Filamentous fungi have a major impact on many aspects of human diets and health more broadly. Their benefits are derived from various mechanisms underlying the production of enzymes, organic acids, and flavors, as well as, more importantly, antibiotic compounds and therapeutic molecules (Satish et al., 2020). Moreover, they are well known as appealing microbial cell factories that possess the industrial capability to secrete a large repertoire of different bioactive secondary metabolites, such as paclitaxel and swainsonine, which have become important clinical therapeutics (Hautbergue et al., 2018; Keller, 2019; Mosunova et al., 2020). There has been a focus on research into secondary metabolites because some secondary metabolites can be used as anti-cancer drugs and anti-bacterial compounds (Lobanovska and Pilla, 2017). A majority of these secondary metabolites can be classified into three chemical categories: polyketides derived from acyl-CoAs, terpenes produced from acyl-CoAs, and small peptides derived from amino acids (Keller, 2019; Mosunova et al., 2020). Most of the genes involved in the biosynthesis of secondary metabolites are frequently clustered together on chromosomes in biosynthetic gene clusters (SM-BGCs), and some are not expressed under standard laboratory culture conditions (Lin et al., 2013; Jiang et al., 2015; Nah et al., 2017; Keller, 2019; Kjaerbolling et al., 2019). Another characteristic of genes in such clusters is that they are not constitutively expressed, and formerly actively expressed genes can become transcriptionally quiescent upon repeated culturing (Kale et al., 1994; Hoffmeister and Keller, 2007). Although secondary metabolite biosynthetic genes in filamentous fungi are generally found in clusters that provide a convenient genetic locus for manipulation (Hoffmeister and Keller, 2007), the exploitation of new bioactive compounds is hindered by intrinsic difficulties involving complex genetic backgrounds and poor efficiency of gene targeting. With increases in genomic insight and gene mining from high-throughput sequencing data and the advancement of genomics and transcriptomics, there has been an acceleration in the identification and utilization of SM-BGCs. Thus, elucidating the genetic basis and the biosynthetic pathways of secondary metabolites has become comparatively easier.

Filamentous fungi, such as Trichoderma reesei, Aspergillus niger, Aspergillus oryzae, and Aspergillus nidulans, are universally used as model eukaryotic microorganisms to produce industrial secondary metabolites. For example, researchers have utilized rice blast fungus Pyricularia oryzae to yield tenuazonic acid, Aspergillus fumigatus to secrete the natural product trypacidin, Fusarium fujikuroi to produce gibberellic acid, Fusarium heterosporum to synthesize the polyketide equisetin, A. nidulans to secrete microperfuranone, and Penicillium chrysogenum to biosynthesize sorbicillin (Gauthier et al., 2012; Kakule et al., 2015; Yun et al., 2017; Shi et al., 2019). However, while the genetic manipulation of secondary metabolites in filamentous fungi is being explored, there are several factors that limit genetic research on secondary metabolite biosynthetic pathways of filamentous fungi. First, filamentous fungi, like the model organism yeast, have complex genetic backgrounds when compared to prokaryotes (Song et al., 2019). Second, it is difficult to apply genetic manipulation and molecular biology tools in filamentous fungi. Furthermore, low homologous recombination efficiency (generally less than 5%) and a lack of suitable selection markers, and plasmids also hamper the exploitation of novel secondary metabolites from filamentous fungi (Mei et al., 2019).

More recently, a variety of genetic engineering methods, such as RNA interference, heterologous expression, gene-targeting strategies, and zinc finger nuclease (ZFN) and transcription-activator-like effector nuclease (TALEN) – based genome editing have been developed to explore and demonstrate the biosynthetic and regulatory mechanisms in filamentous fungi (Boettcher and McManus, 2015; Wang et al., 2017; Dasgupta et al., 2020). Although the existing approaches can be utilized to edit target genes at the genomic level, these do not meet the needs of industrial secondary metabolite production in filamentous fungi owing their low editing efficiency and cumbersome manipulation (Shi et al., 2017). Thus, the exploitation of new secondary metabolites with potential pharmaceutical applications in filamentous fungi is extremely challenging. Fortunately, the emergence of the clustered regularly interspersed short palindromic repeats (CRISPR)/associated protein (Cas) system in recent years has raised hopes of solving the problem presented by the largely inefficient gene editing tools available for use in filamentous fungi. The current CRISPR/Cas systems were discovered in archaea and bacteria and can be classified into three group based on the different Cas effectors (Cas9, Cas13, and Cas12), which can then be further divided into six types and more than 20 subtypes (Makarova et al., 2018; Li et al., 2019). The type-II CRISPR/Cas system from Streptococcus pyogenes has been widely applied across species, including filamentous fungi, as it is much simpler than other CRISPR systems and has proven to be particularly powerful for use in precise DNA modification (Cong et al., 2013; Deng et al., 2017b). Using specific codon optimization and in vitro RNA transcription, Liu et al. (2015) first adopted the CRISPR/Cas9 system in the filamentous fungus T. reesei and achieved relatively high homologous recombination efficiencies (>93%) when the lengths of the homology arms were 200 bp. In the same year, Nodvig et al. (2015) successfully targeted the yA gene by applying this system in the model fungus A. nidulans and obtained a genome-edited phenotype. Additionally, Fuller et al. (2015) demonstrated that the CRISPR/Cas9 system can be applied to high-efficiency gene disruption in A. fumigatus. These instances illustrate that this powerful system has been widely and effectively applied to industrial filamentous fungi. More details about the CRISPR/Cas9 system and its specific application in the biosynthesis of secondary metabolites by filamentous fungi are reviewed in the following sections.

This review introduces and summarizes the current knowledge and applications of the CRISPR/Cas9 system in filamentous fungi. By detailing several examples, we introduce the specific application of the CRISPR/Cas9 system and CRISPRa for precise gene editing and gene cluster activation, respectively. Additionally, we discuss and summarize the challenges and limitations as well as further prospects of this technology in the production of secondary metabolites by filamentous fungi. Our review lays a solid foundation for the exploration of secondary metabolites in filamentous fungi and will be beneficial to future research on activating silent gene clusters involved in secondary metabolites produced by filamentous fungi.

The CRISPR/Cas9 system that has emerged as an advanced technology for genome engineering originated from adaptive immune systems in bacteria as a special defense mechanism against invading viruses and plasmids (Barrangou and Marraffini, 2014; Wang and Coleman, 2019). This genome editing system is composed of two components, a Cas9 nuclease and a guide RNA molecule (gRNA) that targets the nuclease to a specific genomic target site in the genome (Deng et al., 2017b). The single chimeric guide RNA (sgRNA) consisting of a fusion of a CRISPR RNA (crRNA) and a fixed trans-activating crRNA (tracrRNA) are processed by the endogenous bacterial machinery to generate the mature gRNA (Deltcheva et al., 2011). The Cas9 endonuclease is guided to a specific locus by a gRNA, which then forms Watson-Crick base pairs with the target DNA sequence, thereby permitting Cas9 to break the double-stranded DNA at specific sites (Figure 1; Doudna and Charpentier, 2014; Mei et al., 2019). Importantly, to achieve the complementary target-DNA binding and cleavage, Cas9 requires the presence of its major specificity determinant, a well-defined short protospacer adjacent motif (PAM) that is located immediately adjacent to the non-target DNA strand (Mojica et al., 2009; Sternberg et al., 2014). Subsequently, two unique repair mechanisms (Figure 1), non-homologous end-joining (NHEJ) or homology-directed repair (HDR), mend DNA double-strand breaks (DSBs). NHEJ is an error-prone and dominant RNA repair pathway for DSBs via direct ligation of the break ends without using a homologous template. Thus, it can sometimes cause targeted mutations, such as random deletions, insertions, replacement of bases, targeted chromosomal rearrangements, or frameshift mutations at DNA breakage points leading to premature stop codons within the open reading frame (ORF) of the targeted gene (Kim and Kim, 2014; Boettcher and McManus, 2015; Mei et al., 2019). Compared to NHEJ, which is the most common DSB repair mechanism in microorganisms, HDR is a less efficient but high-fidelity pathway. HDR precisely repairs DSBs with the help of a homologous DNA template or exogenous donor fragment, thus having the potential to generate gene modifications by introducing desired nucleotide substitutions or gene insertions (Bortesi and Fischer, 2015; El-Sayed et al., 2017). With the advancement of research and accumulating knowledge on endogenous DNA repair mechanisms, a series of technological tools have been explored to precisely induce DSBs in target genes by using exogenous nucleases. Prior to CRISPR, genome engineering strategies utilizing ZFN or TALEN-based genome editing required the design, generation, and validation of an appropriate protein for a specific DNA locus of interest, thus limiting their widespread application (Doudna and Charpentier, 2014; Boettcher and McManus, 2015; El-Sayed et al., 2017; Leitão et al., 2017). Owing to its high efficiency and the possibility of multi-gene editing, the CRISPR/Cas9 system has rapidly emerged as an extraordinary genome engineering approach has outstripped the performance of earlier technologies (Doudna and Charpentier, 2014; Wang et al., 2017).

The versatility and programmability of Cas9 has made the CRISPR/Cas9 genome editing strategy a revolutionary approach in biological research, and it has been considered useful for creating gene deletions, substitutions, and insertions in filamentous fungi. Here, we gather considerable information regarding the application of the CRISPR/Cas9 system in filamentous fungi over recent years (Table 1). Apart from genetic modification, Cas9 can also modulate transcription without editing the genomic sequence by fusing the enzymatically inactive version of Cas9 (dCas9) with transcriptional repression and activation domains (Gilbert et al., 2013, 2014; Chavez et al., 2015). The former approach, fusion of dCas9 with repression domains (e.g., KRAB/Kox1), has been used to negatively regulate the transcription of specific genomic loci (Gilbert et al., 2013; Leitão et al., 2017). This strategy, commonly termed CRISPR inhibition (CRISPRi), decreases the transcription of target DNA loci mainly by blocking transcriptional elongation, impeding transcription factor binding, or interfering with RNA polymerase transcription initiation (Bikard et al., 2013; Larson et al., 2013; Doudna and Charpentier, 2014; Piatek et al., 2015). Several reports have revealed that this approach can be successfully applied to simultaneously repress the transcription of multiple target genes and that it is reversible (Bikard et al., 2013; Gilbert et al., 2013; Zhao et al., 2014). CRISPR activation (CRISPRa), can be achieved through direct fusing dCas9 with activation domains, such as VP64/p65/HSF1 (Bikard et al., 2013; Gilbert et al., 2014; Konermann et al., 2015; Jia et al., 2018; Strezoska et al., 2020). For example, Cheng et al. (2013) achieved robust endogenous gene activation utilizing VP160 transcriptional activation domain in human and mouse cells. With the intention of enhancing transcriptional activation, Chavez et al. (2015) introduced a unique activation strategy that required the fusion of dCas9 to three activation domains, VP64-p65-Rta (VPR) and thus proved its utility in activating endogenous coding and non-coding genes. CRISPRa also has potential in exploring gene expression in filamentous fungi, particularly, for genes that are closely related to secondary metabolite biosynthesis (Wang and Coleman, 2019). For example, Roux et al. (2020) constructed the first CRISPRa system in A. nidulans by using the same method as Chavez et al. (2015) to improve production of the compound microperfuranone and identify a new metabolite of dehydromicroperfuranone. Filamentous fungi have the capacity to produce a diverse spectrum of valuable secondary metabolites, and the genetic potential of secreting important metabolites has not yet been fully utilized for most SM-BGCs. Therefore, there is little doubt that a strategy that combines the CRISPR/Cas9 technology (i.e., CRISPRa) with the activation of expression of secondary metabolite biosynthetic clusters is on the horizon. This would be particularly useful in developing bioactive products or derivatives for biopharmaceuticals.

In the past few years, the CRISPR/Cas9 system has been introduced into filamentous fungi to explore the potential of this strategy in modulating production of secondary metabolites. From A. oryzae and T. reesei to A. niger and A. nidulans, CRISPR/Cas9 based systems have become versatile platforms for precise genome editing, and great progress has already been made for production of valuable secondary metabolites. Here, we highlight four examples illustrating the application of CRISPR/Cas9 (gene deletion/substitution/insertion) and CRISPRa (gene cluster activation) systems in filamentous fungi.

Filamentous fungi, like other eukaryotes, are extensively used in industrial and pharmaceutical production owing to their ability to secrete a plethora of hydrolytic enzymes, polyunsaturated fatty acids, and, more importantly, bioactive small molecules, including antibiotic agents. Furthermore, filamentous fungi are the most compatible heterologous hosts for expression of compounds such as Penicillium citrinum nuclease P1 in A. niger and fungal BGCs in Fusarium graminearum, owing to their lack of a requirement of intron-removal or codon optimization (He et al., 2018; Chen et al., 2019; Nielsen et al., 2019). With advances in genome sequencing and phylogenetic sleuthing, large repertoires of cryptic or silent secondary metabolite biosynthetic gene clusters are being uncovered. However, the difficulties in genetic manipulation have traditionally impeded secondary metabolite molecular studies on filamentous fungi. Therefore, a powerful and comparatively simple genetic technique for overcoming the obstacle of activating these silent secondary metabolite biosynthetic gene clusters is urgently needed. CRISPR/Cas9 is based on a conservative immune defense mechanism found in bacteria and archaea and has been developed as a convenient and flexible technique for genome editing. The CRISPR/Cas9 genome editing technology has shown great promise in revolutionizing the field of fungal research. The CRISPR/Cas9 genome editing system was first introduced into Saccharomyces cerevisiae (DiCarlo et al., 2013). Subsequently, Liu et al. (2015) applied the CRISPR/Cas9 genome editing system to Trichoderma reesei; Matsu-Ura et al. (2015) and Nodvig et al. (2015) applied this system to the model fungal Neurospora crassa and A. nidulans, respectively. Since then, the CRISPR/Cas9 genome editing system has found wide applications in genetic alteration of many filamentous fungi. These studies strongly confirm that the CRISPR/Cas9 gene-editing system will undoubtedly bring great possibilities for the discovery of new secondary metabolites, on account of its straightforward design, high efficiency, and versatility.

Compared with genome editing approaches that utilize protein-guided programmable nucleases such as ZFN and TALEN-based genome editing tools, nucleic acid-guided nucleases such as that primarily utilized in the CRISPR/Cas9 system have several advantages for genetic engineering to produce secondary metabolites. One advantage is that the CRISPR/Cas9 system can potentially edit almost all genes containing a PAM in their target sequences owing to its simplicity and modularity. Only two components are required in this system: a Cas9 endonuclease and sgRNA (Song et al., 2019). Indeed, there remain challenges and limitations to utilize this approach, such as off-target effects and the need to perform precise editing. As research advances, a well-designed gRNA sequence and specific Cas9 variants can be developed to avoid off-target effects, as can control the amount of intracellular Cas9 or enhance Cas9 for higher specificity through protein engineering (Cho et al., 2014; Tong et al., 2019). For example, Pohl et al. (2016) adopted a strategy for assembling the Cas9-gRNA complex in vitro and co-transformed it with a donor DNA into Penicillium chrysogenum, which proved to be effective in reducing off-target effects. With regard to precise editing, the biggest hurdle is the native NHEJ that intensely affects the efficiency of HDR despite the presence of a homologous template (Tong et al., 2019). By employing the inhibitor Scr7, Maruyama et al. (2015) successfully targeted the DNA ligase IV responsible for the NHEJ pathway, causing suppression of the native NHEJ activity, thus improving the efficiency of precise genome editing. Additionally, several advanced strategies like CRISPRi and FokI-dCas9 have been applied to optimize the Cas9 system to perform more precise gene-editing (Guilinger et al., 2014; Kao and Ng, 2017).

The potential of fungal biosynthetic capabilities in generating secondary metabolites seems virtually unlimited. Researchers have continually explored and developed secondary metabolites from filamentous fungi, with CRISPR/Cas9 technologies now accelerating progress. The powerful advantages of the CRISPR/Cas9 system in studies on filamentous fungal secondary metabolites reduce the application of selective markers. Most secondary metabolites are regulated by gene clusters in filamentous fungi. When three or more genes need to be manipulated at the same time, multiple selection markers were required and thus developed for filamentous fungi. The CRISPR/Cas9 system can edit multiple genes at the same time, and it is possible to obtain mutants with multiple site mutations in a single transformation, which greatly improves the efficiency of genome editing in studies on secondary metabolites of filamentous fungi. However, relatively few reports about the “actual applications” of the CRISPR/Cas9 system in the production of secondary metabolites can be obtained. Thus, the application is still in its initial stages, as most of the research focus is on aspects such as assessing the feasibility of CRISPR/Cas9 systems in fungi (Tong et al., 2019). Apart from CRISPR/Cas9 genome editing strategies, alternative methods, such as CRISPRi and CRISPRa, which do not depend on DSBs, were rarely reported in filamentous fungi, especially with respect to secondary metabolite production. So far, only one application of the CRISPRa technique being used to increase the secondary metabolite contents of a filamentous fungus has been reported (Roux et al., 2020). CRISPRi has not been used in filamentous fungi, but has been successfully applied in other fungi (Roman et al., 2019). In addition, prime editing, an emerging and precise genome editing method that expands the scope of CRISPR genome editing, with few byproducts and without requiring DSBs or donor DNA templates, has attracted great attention (Anzalone et al., 2019). Consequently, this method has potential value in research on secondary metabolites from filamentous fungi. Related technologies and applications of the CRISPR/Cas9 possess great potential and will play a greater role in the discovery of new secondary metabolites and engineering of the strains that produce them in the near future.

In summary, CRISPR/Cas9-based genome editing technology still requires development and improvement in genetic modification of secondary metabolites in filamentous fungi, and the general scope of applications can be expanded further. It is premature to declare that the CRISPR/Cas9 technique is accelerating the metabolic engineering of filamentous fungi for secondary metabolites. However, with the development of full genomes available and metabolomics, knowledge of the secondary metabolite biosynthetic gene clusters of filamentous fungi together with exploitation of CRISPR/Cas9 approaches will help overcome current limitations in increasing production of secondary metabolites. Such advances will also promote the discovery of new bioactive compounds.

GL: conceptualization and writing—original draft preparation. CJ: writing—review and editing and conceptualization. XC and YT: project administration. YD: supervision. BZ: supervision and funding acquisition. BH: writing—review, editing, and funding acquisition. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.