1 Introduction
Cryptococcus neoformans is one of the significant human pathogens
of basidiomycetes because it infects approximately 1 million individuals
per year, with over 600,000 annual mortality attributable to the
opportunistic pathogenic infection, resulting in almost one-third of
AIDS deaths[1]. C. neoformans has two phenotypic
characteristics, the capsule and the synthesis of melanin, which both
protect the yeasts and induce host damage as virulence factors[2].
3,4-dihydroxyphenylalanine (DOPA) melanin of C. neoformans is
synthesized with the exogenous catecholamines, the entire process of
which is catalyzed by laccase, predominantly encoded by the LAC1gene [3]. Recently, a study found that the recognition molecules of
melanin in macrophages elicited metabolic reprogramming and associated
inflammation, which contrasted with the inhibitory effects of melanin in
previous studies [4, 5]. Therefore, we explored two systems that
were constructed quickly and easily for the knock-down/knock-out ofLAC1 : RNAi and CRISPR-Cas9. These two systems are time-saving and
convenient for quickly obtaining small amount of target strains or
rudely screening possible trait-regulating genes at a one-time.
The DOPA melanin of C. neoformans impairs antifungal immune
responses and clearance by weakening immunity response [6, 7] and
lowering the accumulation of antimicrobial substances [8, 9]. DOPA
melanin should anchor to chitin within the cell wall, but
chitinase-inhibited C. neoformans leaked an amount of DOPA
melanin, inducing robust inflammation in mice [10]; moreover, the
immune response induced by the chitosan-deficient strain, including
heat-killed cells of this strain, was sufficient to challenge a virulent
wild-type (WT) strain [11]. This immune response contrasts with the
inhibitory effects of DOPA melanin. But a known mechanism can explain
the immune response: PRRs usually cause inflammation after PAMP
recognition [12], which has been confirmed that
1,8-dihydroxynaphthalene melanin of Aspergillus fumigatus was
recognized by melanin-sensing C-type lectin receptor of macrophages and
induce inflammation mainly by activating glycolysis [4, 5]. However,
further studies are needed to elucidate the specific mechanism of how
DOPA melanin triggers the inflammation of immune cells. In this study,
we used RNAi and CRISPR systems to obtain LAC1 knock-down and
knock-out strains, respectively, and the phenotypic effects in this
study were obvious.
At first, RNAi was found to be a self-protective mechanism for numerous
species (such as plants, animals, fungi, and protists) to prevent the
interference by exogenous genes or endogenous transposon activation and
movement[13-16]. An RNAse III-like endonuclease, Dicer, cleaves
double-strands RNA (dsRNA) into 20–25bp pieces called small
interference RNA (siRNA)[17]. These fragments are incorporated into
Argonaute, the catalytic subunit of RNA-induced silencing complex, and
guide to the complementary homologous mRNA to elicit mRNA degradation.
Meanwhile, RNA-dependent RNA polymerases amplify additional dsRNA
complementary to the target mRNA by siRNA primers, triggering more Dicer
and Argonaute[17]. The mechanism of RNAi allows researchers to
artificially trigger RNAi by dsRNA that has been either directly
introduced into a cell or transcribed intracellularly from transfected
vectors [18]. The dsRNA from vectors has no requirement for in
vitro synthesis of dsRNA [19] and is widely used in many fungal
species[20]. The intracellular transcription of dsRNA is usually
done in two ways: the reverse complementary RNA strands pair to form
dsRNA after being transcribed by two promoters in the opposite
direction, and a single RNA strand consisting of reverse RNA
oligonucleotides matches itself to form a small hairpin RNA (shRNA)
after being driven by a single promoter [18, 19, 21, 22]. Both
forming ways of dsRNA were previously used for the exploration of genic
function (e.g., CAP10 , CAP59 , ADE2 ) of C.
neoformans [18, 19, 22, 23]. The dsRNA from the former way is
relatively inefficient in RNAi compared to hairpin RNA, perhaps due to
the low efficiency of dsRNA formation [20]. However, there was
little research on the interference by shRNA with LAC1 . In this
study, we successfully constructed a plasmid containing the target gene
of LAC1 , which could form a hairpin structure (shRNA) after
transcription (Fig. 1A) and work effectively for RNAi.
CRISPR-Cas9 was firstly discovered as genome protection for bacteria
from the genic invasion of viruses and plasmids, which was gradually
applied to eukaryotic cells for gene editing, including gene knock-out,
gene knock-in, repression or activation[24, 25]. A vector of
CRISPR-Cas9 systems is mainly composed of cas9 gene, single guide
RNA (sgRNA, consist of 17-20 base pairs target sequences and tracrRNA
sequences), selectable marker gene, and their respective operon(s)[24,
25]. After intracellular expression of the vector, Cas9, the type II
RNA-guided endonuclease, is directed by sgRNA to the targeted gene with
a 3-bp protospacer-adjacent motif (PAM) next to it. Then Cas9 introduces
a double-strand break about 5bp away from the PAM, which will be
repaired by nonhomologous end joining, usually resulting in insertions
or deletions and even leading to frameshift of the reading frame and
premature stop codons [26]. However, the expression of valid Cas9
protein and gRNA need specie-specific promoters, which is the main
obstacle to applying CRISPR-Cas9 to C. neoformans , although the
CRISPR-Cas9 system has proved effective for many yeasts and filamentous
fungi [27-32]. Zhang ping and colleagues[33, 34] developed
simplified all-in-one CRISPR-Cas9 vectors specific for C.
neoformans : PNK003 for serotype A strain and PRH003 for serotype D
strain. The difference between PNK003 and PRH003 vectors is present in
their strains-specific promoters of gRNA and Cas9 expression cassettes.
Meanwhile, the all-in-one vectors contain ‘suicide’ systems that can
eliminate Cas9 and gDNA cassettes after gene editing, reducing the
potential cytotoxicity of Cas9 endonuclease and conquering the
difficulty of gene complementation. In this study, we used PNK003 forLAC1 knock-out of serotype D strains (FIG. 2A) and found that
promoters of serotype A strain can drive the expression of gRNA and Cas9
cassettes in serotype D strain.