Simplified and effective RNA interference and CRISPR-Cas9 systems for
Cryptococcus neoformans
Abstract
The 3,4-dihydroxyphenylalanine (DOPA) melanin is one of important
virulence factors for Cryptococcus neoformans, which may trigger immune
responses in the host. It is worth exploring the genetic function of C.
neoformans, by which we may derive more antifungal strategies.
Therefore, 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 used pSilencer 4.1-CMV neo plasmid and
short hairpin RNA to realize the effective transcriptional suppression.
The CRISPR-Cas9 system used the PNK003 vectors to obtain a stable albino
mutant strain. The results of phenotype, qRT-PCR, Transmission Electron
Microscope (TEM) 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 were more powerful and
lasted longer. The CRISPR-Cas9 system constructed an albino strain
completely without the ability to produce melanin. Considering the
weakening of transcriptional suppression, we recommend using a long loop
for the RNAi system and 1st or 2nd passage of knockdown strains for the
subsequent studies. Besides, the different capacities of melanin
production might be useful for exploring the linear relation between
melanin and immunoreaction of the host. In addition, we recommend
applying the PNK003 vectors to other serotypes of C. neoformans for
quick screening of possible trait-regulating genes because of its easy
construction and valid knockout effect.