Recent Advances in Genome Editing Tools in Medical Mycology Research

Abstract

Manipulating fungal genomes is an important tool to understand the function of target genes, pathobiology of fungal infections, virulence potential, and pathogenicity of medically important fungi, and to develop novel diagnostics and therapeutic targets. Here, we provide an overview of recent advances in genetic manipulation techniques used in the field of medical mycology. Fungi use several strategies to cope with stress and adapt themselves against environmental effectors. For instance, mutations in the 14 alpha-demethylase gene may result in azole resistance in Aspergillusfumigatus strains and shield them against fungicide's effects. Over the past few decades, several genome editing methods have been introduced for genetic manipulations in pathogenic fungi. Application of restriction enzymes to target and cut a double-stranded DNA in a pre-defined sequence was the first technique used for cloning in Aspergillus and Candida. Genome editing technologies, including zinc-finger nucleases (ZFNs) and transcriptional activator-like effector nucleases (TALENs), have been also used to engineer a double-stranded DNA molecule. As a result, TALENs were considered more practical to identify single nucleotide polymorphisms. Recently, Class 2 type II Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 technology has emerged as a more useful tool for genome manipulation in fungal research.

Keywords: CRISPR/Cas9; gene editing techniques; medically important fungi.

Figure 1

Hypothetical model of RNA interference (RNAi) pathway in fungi. The Dicer ribonuclease III enzyme (DCR) cleaves exogenous long double-stranded RNA (dsRNAs) into ~21–24 nucleotide small interfering RNAs (siRNAs). The guide siRNA then loaded onto the major catalytic component called Argonaute (Ago) and other proteins generating the RNA-induced silencing complex (RISC). siRNA, along with RISC, complementarily pair with messenger RNA (mRNA) resulting in degradation of mRNAs.

Figure 2

Schematic representation of RNA-guided Cas9 constructs designed for genome editing. Bottom panel: (structure of the vector plasmids used to deliver Cas9-sgRNA components into fungal cells). PromoterX can express NLS-Cas9-NLS protein. PromoterY can express 20 nt guide sequence + sgRNA cassette. Upper panel: (Cas9+sgRNA+genomic DNA). Mechanism of Cas9/gRNA ribonucleoprotein complex action, NGG (PAM site) highlighted in black line. The Cas9 nuclease domain HNH then cleaves the target DNA sequence complementary to the 20 bp guide sequence, while RuvC domain cuts another DNA strand, forming a double stranded break (DSB). DSB must be repaired via either non-homologous end joining (NHEJ) or homologous recombination (HR) immediately to avoid cell death. Insertions and deletion mutations at the target site generated by NHEJ and homology directed repair (HDR) allow disrupting or abolishing the function of a target gene. Moreover, modifications in this system can also be used to silence genes, insert new exogenous DNA, or block RNA transcription.