对Cryptococcus neoformans进行简化和有效的RNA干扰和CRISPR-Cas9系统
Qiying Chen1, Binyi An2, Xinyuan Peng3
1Department of Dermatology, Nanjing Jinling Hospital, The First School of Clinical Medicine, Southern Medical University, Guangzhou, China.
Journal of basic microbiology
|June 13, 2023
概括
研究人员开发了RNA干扰 (RNAi) 和CRISPR-Cas9系统,通过准LAC1基因来控制Cryptococcus neoformans中的黑色素产生. 这些方法产生了具有不同黑色素水平的菌株,有助于研究真菌毒性和宿主免疫力.
科学领域:
- 菌类学 菌类学是指菌类学.
- 分子生物学分子生物学
- 免疫学 免疫学 免疫学
背景情况:
- 3,4-二基烯氨酸 (DOPA) 氨酸是Cryptococcus neoformans中关键的毒性因子,可能调节宿主免疫反应.
- 黑色素的产生主要由LAC1基因编码的laccase进行调节.
研究的目的:
- 建立快速高效的基因系统,以调节C. neoformans.中的LAC1基因表达.
- 为了研究改变黑色素产生的影响真菌表型和宿主相互作用.
主要方法:
- 开发一种RNA干扰 (RNAi) 系统,使用pSilencer 4.1-CMV新等离子体和短毛RNA进行转录抑制.
- 使用PNK003载体的聚类正规间隔短时间的巴林德罗姆重复 (CRISPR-Cas9) 系统,以创建稳定的白色突变菌株.
- 使用表型分析,定量实时聚合酶连锁反应,传输电子显微镜和光谱法评估黑色素的产生.
主要成果:
- RNAi系统证明了有效的LAC1转录抑制,尽管其有效性随着连续的亚培养而下降. 较长的毛RNA结构提供了更强大和持续的抑制作用.
- 克里斯普尔-卡斯9系统成功地产生了一个稳定的白色突变菌株,完全缺乏黑色素合成.
- 这两种系统都产生了C. neoformans菌株,具有明显的黑色素生产能力.
结论:
- RNAi和CRISPR-Cas9系统为操纵C. neoformans中的黑色素产生提供了多功能工具.
- 产生的菌株对于阐明黑色素和宿主免疫反应之间的关系是有价值的.
- 这些系统为选其他C. neoformans血清型中的特征调节基因提供了一种方便的方法.
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