Cas9 studies in S. japonicum.
RNA 聚合酶 III 启动子已广泛用于多种生物（包括曼氏血吸虫）的基因功能分析中表达短发夹 RNA（shRNA）、microRNA（miRNA）和小指导 RNA（sgRNA）。然而，在日本血吸虫中尚未鉴定出内源性 RNA 聚合酶 III 启动子。日本血吸虫缺乏合适的启动子，阻碍了其基因功能分析。因此，迫切需要鉴定日本血吸虫中的功能启动子。
通过序列比对，从日本血吸虫 U6 小核 RNA（snRNA）编码区上游鉴定出一段 347bp 的序列，将其克隆并命名为日本血吸虫 U6（sjU6）启动子。设计了一个名为 sgRNA970 的 sgRNA 序列，并通过体外切割实验证实了其 Cas9 核酸酶导向活性。通过重叠 PCR 将 sjU6 启动子与 sgRNA970 编码序列连接，生成 sjU6-sgRNA970 表达盒。将表达盒插入慢病毒质粒中，构建 pHBLV-sgRNA970 质粒。首先，我们通过转染 HEK293 细胞来测试 pHBLV-sgRNA970 质粒在 HEK293 细胞中的 sjU6 启动子活性。从转染的 HEK293 细胞中提取总 RNA 进行 RT-PCR 扩增，证实了 sgRNA970 转录本的存在，并表明 sjU6 启动子在 HEK293 细胞中能够启动转录。然后，我们将表达 Cas9-ZsGreen 融合蛋白的慢病毒转导至 14 天龄的尾蚴中，以测试慢病毒是否能够在日本血吸虫中诱导外源基因表达。荧光显微镜和 Western blot 结果证实了 Cas9-ZsGreen 融合蛋白在日本血吸虫中的表达。因此，该慢病毒系统被用于测试日本血吸虫中的启动子活性。最后，我们将慢病毒转导至 14 天龄的日本血吸虫中，该慢病毒由 pHBLV-sgRNA970 质粒产生。从转导的尾蚴中提取总 RNA 进行 RT-PCR 扩增，证实了 sgRNA970 转录本的存在，因此表明 sjU6 启动子在日本血吸虫中能够启动转录。
据我们所知，sjU6 启动子将是在日本血吸虫中首次鉴定和验证的内源性 RNA 聚合酶 III 启动子，可用于日本血吸虫的未来 CRISPR/Cas9 研究。
