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.