4 Discussion
We successfully constructed the time-saving and convenient systems of gene knockdown and knockout for C. neoformans by RNAi and CRISPR-Cas9. And the data in this study led to some interesting and useful conclusions.
We applied shRNA expression cassette ligating to pSilencer 4.1-CMV neo for RNAi in C. neoformans . It was found that shRNA containing either the long or short loops could both conduct its interfering function (Fig. 1). However, we found that some gray or even brown colonies appeared in both pS-LAC1-A and pS-LAC1-B groups with continuous passage, indicating that transcriptional interference mediated by siRNA would be gradually attenuated when passed on. The attenuation of transcriptional suppression might be related to variable expression of the transforming plasmids, possibly resulting from variable modification of the exogenous DNA expression[19]. The attenuated suppression also occurred in human cells: only about 50% of transfected clones still showed a significant reduction of target protein after 2 months [38]. More importantly, with continuously passed on 3 times, group pS-LAC1-A appeared to have a higher proportion of white-like colonies (35:39) compared to group pS-LAC1-B (13:22) (Fig. 1D). In addition, group pS-LAC1-A seemed to appear lower mRNA expression of LAC1gene compared to group pS-LAC1-B, although it wasn’t statistically significant. Therefore, we inferred that the interfering effect of long loops might be more pronounced and longer lasting. RNAi in human cells also indicated that the size and nucleotide sequence of the loop had obvious influence to the interference, which was in accord with the results of this study [38]. In addition, other studies also used the shRNA with the same long loops to successfully conduct interference withCAP10 gene of C. neoformans [22, 39]. And we further explored the phenotype of the LAC1 knockdown strains and clarified at which passage the strains would present a visible attenuation of interfering effect.
For effectively genetic editing in CRISPR-Cas9 system,it is crucial to find the specie-specific promoters to initiate the expression of valid Cas9 protein and gRNA. However, our results might indicate that serotypes of the same species share the promoters of arbitrary serotype in the all-in-one CRISPR-Cas9 system. We successfully knocked out theLAC1 gene of C. neoformans B3501A by PNK003, which was supposed to be a specific genetic editor for serotype A of C. neoformans [33]. It might be related to the flexibility of gene expression among closely related species [40, 41]. More importantly, the albino phenotype produced by the PNK003 vector containingLAC1 target gene was stable even after continuous passage for more than 5 generations, with almost no mRNA expression of LAC1gene (Fig.2). These results indicated that the LAC1 knockout strain in this study was without the ability to produce melanin, that is, the albino phenotype was successfully constructed. Therefore, we inferred that the promoters of CAS9 and sgRNA in the PNK003 vectors might apply to other serotypes of C. neoformans ,
By TEM and laccase activity assays, We found that the knockdown strain produced only a small amount of melanin, which cannot be delivered to the cell wall in a few days, while the albino strain produced even no melanin (Fig.3). Meanwhile, the laccase activity of knockdown strain was much lower than that of WT, but also with melanin-accumulation when incubated in DOPA broth (Fig.4). However, the albino strains had even no laccase activity and almost produced no melanin.
Based on this study, we recommend a rapid method to obtain genetic knock-down stains of C. neoformans: transfect strains with pSilencer 4.1-CMV neo plasmids that contain long loops and target genes and, more importantly, it is better to use 1st or 2nd passage of strains after transfection. Besides, the different capacities of melanin production that attributed to the attenuation of transcriptional suppression could be quantified spectrophotometrically, and applied to a linear exploration between melanin and immunoreaction of the host. And the stable genetic knock-out strains produced by PNK003 vectors could refer as a control group of no genetic expression. In addition, we recommended applying the PNK003 vectors to different serotypes of C. neoformans for quick screening of possible trait-regulating genes because of its easy construction and valid knockout effect, so it will be time-saving with no need to rebuild knockout vectors for different variants.
In addition, we transfected the competent yeast cells with vectors containing the LAC1 gene by the Yeast Transformation Kit, whose principle is that the alkaline Li+ can enhance the permeability of the cell membrane, making the cells easier to absorb external DNA. Simultaneously, Polyethylene glycol in the kit protects the cells membrane from chemical damage of high-concentration lithium acetate, and carrier DNA protects the exogenous DNA from degradation by DNase ofC. neoformans . The chemical transformation is easily accessible with no requirement for linearizing the plasmids, and electroporation can harm the cell membrane [42]. Therefore, we applied the chemical transformation to introduce exogenous DNA into C. neoformans . And the results in this study showed that the chemical transformation was effective but probably lowered the transfected efficiency. However, it was low-cost and easily accessible for rough screening of possible trait-regulating genes at a one-time.
Acknowledgement: We thank Ping Zhang and Xudong Zhu from Beijing Key Laboratory of Genetic Engineering Drug and Biotechnology, College of Life Sciences, Beijing Normal University for gifting the PNK003 vectors to us. And we thank the team of ophthalmology department of Jinling hospital, Nanjing, Jiangsu province for the support of experimental equipment. Finally, we thank HOME for Researchers Platform for writing assistance (https://www.home-for-researchers.com/paper) and Figdraw (https://www.figdraw.com) .
Conflict of interests : No conflict of interest exists in the submission of this manuscript, and the manuscript is approved by all authors for publication.
Funding :This work was supported by Jiangsu Dermatology Innovation Team Foundation (grant number CXTDA2017038).