Applications of CRISPR/Cas Gene-Editing Technology in Fungi

Genome editing technology develop fast in recent years. The traditional gene-editing methods, including homologous recombination, zinc nger endonuclease, and transcription activator-like effector nuclease and so on, which have greatly promoted the research of genetics and molecular biology, have gradually showed their limitations such as low eciency, high error rate, and complex design. In 2012,a new gene-editing technology, the CRISPR/Cas9 system, was setup based on the research of the immune responses to viruses from archaea and bacteria. Due to its advantages of high target eciency, simple primer design, and wide application, CRISPR/Cas9 system, whose developers are awared the Nobel Prize in Chemistry this year, has become the dominant genomic editing technology in global academia and some pharmaceuticals. Here we briey introduce the CRISPR/Cas system and its main applications in yeast, lamentous fungi and macrofungi, including single nucleotide, polygene and polyploid editing, yeast chromosome construction, yeast genome and yeast library construction, CRISPRa/CRISPRi-mediated, CRISPR platform of non-traditional yeast and regulation of metabolic pathway, to highlight the possible applications on fungal infection treatment and to promote the transformation and application of the CRISPR/Cas system in fungi. of the endogenous gene. These studies demonstrate that CRISPR/Cas is a powerful tool for fungal genetic screening. CRISPRa/i reversibly regulates the expression of target genes rather than mutation, which reduces errors in gene repair.


Strategies For The Development Of Crispr/cas9 Tools In Various Fungi 2.1 Yeast
Yeast, monocellular eukaryotes, are easy to culture and grow rapidly. Compared with other eukaryotes, yeast have a simpler genetic background. Yeast are also used in industrial fermentation owing to their easy operation and safety. CRISPR technology has been widely used in yeast for the following main objectives: 1) yeast polygene and polyploid knockout, 2) the synthesis of yeast chromosomes, 3) genome-scale engineering and library creation, 4) gene integration, heterologous expression, and metabolic pathway regulation, 5) CRISPR-mediated activation/interactions, 6) non-traditional gene editing and explorations of virulence mechanisms. system in S. cerevisiae. They realized one-step multigene disruption by concatenating different guide RNAs (gRNAs) on a vector, with an e ciency of 27-87%. Mans et al. (2015) transformed three plasmids by the in vitro assembly of plasmids containing two gRNAs, and simultaneously produced six gene deletion strains of S. cerevisiae. The CRISPR/Cas system has also been used for the multiple genome engineering of polyploid industrial yeasts. Zhang et al. (2014) used Cas9 to knock out four genes of S. cerevisiae ATCC 4124 with 15-60% e ciency, and four nutrient-de cient strains were obtained. Lian et al. (2017) expressed gRNA by high-copy plasmids and then transformed the plasmids into a diploid strain (Ethanol Red) and a triploid line (ATCC 4124). Four genes were knocked out in a single step with an e ciency of 100%. Additionally,  found that Cpf1 from Francisella novivida (FnCpf1) can target DNA fragments assembled in vivo for singleplex, doubleplex, and tripleplex genomic integration, with e ciencies of 95%, 52%, and 43%, respectively. Yang et al. (2020) used CRISPR/Cpf1 to target the CAN1 and URA3 genes of Yarrowia lipolytica, obtaining e ciencies of up to 93% and 96%. Zhao  ). The rearrangement of yeast chromosomes clearly establishes the effectiveness of CRISPR/Casbased systems for evaluating the structure and function of genes in eukaryotic evolution.

Genome-scale engineering of yeast and library creation
The programmable control of gene expression is essential for understanding gene function, regulating cell behavior, and developing therapeutic methods. Genetic screening can be used to study the functions of multiple genes simultaneously in a high-throughput manner. CRISPR/Cas9 has been used as a screening strategy to induce mutations and assess gene function. In 2018, Bao et al. (2018) developed CRISPR/Cas9 and homology-directed-repair assisted genome-scale engineering (CHAnGE), which can be used to edit the whole genome of S. cerevisiae at the single nucleotide level. This method can rapidly generate thousands of speci c mutant yeasts. CHAnGE can generate single base pair changes on the whole chromosome and minimize the impact on the function of adjacent genes with unprecedented accuracy. The authors have also established a gene knockout yeast library using the system to improve the production of heterologous natural products. Due to differences in the recognition of promoters among eukaryotic taxa, silent gene clusters cannot be activated when they are directly cloned and transferred into heterologous hosts. The CRISPR/Cas9 system can be used to change the original regulatory elements to achieve the heterologous expression of these genes. Kang et al. (2016) described an improved yeast-based promoter engineering platform (mCRISTAR) that combines CRISPR/Cas9 and the transformation-associated recombination (TAR) (Yamanaka et al. 2014) to replace natural promoters with combinatorial promoters. CRISPR/Cas9 mediates DSBs to form linear DNA fragments at the target promoter and then integrates the homologous arm with biosynthetic gene clusters (BGCs) by homologous recombination in yeast cells. Up to 32 promoters can be inserted into a single natural BGC by four rounds of mCRISTAR using four auxotrophic markers commonly used in yeast, and silent gene clusters can be transcriptionally activated ).

CRISPR activation (CRISPRa)/interaction (CRISPRi) in yeast
Recently, D10A and H804A mutations were introduced into the Ruvc and HNH domains of the Cas9 protein in the CRISPR system to obtain nuclease-de cient Cas9 (dCas9). By fusing dCas9 with different types of transcriptional regulatory domains, such as transcriptional inhibitors, activators, or epigenetic modi cation enzymes, dCas9 proteins can target speci c sites of target genes, resulting in different modes of gene regulation (Larson et 2017) applied the CRISPRi system to Yarrowia lipolytica, designed sgRNAs for the TSS and TATA box of target gene promoter regions, and successfully inhibited 8 of 9 target genes. They also found that dCas9-mxi1 increased the inhibition of KU80 gene transcription from 38% for dCas9 to 87%. Vanegas et al. (2017) combined Cas9 and dCas9 into a SWITCH dynamic CRISPR tool where switching mechanism is based on the recombination of dCas9 after Cas9 is directed to cleave its own gene sequence. The tool enables S. cerevisiae strains to alternate between genetic engineering and metabolic pathway control states, enabling the accurate control of multiple genes of S. cerevisiae. , and multiple transcriptional activation or inhibitory domains and nally constructed a three-phase gene regulatory strategy for S. cerevisiae based on dLbCpf1-VP (CRISPRa), dSpCas9-RD1152 (CRISPRi), and SaCas9 (CRISPRd), known as CRISPR-AID. Using this system, they transformed a single plasmid into yeast to simultaneously induce a 5-fold increase in red uorescent protein, 5-fold inhibition of yellow uorescent protein, and 95% deletion of the endogenous gene. These studies demonstrate that CRISPR/Cas is a powerful tool for fungal genetic screening. CRISPRa/i reversibly regulates the expression of target genes rather than mutation, which reduces errors in gene repair. albicans without stable integration into the genome, addressing the concern that Cas9 may cause long-term adverse effects (such as off-target effects) in the C. albicans genome. Huang et al. (2017) developed a marker recovery strategy using CRISPR/Cas9. Two marker genes can be used to sequentially screen homozygous deletion mutants with three or more genes in the same strain. Nguyen et al. (2017) removed CRISPR and nourseothricin (NAT) markers from the genome by the SAT1 ipper system after determining the target site modi cation of C. albicans, thus allowing the next round of unlabeled genome editing. Grahl  Owing to the limited number of selective marker genes in lamentous fungi and the di culty in multiple rounds of gene manipulation, a single screening marker recycling method in lamentous fungi using the CRISPR/Cas9 system has been established. The self-replicating cas9 plasmid in U. maydis and P. chrysogenum would be lost in the absence of resistance pressure, thus avoiding its in uence on the growth of the strain (Pohl et al. 2016;Schuster et al. 2016). Furthermore, CRISPR/Cas9 has been combined with Cre/loxP to develop a marker-free fungal gene editing system. First, the CRISPR/Cas9 system is used to break a target gene, and the repair template containing the screening marker was integrated into the cleavage site; then, Cre recombinase activated by light illumination could simultaneously delete the selective marker and CRE ). Liu et al. (2019) developed a Vtype CRISPR/Cas12a (AsCpf1) system in M. thermophila. Through three rounds of transformation with two selectable markers, nine genes involved in the cellulase production pathway were targeted. The protein productivity and lignocellulase activity of a mutant (referred to as M9) were 9.0-and 18.5-fold higher than those of the wild type. Cas12a was also used for gene editing in A. nidulans and A. niger (Vanegas et al. 2019).

2.2.2
Interference with metabolic pathways to obtain secondary metabolites and increase yield Some secondary metabolites of microorganisms are important pathogenic factors contributing to human and plant diseases; however, some are also important sources of bioactive substances and drug precursor compounds (Evidente et al. 2014). Filamentous fungi have a strong metabolic capacity and can produce secondary metabolites with diverse structures, such as biocidal agents, drug precursors, and antitumor bioactive substances (Hoffmeister and Keller 2007;Osbourn 2010;). The CRISPR/Cas system can accurately alter gene expression for functional analyses of lamentous fungal metabolic gene clusters, analyses of synthesis and regulatory mechanisms, and the activation of silent gene clusters to interfere with metabolic bypass, which is expected to improve the production and activity of secondary metabolites of lamentous fungi. Kuivanen et al. (2016) combined a transcriptomics approach and CRISPR/Cas technology to delete genes involved in galactaric acid catabolism in A. niger and then heterologously expressed uronate dehydrogenase, yielding a mutant able to convert pectin-rich biomass to galactaric acid in a consolidated bioprocess.
In addition to the conventional gene editing, CRISPR/Cas technology can also knock-in strong promoters or replace promoters upstream of target genes, thus activating silent gene clusters, regulating biosynthetic genes, and synthesizing corresponding metabolites. Matsu-ura et al. (2015) successfully replaced the endogenous promoter of the cellulase related gene CLR-2 with a β-tubulin promoter in N. crassa using the CRISPR/Cas9 system. The mRNA expression of CLR-2 in the mutant strain increased about 200-fold and cellulase production increased signi cantly. Therefore, the CRISPR/Cas system is a powerful synthetic biology tool for the control of the biosynthesis of secondary metabolites in fungi.

Detection And Treatment Of Fungal Infections
Fungal infections are a serious threat to the health of humans, animals, plants, and ecosystems (Fisher et al. 2012). Every year, deaths caused by fungal infections in humans exceed those caused by tuberculosis or malaria (Brown et al. 2012). Diseases in food crops caused by fungi and decreased yields have become a global food security problem (Bebber et al. 2013). Analyses of the pathogenic mechanism of fungi and improvements in diagnostic and antifungal strategies are urgently needed. CRISPR/Cas technology is expected to revolutionize research on pathogenic fungi in humans and plants.
CRISPR/Cas may be used for the rapid detection and identi cation of fungi, which is conducive to the early control of fungal infections. Additionally, it can be applied to target virulence genes, while maintaining the viability of fungi, thus preventing infection and avoiding the destruction of the normal ora (Fig 3). It is also a promising method to eliminate drug-resistant genes ( These studies demonstrate that the CRISPR/Cas system can be used to precisely destroy pathogenic genes in harmful lamentous fungi, providing a new strategy for the prevention and control of fungal diseases. The CRISPR/Cas system can also be introduced into human pathogenic fungi for targeted intervention. However, methods to transfer gRNAs and Cas proteins with different targets safely and effectively in vivo are still needed. At present, delivery methods include physical, viral vector, and non-viral vector methods  ). Viral vectors, including adenoviruses and adeno-associated viruses and lentiviruses, can insert genes encoding Cas9 and sgRNA into a single vector for delivery, although concerns about potential carcinogenesis and immunogenicity remain (Li et al. 2015). Non-viral vectors, including lipid nanoparticles and inorganic nanoparticles, have broad potential application because they are relatively safe and easy to package (Li et al. 2015). There is no doubt that CRISPR/Cas technology will promote explorations of the evolution of fungal virulence and host-pathogen interactions and contribute to the development of accurate diagnostic tools and new antifungal drugs.

Conclusion And Prospects
CRISPR/Cas is revolutionizing fungal genetic research. In addition to gene knockouts induced by CRISPR/Cas nuclease, a series of distinct optimizations and combinations have been reported, such as CRISPR/Cas9-TAR, CRISPRa/CRISPRi, and CRISPR-Cre recombinase for accurate genome modi cations.
These tools have unparalleled gene editing ability and are expected to initiate a revolution in fungal research based on CRISPR technology. However, these multi-functional tools still need to be optimized for fungal genome modi cation. For example, the gene editing e ciency is still very low in macrofungi, and gene manipulation vectors need to be further simpli ed in yeast and lamentous fungi to improve the knockout e ciency of multiple genes. Using CRISPR technology to target drug resistance genes may be a weapon against fungal drug resistance. For the development of drugs against pathogenic fungi, it is necessary to accurately study the fungal genome, and methods to reduce off-target effects are needed.  The main application of CRISPR/Cas gene-editing technology in various fungi  Detect and treat fungal infections. (a) CRISPR used for rapid detection and identi cation of fungi. The dCas-sgRNA complex with signal tags can identify and combine the speci c sequences of fungal virulence or drug resistance genes in pathogenic microorganisms to be tested, so that fungi can be rapidly detected and identi ed by detecting signals. (b) CRISPR to treat fungal infections or eliminate drug-resistant fungi. The Cas-sgRNA complex identi es and binds virulence genes of fungi, aiming to transform highly virulent pathogenic fungi into non-pathogenic fungi, or target drug-resistant genes to restore the susceptibility of fungi to drugs, or even target some essential genes to kill pathogenic fungi directly.