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).