3,4-二羟基苯丙氨酸(DOPA)黑色素是新生隐球菌的重要毒力因子之一,这可能会触发宿主的免疫反应。而DOPA黑色素的产生是由LAC1基因主要编码的漆酶催化的。因此,调控新生梭菌的基因表达有利于探索感兴趣的分子对宿主的影响。在这项工作中,我们建立了两个系统,这些系统可以快速轻松地构建LAC1基因的敲除/敲除:RNA干扰(RNAi)和成簇规则间隔的短回文重复序列CRISPR-Cas9。通过pSilencer4.1-CMVneo质粒和短发夹RNA构建RNAi系统以实现有效的转录抑制。CRISPR-Cas9系统用于PNK003载体以获得稳定的白化突变菌株。表型的结果,定量实时聚合酶链反应,透射电子显微镜,和分光光度法用于评估黑色素产生的能力。因此,当转化体在新平板上连续传递时,RNAi系统显示转录抑制的减弱。然而,短发夹RNA中长环的转录抑制作用更强,持续时间更长。由CRISPR-Cas9产生的白化菌株完全不能合成黑色素。总之,通过RNAi和CRISPR-Cas9系统获得具有不同黑色素生产能力的菌株,这可能有助于探索黑色素与宿主免疫反应之间的线性关系。此外,本文中的这两个系统可能方便快速筛选其他血清型新型梭菌的可能性状调节基因。
The 3,4-dihydroxyphenylalanine (DOPA) melanin is one of the important virulence factors for Cryptococcus neoformans, which may trigger immune responses in the host. While the production of DOPA melanin is catalyzed by laccase that is predominantly encoded by
LAC1 gene. Therefore, regulating the genetic expression of C. neoformans is conducive to exploring the impact of interested molecules on the host. In this work, we established two systems that were constructed quickly and easily for the knock-down/knock-out of
LAC1 gene: RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats CRISPR-Cas9. The RNAi system was constructed by pSilencer 4.1-CMV neo plasmid and short hairpin RNA to achieve effective transcriptional suppression. The CRISPR-Cas9 system was used the PNK003 vectors to obtain a stable albino mutant strain. The results of phenotype, quantitative real-time polymerase chain reaction, transmission electron microscope, and spectrophotometry were used to assess the ability of melanin production. As a result, the RNAi system displayed attenuation of transcriptional suppression when the transformants continuously passed on new plates. However, the transcriptional suppression of long loop in short hairpin RNA was more powerful and lasted longer. An albino strain produced by CRISPR-Cas9 was completely unable to synthesize melanin. In conclusion, strains with different capacities of melanin production were obtained by RNAi and CRISPR-Cas9 systems, which might be useful for exploring the linear relation between melanin and immunoreaction of the host. In addition, the two systems in this article might be convenient to quickly screen the possible trait-regulating genes of other serotypes of C. neoformans.