Advances and Challenges in CRISPR/Cas-Based Fungal Genome Engineering for Secondary Metabolite Production: A Review

Abstract

Fungi represent an important source of bioactive secondary metabolites (SMs), which have wide applications in many fields, including medicine, agriculture, human health, and many other industries. The genes involved in SM biosynthesis are usually clustered adjacent to each other into a region known as a biosynthetic gene cluster (BGC). The recent advent of a diversity of genetic and genomic technologies has facilitated the identification of many cryptic or uncharacterized BGCs and their associated SMs. However, there are still many challenges that hamper the broader exploration of industrially important secondary metabolites. The recent advanced CRISPR/Cas system has revolutionized fungal genetic engineering and enabled the discovery of novel bioactive compounds. In this review, we firstly introduce fungal BGCs and their relationships with associated SMs, followed by a brief summary of the conventional strategies for fungal genetic engineering. Next, we introduce a range of state-of-the-art CRISPR/Cas-based tools that have been developed and review recent applications of these methods in fungi for research on the biosynthesis of SMs. Finally, the challenges and limitations of these CRISPR/Cas-based systems are discussed and directions for future research are proposed in order to expand their applications and improve efficiency for fungal genetic engineering.

Terpenoid balance in Aspergillus nidulans unveiled by heterologous squalene synthase expression

“…The activation of silent gene clusters in fungi for production of uncharacterized secondary metabolites has garnered substantial attention in recent years ( 36 ). Genetic tools play a crucial role in this pursuit by providing means to unlock the hidden potential of fungal genomes ( 37 ). One such tool is the implementation of FAC technology that can specifically target and activate silent BGCs in heterologous hosts ( 10 ).…”

Secondary Metabolites from Fungi Microsphaeropsis spp.: Chemistry and Bioactivities

“…Accordingly, the following recommendations were proposed: (i) Employ non-directional activation strategies to activate the biosynthesis of secondary metabolites, such as the OSMAC (one strain many compounds) strategy [49], co-culture strategy [50], and epigenetic regulation strategy [51]. (ii) Harness targeted activation strategies to activate numerous dormant gene clusters within fungal genomes, such as target sequence promoter replacements [52], transcription regulatory factor knockouts [53], the heterologous expression of biosynthetic gene clusters [54], DNA-assembly technology [55], and ribosome engineering [56]. However, the genomic information of Microsphaeropsis has not been reported in the literature so far.…”

On the ever‐growing functional versatility of the CRISPR‐Cas13 system

“…In bacteria, a controlled expression (then toxicity) of Cas13d through a low copy number vector, a tightly regulated promoter and a weak ribosome binding site allowed its use to repress gene expression (Zhang et al, 2020). Furthermore, the CRISPR-Cas13d system was harnessed to induce RNA silencing in fission yeast using a CRISPR array (Chen et al, 2023).…”

“…Besides, research was conducted to harness a Cas13a to promote the edition of endogenous and exogenous transcripts in Schizosaccharomyces pombe (Jing et al., 2018 ). Such advances in RNA editing and silencing may lead to new metabolic control implementations in yeast for the efficient bioproduction of compounds of clinical and industrial importance (Wang et al., 2023 ). Overall, CRISPR‐Cas13‐mediated RNA editing provides a promising opportunity to exert therapeutic actions aimed at correcting undesired mutations and to engineer synthetic gene circuits with sophisticated posttranscriptional regulations.…”