Two CRISPR/Cas9 Systems Developed in Thermomyces dupontii and Characterization of Key Gene Functions in Thermolide Biosynthesis and Fungal Adaptation

Wei-Ping Huang; Yuan-Jiang Du; Yun Yang; Jia-Ning He; Qian Lei; Xiao-Yu Yang; Ke-Qin Zhang; Xue-Mei Niu

doi:10.1128/AEM.01486-20

. 2020 Oct 1;86(20):e01486-20. doi: 10.1128/AEM.01486-20

Two CRISPR/Cas9 Systems Developed in Thermomyces dupontii and Characterization of Key Gene Functions in Thermolide Biosynthesis and Fungal Adaptation

Wei-Ping Huang ^a, Yuan-Jiang Du ^a, Yun Yang ^a, Jia-Ning He ^a, Qian Lei ^a, Xiao-Yu Yang ^a, Ke-Qin Zhang ^a, Xue-Mei Niu ^a,^✉

Editor: Harold L Drake^b

PMCID: PMC7531979 PMID: 32769197

Thermomyces represents a unique ecological taxon in fungi, but a lack of flexible genetic tools has greatly hampered the study of gene function in this taxon. The biosynthesis of potent nematicidal thermolides in T. dupontii remains largely unknown. In this study, mesophilic and thermophilic CRISPR/Cas9 gene editing systems were successfully established for both disrupting and activating genes in T. dupontii. In this study, a usable thermophilic CRISPR/Cas9 gene editing system derived from bacteria was constructed in thermophilic fungi. Chemical analysis of the mutants generated by these two gene editing systems identified the key biosynthetic genes and pathway for the biosynthesis of nematocidal thermolides in T. dupontii. Phenotype analysis and chemical stress experiments revealed potential roles of secondary metabolites or their biosynthetic genes in fungal development and adaption to chemical stress conditions. These two genomic editing systems will not only accelerate investigations into the biosynthetic mechanisms of unique natural products and functions of cryptic genes in T. dupontii but also offer an example for setting up CRISPR/Cas9 systems in other thermophilic fungi.

KEYWORDS: CRISPR/Cas9, Thermomyces dupontii, biosynthetic pathway, functions, gene editing, thermolides, thermophilic fungi

ABSTRACT

Thermomyces dupontii, a widely distributed thermophilic fungus, is an ideal organism for investigating the mechanism of thermophilic fungal adaptation to diverse environments. However, genetic analysis of this fungus is hindered by a lack of available and efficient gene-manipulating tools. In this study, two different Cas9 proteins from mesophilic and thermophilic bacteria, with in vivo expression of a single guide RNA (sgRNA) under the control of tRNA^Gly, were successfully adapted for genome editing in T. dupontii. We demonstrated the feasibility of applying these two gene editing systems to edit one or two genes in T. dupontii. The mesophilic CRISPR/Cas9 system displayed higher editing efficiency (50 to 86%) than the thermophilic CRISPR/Cas9 system (40 to 67%). However, the thermophilic CRISPR/Cas9 system was much less time-consuming than the mesophilic CRISPR/Cas9 system. Combining the CRISPR/Cas9 systems with homologous recombination, a constitutive promoter was precisely knocked in to activate a silent polyketide synthase-nonribosomal peptide synthase (PKS-NRPS) biosynthetic gene, leading to the production of extra metabolites that did not exist in the parental strains. Metabolic analysis of the generated biosynthetic gene mutants suggested that a key biosynthetic pathway existed for the biosynthesis of thermolides in T. dupontii, with the last two steps being different from those in the heterologous host Aspergillus. Further analysis suggested that these biosynthetic genes might be involved in fungal mycelial growth, conidiation, and spore germination, as well as in fungal adaptation to osmotic, oxidative, and cell wall-perturbing agents.

IMPORTANCE Thermomyces represents a unique ecological taxon in fungi, but a lack of flexible genetic tools has greatly hampered the study of gene function in this taxon. The biosynthesis of potent nematicidal thermolides in T. dupontii remains largely unknown. In this study, mesophilic and thermophilic CRISPR/Cas9 gene editing systems were successfully established for both disrupting and activating genes in T. dupontii. In this study, a usable thermophilic CRISPR/Cas9 gene editing system derived from bacteria was constructed in thermophilic fungi. Chemical analysis of the mutants generated by these two gene editing systems identified the key biosynthetic genes and pathway for the biosynthesis of nematocidal thermolides in T. dupontii. Phenotype analysis and chemical stress experiments revealed potential roles of secondary metabolites or their biosynthetic genes in fungal development and adaption to chemical stress conditions. These two genomic editing systems will not only accelerate investigations into the biosynthetic mechanisms of unique natural products and functions of cryptic genes in T. dupontii but also offer an example for setting up CRISPR/Cas9 systems in other thermophilic fungi.

INTRODUCTION

Thermophilic fungi are the only eukaryotes that possess the exceptional ability to grow at the high temperatures of 50° to 60°C, often as the chief components of the mycoflora in diverse high-temperature environments (1). Due to their capability to survive and reproduce in high-temperature habitats, thermophilic fungi represent a unique reservoir of valuable thermophilic biocatalysts and biologically active compounds for industrial applications (2).

Thermomyces dupontii Griffon & Maubl. (formerly known as Talaromyces thermophilus) that has an optimal growth temperature of 45 to 50°C is a widely distributed thermophilic fungus in hot springs, composts, and geothermal soil (1). Comparative genomic analysis revealed that the T. dupontii genome contained only 18.9 Mb of assembled DNA sequence with 7,940 predicted genes (3), a genome size much smaller than the genome sizes of industrial enzyme producers Rhizomucor miehei (27.6 Mb) and Malbranchea cinnamomea (25 Mb) (4, 5) and only about half the size of the best-characterized thermophilic fungi, Myceliophthora thermophila (36.9 Mb) and Thielavia terrestris (38.7 Mb) (6). Phylogenetic analyses showed that T. dupontii and its sister thermophilic fungus Thermomyces lanuginosus formed a monophyletic group, nesting in Eurotiales, which includes mesophilic fungi Aspergillus and Penicillium (7). It is interesting that T. dupontii also has a much smaller genome than its closest mesophilic relatives in Aspergillus and Penicillium (36 to 45 Mb) (8, 9). The small genome size has led to the hypothesis that T. dupontii contains the core genes for the growth and reproduction of fungi in elevated temperature environments, thus rendering T. dupontii an ideal material for investigating the underlying evolution mechanism of thermophilic fungi and potential novel host development for future biotechnological use (10, 11).

Recently, we purified a unique class of polyketide synthase-nonribosomal peptide synthase (PKS-NRPS) hybrid macrolides (called thermolides), with potent nematocidal activities from T. dupontii (10). Interestingly, the structural features of these metabolites are more reminiscent of those from a bacterial origin, as opposed to a fungal origin (10). The homologous recombination (HR)-based genetic manipulating system that was recently developed by our group was used for the sequenced T. dupontii strain NRRL 2155 and was applied for constructing gene deletion strains (3, 11). However, this approach of using homologous integration with flanking regions longer than 1,500 bp is technically complicated and time intensive, with a success rate of less than 20%. Specifically, double and multiple gene manipulation in T. dupontii remains a challenge at present due to there being only one selectable marker available for T. dupontii (11). Therefore, the lack of versatile genetic manipulation tools for T. dupontii has impeded the study of its gene function. To circumnavigate this obstacle, heterologous expression and in vitro experiments were applied for elucidating the biosynthesis of thermolides (12). However, heterologous hosts may lack the correct precursors, intermediates, and/or auxiliary enzymes of a specific biosynthetic pathway. Furthermore, in vivo evidence is necessary for better understanding the native biosynthetic pathway and natural functions of fungal secondary metabolites in T. dupontii. For these reasons, flexible genetic manipulation tools are vital for the molecular study of T. dupontii.

The CRISPR/Cas9 system for gene editing has become popular for genetic function study through gene disruption, activation, or suppression (13 –19). In the CRISPR/Cas9 system, a single guide RNA (sgRNA) containing a spacer sequence complementary to the protospacer of the target DNA is required to lead nuclease Cas9 and cleave the target sequences upstream of the protospacer-adjacent motif (PAM) (13 –19). An error-prone nonhomologous end joining (NHEJ) or high-fidelity homology-directed repair (HDR) strategy would then be recruited to repair the double-stranded DNA breaks (DSBs), leading to a nonsense gene mutation or gene disruption (13 –19).

In an effective CRISPR/Cas9 system, the precise expression of both Cas9 and sgRNA is vital for successful gene editing. Generally, three different processes have been employed for sgRNA expression in various types of fungi. First, an RNA polymerase II promoter in combination with the hammerhead ribozyme sequences of hepatitis delta virus (here termed HH-HDV) was successfully introduced into the fungi Phytophthora sojae (20) and Blastomyces dermatitidis (21). The second process introduced an RNA polymerase III (RNAP III) promoter such as U6 in Aspergillus fumigatus (22), Myceliophthora thermophila (23), and Yarrowia lipolytica (24). The third process used a tRNA^Gly self-processing system in the fungus Aspergillus nidulans (25). Currently, the Cas9 from mesophilic bacterium Streptococcus pyogenes (SpCas9) is the most widely used nuclease in CRISPR/Cas9 gene editing systems as it has high efficiency for many mesophilic fungi (20, 21, 26 –31), including human-pathogenic fungi Blastomyces dermatitidis (21, 31) and Aspergillus fumigatus (22), plant-pathogenic Phytophthora sojae (20), and the industrial filamentous fungi Trichoderma reesei (26) and Talaromyces atroroseus (27). In 2017, two thermostable Cas9 nucleases from the thermophilic bacteria Geobacillus stearothermophilus (GeoCas9) and Geobacillus thermodenitrificans (ThermoCas9) were reported to display high genome editing efficiencies at elevated temperatures as high as 70°C (32, 33). Furthermore, the GeoCas9 nuclease showed efficient gene editing activity in mesophilic eukaryotic cells (32). Although the mesophilic CRISPR/Cas9 system succeeded in editing the genome of thermophilic fungi Myceliophthora thermophila and Myceliophthora heterothallica at 35°C (23), the working temperature was 10 to 15°C lower than the optimal growth temperature of M. thermophila. Whether the mesophilic CRISPR/Cas9 system could also work in thermophilic fungi at their optimal growth temperatures (i.e., >37°C) still remained unknown.

In this work, we succeeded in editing the genome of T. dupontii with both mesophilic SpCas9 and thermophilic GeoCas9. Mesophilic SpCas9 displayed genomic editing activity in T. dupontii at a growth temperature of 37°C but not at the optimal fungal temperature of 45°C. On the other hand, GeoCas9 could generate gene editing in T. dupontii at 45°C. Simultaneous CRISPR/Cas9-mediated two-locus targeting and HDR were also successful in this fungus. A silent PKS-NRPS hybrid gene of T. dupontii was also activated by the CRISPR/Cas9-mediated HDR, which had much shorter homologous templates. Our results indicated that CRISPR/Cas9 systems were useful not only in identifying native biosynthetic pathways of secondary metabolites such as thermolides in T. dupontii but also in exploring the natural products of thermophilic fungi. Furthermore, our results showed that these secondary metabolites and biosynthetic genes might play a crucial function in fungal morphology and its adaptation to chemical stress. The systems developed here should provide a more detailed and thorough understanding of the biosynthetic gene functions in fungi.

RESULTS

Expression and localization of mesophilic SpCas9 in T. dupontii.

To test whether the mesophilic CRISPR/Cas9 system could work with our thermophilic fungus, the Spcas9 expression plasmid p-trpC-NLS-coSpcas9-NLS (Fig. 1A), which was composed of a selective marker hygromycin (HygB) and the codon-optimized Spcas9 gene (coSpcas9) with nuclear localization signal sequences (NLS) under the control of fungal constitutive promoter trpC, was constructed and introduced into T. dupontii NRRL 2155. Phenotype analysis of the resulting mutants at 45°C revealed that their morphology and growth were indistinguishable from those of the wild type (WT) (Fig. 1B and C). The Western blot experiment detected the expression of SpCas9 in T. dupontii grown at both 45°C and 37°C (Fig. 1D). In order to test whether the NLS could guide SpCas9 into the fungal nucleus, a second plasmid with an enhanced green fluorescent protein (eGFP) gene fused to the coSpcas9 open reading frame (ORF) in p-trpC-NLS-coSpcas9-eGFP-NLS was constructed and transformed into T. dupontii (Fig. 1A). Microscopic analysis confirmed the nuclear localization of SpCas9 in transformed strains (Fig. 1E).

FIG 1 — Expression of fungal codon-optimized *Spcas9* gene (*coSpcas9*) in *T. dupontii*. (A) Schematic illustrating the SpCas9 expression plasmids. The red boxes represent the nuclear localization signals (NLS). (B and C) Colony phenotype and growth rates of the SpCas9 expression strain (*coSpcas9* strain) and wild-type (WT) strain on PDA medium at 45°C for 8 days. (D) Western blot results of the expression of SpCas9 in *T. dupontii* at both 37°C and 45°C. (E) Fluorescence microscopic assessment of SpCas9 localization in the coSpCas9-eGFP expression strain grown at 45°C. Nuclei were stained with 4′,6′-diamidino-2-phenylindole (DAPI).

Expression of sgRNA in T. dupontii.

Three sgRNA expression systems (Fig. 2A), HH-HDV, U6, and tRNA^Gly, were evaluated for the expression of sgRNA in T. dupontii. Bioinformatic analysis showed that there were two native U6 systems, U6₁ and U6₂, and one tRNA^Gly system containing a promoter ptRNA^Gly in T. dupontii (see Materials and Methods and Fig. S1 in the supplemental material).

A single PKS gene, PKS1 (Talth1p4_003117), and an aldehyde dehydrogenase (AD) gene (Talth1p4_005402) in different biosynthetic clusters of T. dupontii were selected as target genes for editing (Fig. 2B). The protospacer and the corresponding PAM sequences for each sgRNA were chosen from the 5′ part of the putative catalytic domain in each target gene and are listed in Table S1. BLASTN analysis was performed to guarantee the uniqueness of every protospacer in the T. dupontii genome. Eight different sgRNA expression plasmids were constructed and transformed into T. dupontii protoplasts (Fig. 2B; also Materials and Methods). Reverse transcription-PCR (RT-PCR) analysis of the transformants demonstrated that all of the target sgRNAs were successfully transcribed in both HH-HDV and tRNA^Gly groups (Fig. 2B). Meanwhile, no sgRNA transcriptions were identified in U6₁, U6₂, or WT groups (Fig. 2B). These results suggested that HH-HDV and tRNA^Gly were feasible for expressing sgRNA in T. dupontii but that the U6 groups were not.

CRISPR/SpCas9 gene editing in T. dupontii.

An SpCas9 expression plasmid and sgRNA expression plasmid (HH-HDV and tRNA^Gly) were cotransformed into T. dupontii protoplasts to target the AD gene for editing. Transformed protoplasts were then cultivated at 45°C on plates containing hygromycin. PCR analysis demonstrated that all of the randomly picked transformants harbored both SpCas9 and sgRNA expression cassettes. Although both SpCas9 and sgRNA were successfully integrated into the fungal genome, no AD gene editing mutants were detected in the HH-HDV/SpCas9 or tRNA^Gly/SpCas9 group.

Previous studies reported that SpCas9 had maximum nuclease activity at 37°C but that elevated temperature (i.e., ≥42°C) would impair and even stop its activity (33 –35). Presumably, the optimum cultivation temperature of 45°C of T. dupontii might cause SpCas9 to lose its nuclease activity. Therefore, the temperature for the editing experiments with SpCas9 was adjusted to 37°C even though the lower temperature greatly slowed fungal growth. Intriguingly, when T. dupontii was grown at 37°C, 86% (12/14) of randomly picked transformants in the tRNA^Gly/SpCas9 group displayed AD gene mutagenesis at the expected locus (Table 1 and Fig. S2A), and 5% displayed AD gene mutagenesis (1/20) in the HH-HDV/SpCas9 group (Table 1 and Fig. S2B). Most AD gene mutants contained deletions from 1 bp to 283 bp (Table 1 and Fig. S2).

TABLE 1.

The effect of Cas9/tRNA^Gly gene editing in T. dupontii

Cas9 type	Target locus	Mutation(s) (no. of events)^a	Efficiency (%)^b
SpCas9	AD	del C (10), ins C (1), del 283 bp (1)	12/14 (86)
	PKS1-1	del G (9), del TGCGCGC (1), ins ∼2,000 bp (1)	11/14 (79)
	PKS1-2	del C (2), ins C (2), ins CC (1), ins ∼149 bp (1), del 13 bp (1)	7/10 (70)
	HLHTF1	del A (2), ins A (1), del AAGAGG (1), ins GAG (1)	5/10 (50)
	HLHTF2	del A (1), ins T (1), del CCAACAA (1)	3/5 (60)
	AD PKS1	del C in AD + del G PKS1 (1), del C in AD + del G and ins ∼2,000 bp in PKS1 (1), ins ∼4,000 bp in AD + del G in PKS1 (1)	3/10 (30)
GeoCas9	AD	del C (5), ins C (1), G to A (1), ins ∼1,500 bp (1)	8/12 (67)
	HLHTF3	del 14 bp (2), del CCGGCGG (1), del CC +ins ∼5,000 bp (1), ins ∼5,000 bp (1)	5/10 (50)
	HLHTF4	ins ∼700–2,000 bp (4)	4/10 (40)
	SDR	del A (2), ins C (1), del ∼800 bp (1)	4/7 (57)

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del, deletion; ins, insertion. The number of the same type of editing event is indicated in parentheses. For the ΔAD ΔPKS1 mutants, dual gene editing events are shown for each mutant.

Gene editing efficiencies were calculated only for homologous mutation events. Values are for single-gene mutants except for the values for the ΔAD ΔPKS1 dual-gene mutants.

For further comparison of the gene editing efficiencies of tRNA^Gly/SpCas9 and HH-HDV/SpCas9, PKS1 was selected as the second target gene for the editing experiments. Two different sgRNAs, sgRNA_PKS1-1 and sgRNA_PKS1-2, were designed, respectively, from the keto-synthase (KS) and enoyl reductase (ER) domains in the PKS1 gene (Fig. 3A). Four sgRNA expression plasmids were constructed and cotransformed with an SpCas9 plasmid into T. dupontii protoplasts. Our results (Fig. 3B) showed that in the KS domain- targeting experiment, 11 of the 14 transformants (79%) exhibited KS domain editing evidence in the tRNA^Gly/SpCas9 group (Table 1 and Fig. S3A), while in the HH-HDV/SpCas9 group, only 1 of the 15 transformants (7%) displayed KS domain editing events. These KS domain editing events exhibited multiple sequence peaks within the expected editing region (Fig. S3B). In the ER domain-targeting experiment, 70% of transformants (7/10) displayed mutational ER events in the tRNA^Gly/SpCas9 group (Table 1 and Fig. S3C), while no ER editing event occurred in the HH-HDV/SpCas9 group (Fig. 3B). The most common gene editing patterns in the PKS1 mutants were short indels (Fig. S3). Large fragment insertions (up to 2,000 bp) were also identified in some mutants at a relatively low frequency (Table 1). Sanger sequencing analysis showed that these large inserted sequences were derived from expression plasmids. Together with the AD gene-targeting experiment (Fig. 3B), the tRNA^Gly strategy achieved significantly high efficiency (>50%) in all of the SpCas9-mediated gene editing experiments while the HH-HDV strategy failed in T. dupontii.

FIG 3 — (A) Schematic illustration of the target *PKS1* gene cluster and the two protospacer sites of the KS and ER domains in *PKS1*. KS, keto-synthase domain; AT, acyltransferase domain; DH, dehydratase domain; MT, methyltransferase domain; ER, enoyl reductase domain; KR, keto-reductase domain; PP, phosphopantetheine binding site. (B and C) Comparison of the gene editing frequencies of the SpCas9-mediated system and GeoCas9/tRNA^Gly system, as indicated, in *T. dupontii*. tRNA, tRNA^Gly.

From the previously mentioned AD gene and PKS1 editing experiments, the tRNA^Gly system for expressing sgRNA was clearly more feasible than the HH-HDV system for SpCas9-mediated gene editing in T. dupontii (Fig. 3B). Moreover, the tRNA^Gly/SpCas9 gene editing system displayed a much higher efficiency than traditional HR (86% to 70% versus <20%) (10, 11). To further assess the application of tRNA^Gly/SpCas9 in T. dupontii genome editing, two basic helix-loop-helix transcription factors (HLHTF), HLHTF1 (Talth1p4_003063) and HLHTF2 (Talth1p4_006974) (36), were selected for gene editing. The gene editing efficiencies of the tRNA^Gly/SpCas9 system for HLHTF1 and HLHTF2 were 50% and 60%, respectively, and short indels were observed in all the mutants (Fig. 3B; Table 1; Fig. S4).

Double-locus editing in T. dupontii via a tandem array in a single architecture.

In order to assess the multiplex genome editing ability of the tRNA^Gly/SpCas9 system in T. dupontii, both PKS1 and the AD gene were used for double gene editing experiments. A tandem arrangement of tRNA^Gly-sgRNA in a single architecture, which consisted of tandem repeats of tRNA^Gly-gRNA under the control of one tRNA^Gly promoter (Fig. 2A), was designed and constructed according to a previous report (25). This dual-sgRNA expression plasmid p-PtRNA^Gly-tRNA^Gly-sgRNA_AD-tRNA^Gly-sgRNA_PKS1 was cotransformed with an SpCas9 expression vector into T. dupontii. Sanger sequencing results revealed that 3 of the 10 (30%) transformants were AD gene and PKS1 double mutants (Fig. 3B and Table 1).

Activation of a silent gene via tRNA^Gly/SpCas9-meditated HR in T. dupontii.

Our previous study revealed that only the PKS-NRPS hybrid gene (PNH in T. dupontii remained silent under laboratory conditions (11). In order to figure out whether this cryptic gene could be capable of natural product biosynthesis under laboratory conditions, the induction of PNH expression with insertion of the constitutive promoter trpC upstream via tRNA^Gly/SpCas9-mediated HR in T. dupontii was performed (Fig. 4). An sgRNA expression plasmid (p-coSpcas9-tRNA^Gly-sgRNA) was constructed, and a donor plasmid, p-trpC-donor (Fig. 4A), containing the promoter trpC flanked with 420-bp and 430-bp homology sequences from the PNH gene and its upstream region were created. These two plasmids were cotransformed with the SpCas9 expression plasmid into T. dupontii. Among the 15 candidate transformants, 12 strains (80%) were detected to have the promoter trpC gene inserted upstream of PNH (Fig. 4B), while no mutant was found in the control group that was transformed with only the p-trpC-donor. Sanger sequencing analysis confirmed the insertion of a trpC promoter upstream of the ATG start codon of PNH (Fig. S5) in the mutant overexpressing PNH (PNH-OE mutant). Further RT-PCR analysis showed a high transcription level of PNH in the PNH-OE mutant (Fig. 4B), suggesting that the insertion of a trpC promoter induced in vivo expression of PNH. High-performance liquid chromatography mass spectrometry (HPLC-MS) analysis of the culture broths revealed that in comparison with WT, the PNH-OE mutant exhibited extra metabolites (Fig. 4C) with UV absorption similar to that of yellowish fusarin C (12) at a retention time (RT) of 10 to 12 min (424 m/z under positive mode) (Fig. 4D).

FIG 4 — Production of extra metabolites in a *T. dupontii* mutant with activation of the hybrid *PKS-NRPS* gene *PHN* (*PNH*-OE). (A) Schematic illustration of the SpCas9/tRNA^Gly-mediated insertion of the *trpC* promoter upstream of the gene *PNH*. Primers are indicated. (B) Verification of the *PNH* strain and putative *PNH*-OE mutants as determined by RT-PCR. M, molecular size marker. (C) HPLC-MS profiles of methanol extracts from WT and PNH-OE strains under positive mode. The target peaks are highlighted in the dashed box. (D) Extracted ion chromatography (EIC) of the target peaks at *m/z* 424.

Thermostable CRISPR/Cas9 for gene editing in T. dupontii at 45°C.

As mentioned previously, the success of mesophilic SpCas9-mediated genome editing in T. dupontii requires an experimental temperature of no more than 37°C. A lower temperature greatly decreases fungal growth and makes the experimental process considerably longer. Furthermore, we tried to evaluate whether the latest bacterial thermostable CRISPR/Cas9 system could be functionally developed in thermophilic fungi at the optimum growth temperature of 45°C. One plasmid p-trpC-NLS-coGeocas9-NLS carrying the fungal codon-optimized Geocas9 (coGeocas9) from the thermophilic bacterium G. stearothermophilus was constructed according to a method similar to that used for the SpCas9 expression plasmid (Fig. 1A). In order to compare SpCas9 and GeoCas9, the same AD gene was also selected as the target gene for the GeoCas9 experiment. Previous studies suggested that GeoCas9 preferred to use NNNNCGAA as the PAM sequence (32, 33). For that reason, the previously mentioned protospacer sequence that was used as a guide sequence for AD gene editing in the SpCas9 experiment had to be modified for the GeoCas9 experiment by extending two more residues at the 3′ end (Table S1 and Fig. S6). The tRNA^Gly-sgRNA expression plasmid for the GeoCas9 experiment was constructed with using a method similar to that of the SpCas9 experiment and was cotransformed with the GeoCas9 expression plasmid into T. dupontii WT. Transformed protoplasts were then cultivated at 45°C for 7 to 9 days. PCR and sequencing analysis revealed that 8 of the 12 transformants (67%) displayed gene editing events in the GeoCas9-mediated AD gene editing experiment (Table 1 and Fig. 3C), a result that is slightly lower than that of SpCas9. Similar to the SpCas9 experimental results, short indels were the most commonly found mutations in the group of GeoCas9-generated AD gene mutants (6/8) (Table 1).

In order to further evaluate the application of the tRNA^Gly/GeoCas9 genome editing system in T. dupontii, three additional genes, including two HLH-type genes, HLHTF3 (Talth1p4_001383) and HLHTF4 (Talth1p4_005393), and one short-chain dehydrogenase (SDR) gene (Talth1p4_003119) in the PKS1 gene cluster (Fig. 3C) were chosen as target genes for editing. Our results showed that gene editing efficiencies for HLHTF3, HLHTF4, and SDR were 50%, 40%, and 57%, respectively (Table 1 and Fig. 3C). Short indels, large sequence insertions (700 to 5,000 bp), and deletions (∼300 bp) were all observed in these gene editing mutants (Table 1; Fig. S4), suggesting that the gene editing results from the thermophilic GeoCas9 were similar to those from the mesophilic SpCas9. Despite this, the GeoCas9-mediated gene editing system was a more efficient mutagenesis tool for T. dupontii.

Elucidation of the key biosynthetic step for production of potent nematocidal thermolides in T. dupontii.

T. dupontii can produce a unique class of PKS-NRPS hybrid thermolides with structural features more reminiscent of those from bacterial origin, as opposed to those of fungi. Among them, the thermolides A to C (compounds 1 to 3) were acetyl derivatives of thermolide D (4), and thermolides A and B (compounds 1 and 2) showed potent nematocidal activities (10). A recent study reported that through heterologous expression methods, four genes in the PKS1 gene cluster, including the NRPS, PKS1, and SDR genes and an acetyltransferase (AT) gene (Talth1p4_003115), were characterized to be responsible for the biosynthesis of thermolides in the heterologous host Aspergillus (12). However, heterologous reconstitution of the biosynthetic pathway often was not able to elucidate the original biosynthetic pathway in the natural host, and the final two steps for biosynthesis of thermolides A to C (compounds 1 to 3) in the heterologous host Aspergillus still remained questionable (12). Furthermore, was the leftover monooxygenase (MO) gene (Talth1p4_003120) in the PKS1 gene cluster involved in the biosynthesis of thermolides in T. dupontii? To clarify the original key biosynthetic pathway of compounds 1 to 3 in the native host of T. dupontii, we deleted the core genes in the PKS1 cluster, including the SDR and AT genes that were, respectively, involved in the last two steps for thermolide biosynthesis, and the MO gene. Through the above-mentioned tRNA^Gly/GeoCas9 method, the constructions of ΔSDR, ΔAT, and ΔMO mutants were successfully achieved. HPLC-MS analysis was applied to investigate the metabolic profiles of all of the mutants related to the biosynthesis of thermolides. Compared to the WT, all of the ΔPKS1 and ΔAD ΔPKS1 mutants with the disruption of the PKS1 gene lacked production of thermolides A to E (compounds 1 to 5) (Fig. 5A). This confirms that PKS1 is involved in the biosynthesis of the backbones of thermolides. The result showed that all of the thermolides were detected in the ΔMO mutant (Fig. 5A), suggesting that the MO gene does not participate in thermolide production.

However, it was interesting that the ΔSDR mutant also stopped the production of all thermolides (Fig. 5A). In addition to this, the ΔAT mutant caused the production of only thermolides 4 and 5 without thermolides A to C (compounds 1 to 3) (Fig. 5A). These results suggested that the AT gene was responsible for the last step in the biosynthesis of thermolides A to C (compounds 1 to 3) in the native host T. dupontii (Fig. 5B). This was in sharp contrast to the result from the heterologous expression study stating that SDR, not the AT gene, was responsible for the last step in the biosynthesis of thermolides A to C (compounds 1 to 3) in the heterologous host Aspergillus (Fig. 5C).

Influence of biosynthetic genes on fungal growth, conidiation, and conidial germination.

In order to evaluate the influence of the previously mentioned biosynthetic genes in this study on fungal phenotype and development, all of the mutants derived from biosynthetic gene editing were compared with the WT on tryptone, yeast extract, glucose, and agar (TYGA), yeast-mannitol agar (YMA), and potato dextrose agar (PDA) at 45°C. The PNH-OE mutant displayed a dark color on YMA (Fig. 6), which was consistent with its production of the extra yellow metabolites. On PDA, all mutants with the exception of the ΔSDR mutant exhibited slightly slowed colony growth (≤12%). Meanwhile, on both TYGA and YMA all of the mutants with the exception of the ΔAD mutant displayed slightly faster growth than the WT, with the ΔPKS1 mutant showing up to a 28% increase in growth rate (Fig. 6A). Microscopy analysis revealed that ΔAD, ΔAD ΔPKS1, and ΔMO mutants displayed abnormally shaped hypha and strange tesla coils, whereas the ΔAT mutant yielded cells that were far more swollen than those of the WT (Fig. 6).

In comparison to the WT, the PNH-OE, ΔAD, and ΔPKS1 mutants all displayed significantly increased conidium production, with up to 31%, 78%, and 54% increases, respectively (Fig. 6B). However, the ΔAD ΔPKS1, ΔMO, ΔSDR, and ΔAT mutants all exhibited drastically reduced conidium production by up to 70%, 73%, 96%, and 83%, respectively (Fig. 6B). It was very surprising that disruption of the PKS1 or AD gene could increase the rate of conidiation, while the dual disruption of both the PKS1 and AD genes had the opposite effect (Fig. 6B).

Within 12 h, conidial germination rates of ΔPKS1 (53.1%), PNH-OE (48.4%), and ΔSDR (33%) mutants were 2.7, 2.5, and 1.7 times the rate of the WT (19.1%) (Fig. 6C). On the other hand, conidial germination rates of the ΔAD ΔPKS1 (9.7%) and ΔAT (14.4%) mutants were only 50% and 75% that of WT (Fig. 6C).

Contributions of biosynthetic genes to chemical stress responses.

To assess the involvement of the biosynthetic genes in fungal responses to chemical stress, all of the biosynthetic gene mutants were compared with the WT grown on PDA medium with three types of chemical agents. These chemical agents included the osmotic agent NaCl, the cell wall-perturbing agent Congo red, and the oxidant H₂O₂. Our results showed that all of the strains were inhibited by these three stress chemicals, and several distinguishable phenotypes for mutants were observed: (i) the PNH-OE strain appeared yellowish brown when it was exposed to 0.6 M NaCl (Fig. 7); (ii) all of the mutants with the exception of the ΔSDR mutant displayed a higher growth rate than the WT when exposed to 0.3 to 0.9 M NaCl (Fig. 7A), and among the mutants, the ΔMO mutant increased its growth rate by 25% to 57%; (iii) when exposed to 0.1 to 0.3 mM Congo red, the ΔAD and ΔAD ΔPKS1 mutants displayed increases in colony growth rates by up to 118% and 54%, respectively (Fig. 7B); (iv) when exposed to 10 to 20 mM H₂O_2, the ΔMO and ΔSDR mutants increased fungal growth by up to 20%, while the PNH-OE strain had a decrease in growth rate by up to 18 to 25% in comparison to the WT level (Fig. 7C).

FIG 7 — Comparison of the tolerances to osmotic agent NaCl, the cell wall-perturbing agent Congo red, and the oxidant H₂O₂ between WT and mutant lines. Colony morphologies and relative colony growth rates of the WT and mutants were analyzed following a 6-day incubation at 45°C on PDA supplemented with NaCl (A), Congo red (B), or H₂O₂ (C) at the indicated concentrations. WT, wild-type; PN, *PNH*-OE mutant; AD, ΔAD mutant; A/P, ΔAD ΔPKS1 mutant; PK, ΔPKS1 mutant; MO, SD, and AT represent the ΔMO, ΔSDR, and ΔAT mutants, respectively.

To summarize our findings, our results showed that the disruption of the MO gene strongly increased fungal tolerance to the osmotic agent NaCl and the oxidant H₂O₂. Furthermore, the disruption of the AD gene led to an increase in fungal tolerance to the cell wall-perturbing agent Congo red. The activation of the PNH gene significantly decreased fungal tolerance to the oxidant H₂O₂.

DISCUSSION

Even though the CRISPR/Cas9 system appears to work universally, individual components of this gene editing system must be optimized to achieve a gene mutation in a particular species (13, 37 –42). In contrast to most previous reports that stated that the native U6 promoter was efficient for sgRNA expression in mesophilic fungi and in the thermophilic fungus M. thermophila (23), it was found that the native U6 promoters failed to drive sgRNA expression in T. dupontii. A similar result occurred in the oomycete Phytophthora (20). As U6 promoters are still poorly characterized in nonmodel fungal species (21, 43), more sgRNA expression strategies should be exploited for the development of the CRISPR/Cas9 system in nonmodel fungi.

A previous study indicated that both sgRNA levels and nuclear localization are key factors for Cas9-mediated mutagenesis in eukaryotic cells (31). In T. dupontii, even though both HH-HDV and tRNA^Gly possess the ability to drive sgRNA expression, only tRNA^Gly was found to be feasible for the development of effective CRISPR/Cas9 systems in T. dupontii. It was highly likely that HH-HDV would fail to properly process the uncleaved 5′ cap and 3′ poly(A) tail of the transcribed sgRNA, which would lead to the impairment of sgRNA specificity and nuclear nonlocalization. In turn, this would impede the guide activity of sgRNAs (44, 45).

To our knowledge, mesophilic SpCas9 has not been applied in thermophilic eukaryotes for genome editing. Our results showed that when expressed at 37°C, the mesophilic SpCas9 could function well. However, at 45°C, it was unable to edit the genome of T. dupontii. This result was consistent with previous reports that stated that SpCas9 showed maximum activity in plants at 37°C while a temperature higher than 42°C would destroy its nuclease activity (33, 35). This result further suggested that temperature limiting should be considered when an SpCas9-mediated gene editing system in thermophilic fungi is being developed.

While mutating a single gene of T. dupontii at 37°C, the SpCas9/tRNA^Gly system showed 50 to 86% efficiency, similar to the levels in the mesophilic fungi Aspergillus oryzae, Fusarium oxysporum, and Blastomyces dermatitidis (50% to 100%) (1 –3). However, this efficiency is much higher than that of traditional mutation methods for T. dupontii (2 to 20%). Among all of the gene editing mutants of T. dupontii with mutations of single genes, short indel mutations were dominant. These mutations are commonly observed in gene editing mutants of fungi, plants, and animals via CRISPR/Cas9 (20, 21, 23, 26). Interestingly, large sequence insertions in the expected gene editing region were also observed in this study. This can lead to gene disruption. These inserted sequences were characterized to be derived from the expression plasmids. This phenomenon was consistent with findings in the pathogenic fungus Sclerotinia sclerotiorum, in which a large sequence insertion from transformed plasmids was identified in all gene editing mutants. The authors suspected that the transformation plasmids were cleaved by the endogenous nuclease, after which fragments were generated and then inserted in the DSBs (29). In our CRISPR/Cas9 experiments, we also observed several heterokaryotic edited mutants displaying multiple sequence peaks at the expected Cas9 cleavage sites. These cleavage sites were also commonly found in SpCas9 editing strains of multinucleate Blastomyces (21).

By constructing a single polycistronic sgRNA expression plasmid, we also succeeded in the simultaneous editing of two genes in T. dupontii with an efficiency of 30%, which was lower than that (68%) of the polycistronic sgRNA expression plasmid for Aspergillus oryzae (3). However, in Aspergillus oryzae’s polycistronic sgRNA expression plasmid, each sgRNA was expressed by its own U6 promoter (3). In this study, the two sgRNAs were controlled by only one single tRNA^Gly promoter. Nevertheless, as HygB is the only available selective marker for T. dupontii, the simultaneous editing achieved in this study would greatly benefit study of gene function in T. dupontii in the future.

In the presence of a donor template, efficient gene editing via SpCas9-mediated HR in T. dupontii (80%) was achieved. Furthermore, the knocked-in constitutive promoter succeeded in inducing the expression of the silent biosynthesis gene, PNH, which in turn led to the production of new compounds. Fungal biosynthetic gene clusters (BGCs) are commonly silent or expressed at minimal levels under laboratory conditions. Activating their expression would likely stimulate new metabolite biosynthesis. It would then become critical to study these potential chemical treasures (46). Our study indicated that this CRISPR/Cas9-meditated promoter knock-in strategy was a feasible option for awakening silent biosynthesis genes and mining the potential natural products of T. dupontii. Furthermore, homologous arms used for HR in this study were only about 400 bp long, far shorter than those of traditional HR (>1,500 bp). This was also consistent with previous reports that stated that long homologous arms were not necessary for CRISPR/Cas9-mediated HR, as homologous sequences dozens to several hundred base pairs long were sufficient for the CRISPR/Cas9-mediated HR for fungi (20, 43, 47). Because the short homology arms were much easier for plasmid construction, Cas9/tRNA^Gly-mediated HR would be a superior option to traditional HR, with the purpose of genetic manipulating in Thermomyces.

Our results showed that the thermostable GeoCas9/tRNA^Gly system was very flexible for generating gene editing mutants of T. dupontii. Most notably, GeoCas9/tRNA^Gly saved a lot of time in the process of gene editing in the thermophilic fungus T. dupontii compared to use of SpCas9/tRNA^Gly as the temperature limitation of no more than 37°C for SpCas9 was not necessary for GeoCas9. With the disappearance of the temperature limitation, the thermophilic fungus grows faster at the optimal temperature of 45°C than at 37°C. Furthermore, the risk of contamination with mesophilic microorganisms such as Escherichia coli and Bacillus subtilis significantly increases when T. dupontii is grown at 37°C. Therefore, the GeoCas9/tRNA^Gly system represents not only an alternative and time-saving option to SpCas9/tRNA^Gly but also a safe genomic editing tool for T. dupontii.

Through a metabolic analysis of the T. dupontii mutants with HPLC-MS, the key biosynthetic steps were characterized for the biosynthesis of well-known nematicidal thermolides in the native host. Our results indicated that the AT gene in this gene cluster played a key role in the last biosynthetic step for the biosynthesis of potent nematicidal thermolides A and B as it was responsible for transferring an acetyl group to nonnematocidal thermolide D and yielding thermolides A to C in T. dupontii (Fig. 5B). It was interesting that this result sharply contrasted with the result of the most recent study (12). Specifically, in the heterologous host Aspergillus, acetylation via AT occurred prior to reduction via SDR (Fig. 5C) (12). Presumably, the heterologous host and the in vitro experiment might lack the host factors (48), resulting in different biosynthetic pathways for the same metabolites (49). This would render the characterization of fungal biosynthetic pathways in a heterologous host unknown (50).

Our study results also showed that the disruption of biosynthetic genes could affect conidiation and spore germination of T. dupontii. The underlying mechanism that explains how these secondary biosynthetic genes regulate fungal development still remains elusive (51, 52).

It has been widely assumed that secondary metabolites can benefit hosts in tolerating, surviving, or favoring harsh growth conditions (51). However, there is little solid evidence that has been reported on thermophilic fungi. In this study, we found that the loss of particular biosynthetic genes made mutants gain or lose their tolerance toward the stress compounds NaCl, Congo red, and H₂O₂. This result suggests that secondary metabolites might play roles in the chemical stress response of thermophilic fungi.

In summary, two genomic editing systems that are based on Cas9 of mesophilic S. pyogenes bacteria and thermophilic G. stearothermophilus bacteria were developed in the thermophilic fungus T. dupontii. Reducing the temperature from 45°C to 37°C is vital for achieving SpCas9-mediated gene editing in T. dupontii although it is time-consuming. In contrast, GeoCas9 could function at the elevated temperature of 45°C without delaying fungal growth. Both genome editing systems could generate a large number of mutants, which was ideal for study of gene functions. CRISPR/Cas9 systems developed in T. dupontii offer an example for developing genomic editing systems in other thermophilic fungi. Morphological characteristic data of all the mutants provide the characterization of biological functions of silent and unexploited biosynthetic genes in a nonmodel thermophilic species, T. dupontii. Our results demonstrated that the SpCas9 and GeoCas9 genome editing systems developed in T. dupontii could greatly advance our study, illuminating the natural products of biosynthesis in native hosts and mining the potential of thermophilic secondary metabolites. This benefits our understanding of the influence of biosynthetic genes or products on thermophilic fungal development and stress tolerance.

MATERIALS AND METHODS

Fungal strain and cultural conditions.

Thermomyces dupontii NRRL 2155 was obtained from the State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan at Yunnan University. The WT strain was maintained and grown on PDA medium (200 g of potato, 20 g of glucose, and 20 g of agar per liter) at 37°C or 45°C, while mutants were cultured on either a PDA or TB3 regeneration medium (200 g of sucrose, 3 g of yeast extract, 3 g of tryptone, and 8 g of agar per liter) either unsupplemented or supplemented with 200 μg/ml hygromycin B (Sangon Biotech Shanghai Co., Ltd.). For the flask culture, strains were first cultured on PDA at 45°C for 10 days to obtain conidia, and then the conidia were inoculated in 250 ml of liquid YPG medium (5 g of yeast extract, 5 g of peptone, and 20 g of glucose per liter) with a final concentration of 1 × 10⁵ conidia ml⁻¹ at 45°C at 180 rpm. For fungal growth analysis, 9-mm agar blocks containing cultivated fungal cells were inoculated onto a 9-cm PDA petri dish for 10 days. Fungal colony diameter was measured every day until the hyphae completely covered the petri dish.

Construction of CRISPR/Cas9 system.

The hygromycin B (HygB) gene was amplified from pCAMBIA1300 (Marker Gene Technologies, Inc.) and fused with a TrpC promoter and terminator by overlap PCR and then assembled into the KpnI- and HindIII-digested p-UC19 (TaKaRa) in order to create plasmid p-HygB. A codon-optimized Spcas9 gene with a nuclear localization signal (NLS) [derived from pSpCas9(BB)-2A-GFP, plasmid 48138; Addgene) was synthesized by TSINGKE Biological Technology (Beijing) for expression in T. dupontii. This was then inserted into p-HygB to replace the HygB ORF based on an In-Fusion clone (TaKaRa), creating the Spcas9 expression plasmid p-trpC-NLS-coSpcas9-NLS. The eGFP gene fragment of plasmid pSpCas9(BB)-2A-GFP was amplified and fused with coSpcas9 to create plasmid p-trpC-NLS-coSpcas9-eGFP-NLS. The same process was used to construct the Geocas9 expression plasmid p-trpC-NLS-Geocas9-NLS. All primer sequences are listed in Table S2 in the supplemental material, and the sequences of two Cas9 expression plasmids are described in Fig. S7 and S8 in the supplemental material.

The sgRNA scaffold was amplified from plasmid p-SpCas9(BB)-2A-GFP. The sgRNA_PKS1 expression plasmid was constructed as follows. (i) The native promoter of putative U6 genes was searched for within the genome of T. dupontii (https://genome.fungalgenomics.ca/) using U6 genes from human and Aspergillus and Myceliophthora fungi as references. Putative U6 promoters (U6₁ and U6₂) were amplified from T. dupontii genomic DNA using the primer pairs U6-F1/R1 and U6-F2/R2 and then fused to the sgRNA scaffold with a 20-bp PKS1 protospacer by overlap PCR to create fragments U6₁-sgRNA_PKS1 and U6₂-sgRNA_PKS1. The resulting fusion fragments were cloned into pHygB via a restriction clone to create plasmids pU6₁-sgRNA_PKS1 and pU6₂-sgRNA_PKS1. (ii) sgRNA_PKS1 was synthesized to be flanked by hammerhead and hepatitis delta virus ribozymes (20, 21) and inserted between the trpC promoter and trpC terminator by overlap PCR. It was then then cloned into vector pHygB via a restriction clone to create p-trpC-HH-sgRNA_PKS1-HDV. The tRNA^Gly gene and the putative promoter of T. dupontii were determined based on their alignment with tRNA^Gly genes from rice and Drosophila. The putative 117-bp tRNA^Gl^y promoter, the 71-bp tRNA^Gly gene, and PKS1 protospacer 1 were fused with the sgRNA scaffold by overlap PCR and then inserted to SpeI/HindIII-cut pHygB to create p-tRNA^Gly-sgRNA_PKS1. All other sgRNA expression plasmids were created according to the construction of sgRNA_PKS1 with the primers listed in Table S1. The Golden Gate clone method was applied to construct a dual-targeting vector according to a previous study (25). The sgRNA expression plasmids for GeoCas9 experiments were constructed as previously described (32). All of the protospacer sequences were blasted against the T. dupontii genome to verify their uniqueness in this fungus. Protospacer sequences in targeting plasmids were further confirmed by Sanger sequencing.

PCR amplification was performed with PrimeSTAR HS DNA polymerase (TaKaRa). PCR products were cleaned up with a TaKaRa MiniBEST agarose gel DNA extraction kit or TaKaRa MiniBEST DNA fragment purification kit for further use.

Donor plasmid construction.

The 5′ and 3′ flanking fragments of the PNH gene were amplified from genomic DNA and then fused with the TrpC promoter (amplified from p-HygB) by overlap PCR to create the donor DNA cassette. The donor cassette was then inserted into p-HygB via restriction to form donor plasmid p-trpC-donor.

Protoplasts transformation of T. dupontii.

Protoplasts were generated and transformed as described previously (11). Briefly, fungal conidia were germinated in YPG medium for 16 to 18 h to obtain hyphae, and the collected hyphae were digested by lysing enzymes from Trichoderma harzianum (L1412; Sigma). A total of 5 to 10 μg of each plasmid was used in transformation. Protoplasts that were screened on TB3 medium were supplied with 200 μg ml⁻¹ hygromycin B at 45°C or 37°C.

Subcellular localization of SpCas9 in T. dupontii.

PCR-screened positive T. dupontii colonies transformed with p-trpC-NLS-coSpcas9-eGFP-NLS were examined for SpCas9 localization as previously described in Phytophthora (20). Imaging fluorescence was obtained with a Zeiss Axioskop 2 Plus fluorescence microscope. Linear adjustments to contrast and brightness of pictures was done with the software ImageJ2 (https://imagej.net/ImageJ2).

Screening of targeted clones.

T. dupontii transformants were screened by PCR analysis. Total genomic DNA (gDNA) was extracted from hygromycin-resistant colonies and the WT with DNAiso reagent (TaKaRa), according to the manufacturer’s protocol. Gene target regions were amplified and purified for Sanger sequencing. Sequences from transformants and the WT reference were aligned with BioEdit and SnapGene software.

RNA preparation and RT-PCR.

A 3-day-old flask culture of transformants and the WT that grew at 45°C was collected via centrifugation for 10 min at 4,000 × g, washed with cold double-distilled H₂O (ddH₂O) three times, and then frozen in liquid nitrogen. Frozen fungal pellets were used for total RNA extraction with RNAiso Plus (TaKaRa), according to the manufacturer’s protocol. Two micrograms of extracted RNA of each sample was treated with gDNA wiper mix (Vazyme Biotech, Nanjing, China) to remove contaminant gDNA, and cDNA synthesis was performed with a HiScript II 1st Strand cDNA Synthesis kit (Vazyme Biotech, Nanjing, China). The cDNA that was diluted at a ratio of 1:20 with ddH₂O was used as the template in an RT-PCR experiment. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was selected as a control. Primers used for RT-PCR are listed in Table S1.

Western blotting.

coSpCas9 strains that were cultivated at 37°C and 45°C were harvested in the same manner as in RT-PCR experiments and stored at −80°C in a refrigerator. Frozen pellets were used for crude protein extraction with radioimmunoprecipitation assay (RIPA) lysis buffer (strong; Beyotime Biotechnology, Shanghai, China). Equalized protein was electrophoresed on a polyacrylamide gel and then wet transferred onto polyvinylidene difluoride (PVDF) membrane. This was followed by 1 h of blocking with 5% bovine serum albumin (BSA) in Tris-buffered saline with 0.5% Tween 20 (TBST) at room temperature. The membrane was then incubated with primary antibodies diluted in TBST (3% BSA, wt/vol) as follows: SpCas9 mouse monoclonal antibody (MAb) Cas9 (7A9-3A3) (1:3,000 dilution; Cell Signaling Technology) to detect coSpCas9 and ProteinFind anti-β-tubulin mouse MAb (1:10,000 dilution; TransGene, Beijing TransGen Biotech Co., China) as loading control. The membranes were then washed properly with TBST and incubated with ProteinFind horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG(H+L) secondary antibody (1:10,000 dilution; TransGene). The membranes were then washed thoroughly with TBST, stained with BeyoECL Plus, and then imaged by a GE Amersham Imager 600 (General Electric Company).

LC-MS analysis.

Strains were inoculated in a 250-ml PDB flask culture at 45°C (180 rpm) for 10 days before analysis by ultraperformance liquid chromatography with diode array detection MS (UPLC-DAD-MS). The fermentation broths were extracted with the same volume of ethyl acetate overnight, and the organic layers were evaporated and dissolved in 1 ml of menthol and filtered through 0.22-μm-pore-size membranes. All of the samples were then analyzed using a UPLC system coupled to a Q Exactive Focus Orbitrap mass spectrometer (Thermo Fisher) equipped with an Agilent Zorbax ODS 4.6- by 250-mm column (Agilent, Santa Clara, CA) using the electrospray ionization (ESI) mode. The mobile phase was composed of acetonitrile (elution A) and water (elution B), both containing 0.1% formic acid. The flow rate was 1 ml/min with a linear gradient program. Mobile phase composition was held at 10% A for 2 min and was followed by a linear gradient to 100% A within 42 min. Column temperature was set to 40°C, and the injection volume was 10 μl. Full-scan mode with a scan range from m/z 100 to 1,000 was set up for the Orbitrap mass analyzer.

Fungal morphology, conidial production, and germination.

Mycelium plugs with an estimated 9-mm diameter from 7-day fungal colonies of the wild-type and mutant strains were inoculated onto 9-cm plates with several different media (5 replicates/strain) and then incubated at 45°C. Radial colony growth diameter was measured every 2 days. Fungal strains were stained with calcofluor white (Sigma-Aldrich, St. Louis, MO, USA) and observed under a Zeiss Axioskop 2 Plus fluorescence microscope.

To assess the sporulation capacities of the wild-type and mutant strains, 100-μl aliquots of suspensions of 10⁶ conidia were spread on 9-cm plates with PDA medium and incubated for 10 days at 45°C. The conidia were washed in 10 ml of sterile water, followed by filtration through four layers of lens tissues to remove mycelium debris. The concentrations of the conidial suspensions were determined by microscopic counts on a hemocytometer and converted to the number of conidia per square centimeter of plate culture. This served as an estimate of conidial yield, and conidial formation was observed as previously described (53, 54). To calculate the conidial germination rates, 50-μl aliquots of suspensions of 10⁵ conidia ml⁻¹ of the WT strain and mutants were inoculated on water agar medium. Samples were incubated at 45°C. The germinated conidia were counted at 12 h postinoculation. The phenotypic analyses were repeated at least three times.

Stress assays.

To evaluate the effects of biosynthetic genes on the resistance of the thermophilic fungus T. dupontii to environmental stress, the colonies of each strain were initiated with hyphal mass discs (9-mm diameter), incubated at 45°C for 7 to 10 days on either PDA medium alone (control) or supplemented with the following chemical stressors: (i) NaCl for osmotic stress, (ii) Congo red for cell wall perturbation, and (iii) H₂O₂ for oxidative stress (53, 54). These experiments were performed three times.

Data availability.

The sequences of the PKS1 (Talth1p4_003117), AD (Talth1p4_005402), SDR (Talth1p4_003119), MO (Talth1p4_003120), AT (Talth1p4_003115), PNH (Talth1p4_005395), HLHTF1 (Talth1p4_003063), HLHTF2 (Talth1p4_006974), HLHTF3 (Talth1p4_001383), and HLHTF4 (Talth1p4_005393) genes of Thermomyces dupontii NRRL 2155 were deposited in the database of the Centre for Structural and Functional Genomics (CSFG) (https://gb.fungalgenomics.ca/fgb2/gbrowse/Talth_public/).

Supplementary Material

Supplemental file 1

AEM.01486-20-s0001.pdf^{(2.1MB, pdf)}

ACKNOWLEDGMENTS

We thank Jianping Xu for editing.

This work was sponsored by NSFC grants 21977086 to X.-M.N. and 21867018 to W.-P. Huang, a YNU Program award to X.-M.N. (XT412003), and the Yunnan Innovative Research Team for Discovery and Biosynthesis of Bioactive Natural Products (2018HC012).

W.-P.H. and X.-M.N. conceived the study. W.-P.H., Y.-J.D., Y.Y., J.-N.H., and Q.L. conceived the experiments, and W.-P.H. and X.-M.N. analyzed data. K.-Q.Z. and X.-M.N. supervised the study. W.-P.H. and X.-M.N. wrote the article with contributions from all authors.

We declare that we have no competing interests.

Footnotes

Supplemental material is available online only.

REFERENCES

1.Maheshwari R, Bharadwaj G, Bhat MK. 2000. Thermophilic fungi: their physiology and enzymes. Microbiol Mol Biol Rev 64:461–488. doi: 10.1128/mmbr.64.3.461-488.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
2.de Oliveira TB, Gomes E, Rodrigues A. 2015. Thermophilic fungi in the new age of fungal taxonomy. Extremophiles 19:31–37. doi: 10.1007/s00792-014-0707-0. [DOI] [PubMed] [Google Scholar]
3.Niu X, Chen L, Yue Q, Wang B, Zhang J, Zhu C, Zhang K, Bills GF, An Z. 2014. Characterization of thermolide biosynthetic genes and a new thermolide from sister thermophilic fungi. Org Lett 16:3744–3747. doi: 10.1021/ol501595z. [DOI] [PubMed] [Google Scholar]
4.Zhou P, Zhang G, Chen S, Jiang Z, Tang Y, Henrissat B, Yan Q, Yang S, Chen C, Zhang B, Du Z. 2014. Genome sequence and transcriptome analyses of the thermophilic zygomycete fungus Rhizomucor miehei. BMC Genomics 15:294. doi: 10.1186/1471-2164-15-294. [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Huttner S, Nguyen TT, Granchi Z, Chin AWT, Ahren D, Larsbrink J, Thanh VN, Olsson L. 2017. Combined genome and transcriptome sequencing to investigate the plant cell wall degrading enzyme system in the thermophilic fungus Malbranchea cinnamomea. Biotechnol Biofuels 10:265. doi: 10.1186/s13068-017-0956-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Berka RM, Grigoriev IV, Otillar R, Salamov A, Grimwood J, Reid I, Ishmael N, John T, Darmond C, Moisan M-C, Henrissat B, Coutinho PM, Lombard V, Natvig DO, Lindquist E, Schmutz J, Lucas S, Harris P, Powlowski J, Bellemare A, Taylor D, Butler G, de Vries RP, Allijn IE, van den Brink J, Ushinsky S, Storms R, Powell AJ, Paulsen IT, Elbourne LDH, Baker SE, Magnuson J, Laboissiere S, Clutterbuck AJ, Martinez D, Wogulis M, de Leon AL, Rey MW, Tsang A. 2011. Comparative genomic analysis of the thermophilic biomass-degrading fungi Myceliophthora thermophila and Thielavia terrestris. Nat Biotechnol 29:922–927. doi: 10.1038/nbt.1976. [DOI] [PubMed] [Google Scholar]
7.Morgenstern I, Powlowski J, Ishmael N, Darmond C, Marqueteau S, Moisan MC, Quenneville G, Tsang A. 2012. A molecular phylogeny of thermophilic fungi. Fungal Biol 116:489–502. doi: 10.1016/j.funbio.2012.01.010. [DOI] [PubMed] [Google Scholar]
8.Machida M, Asai K, Sano M, Tanaka T, Kumagai T, Terai G, Kusumoto K-I, Arima T, Akita O, Kashiwagi Y, Abe K, Gomi K, Horiuchi H, Kitamoto K, Kobayashi T, Takeuchi M, Denning DW, Galagan JE, Nierman WC, Yu J, Archer DB, Bennett JW, Bhatnagar D, Cleveland TE, Fedorova ND, Gotoh O, Horikawa H, Hosoyama A, Ichinomiya M, Igarashi R, Iwashita K, Juvvadi PR, Kato M, Kato Y, Kin T, Kokubun A, Maeda H, Maeyama N, Maruyama J-I, Nagasaki H, Nakajima T, Oda K, Okada K, Paulsen I, Sakamoto K, Sawano T, Takahashi M, Takase K, Terabayashi Y, Wortman JR, Yamada O, Yamagata Y, Anazawa H, Hata Y, Koide Y, Komori T, Koyama Y, Minetoki T, Suharnan S, Tanaka A, Isono K, Kuhara S, Ogasawara N, Kikuchi H. 2005. Genome sequencing and analysis of Aspergillus oryzae. Nature 438:1157–1161. doi: 10.1038/nature04300. [DOI] [PubMed] [Google Scholar]
9.van den Berg MA, Albang R, Albermann K, Badger JH, Daran J-M, Driessen AJM, Garcia-Estrada C, Fedorova ND, Harris DM, Heijne WHM, Joardar V, Kiel JAKW, Kovalchuk A, Martín JF, Nierman WC, Nijland JG, Pronk JT, Roubos JA, van der Klei IJ, van Peij NNME, Veenhuis M, von Döhren H, Wagner C, Wortman J, Bovenberg RAL. 2008. Genome sequencing and analysis of the filamentous fungus Penicillium chrysogenum. Nat Biotechnol 26:1161–1168. doi: 10.1038/nbt.1498. [DOI] [PubMed] [Google Scholar]
10.Guo J-P, Zhu C-Y, Zhang C-P, Chu Y-S, Wang Y-L, Zhang J-X, Wu D-K, Zhang K-Q, Niu X-M. 2012. Thermolides, potent nematocidal PKS-NRPS hybrid metabolites from thermophilic fungus Talaromyces thermophilus. J Am Chem Soc 134:20306–20309. doi: 10.1021/ja3104044. [DOI] [PubMed] [Google Scholar]
11.He J, Niu X. 2019. An efficient system for targeted gene replacement in the thermophilus fungus Thermomyces dupontii. Mycosystema 38:230–241. [Google Scholar]
12.Zhang J-M, Wang H-H, Liu X, Hu C-H, Zou Y. 2020. Heterologous and engineered biosynthesis of nematocidal polyketidenonribosomal peptide hybrid macrolactone from extreme thermophilic fungi. J Am Chem Soc 142:1957–1965. doi: 10.1021/jacs.9b11410. [DOI] [PubMed] [Google Scholar]
13.Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. 2013. Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823. doi: 10.1126/science.1231143. [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Tapscott T, Guarnieri MT, Henard CA. 2019. Development of a CRISPR/Cas9 system for Methylococcus capsulatus in vivo gene editing. Appl Environ Microbiol 85:e00340-19. doi: 10.1128/AEM.00340-19. [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Zhou D, Jiang Z, Pang Q, Zhu Y, Wang Q, Qi Q. 2019. CRISPR/Cas9-assisted seamless genome editing in Lactobacillus plantarum and its application in N-acetylglucosamine production. Appl Environ Microbiol 85:e01367-19. doi: 10.1128/AEM.01367-19. [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S. 2015. Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol 81:2506–2514. doi: 10.1128/AEM.04023-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Wang Y, Wang S, Chen W, Song L, Zhang Y, Shen Z, Yu F, Li M, Ji Q. 2018. CRISPR-Cas9 and CRISPR-assisted cytidine deaminase enable precise and efficient genome editing in Klebsiella pneumoniae. Appl Environ Microbiol 84:e01834-18. doi: 10.1128/AEM.01834-18. [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Wasels F, Chartier G, Hocq R, Ferreira NL. 2020. A CRISPR/anti-CRISPR genome editing approach underlines the synergy of butanol dehydrogenases in Clostridium acetobutylicum DSM 792. Appl Environ Microbiol 86:AEM.00408-20. doi: 10.1128/AEM.00408-20. [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. 2012. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821. doi: 10.1126/science.1225829. [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Fang YF, Tyler BM. 2016. Efficient disruption and replacement of an effector gene in the oomycete Phytophthora sojae using CRISPR/Cas9. Mol Plant Pathol 17:127–139. doi: 10.1111/mpp.12318. [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Kujoth GC, Sullivan TD, Merkhofer R, Lee TJ, Wang H, Brandhorst T, Wuthrich M, Klein BS. 2018. CRISPR/Cas9-Mediated gene disruption reveals the importance of zinc metabolism for fitness of the dimorphic fungal pathogen Blastomyces dermatitidis. mBio 9:e00412-18. doi: 10.1128/mBio.00412-18. [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Fuller KK, Chen S, Loros JJ, Dunlap JC. 2015. Development of the CRISPR/Cas9 system for targeted gene disruption in Aspergillus fumigatus. Eukaryot Cell 14:1073–1080. doi: 10.1128/EC.00107-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Liu Q, Gao R, Li J, Lin L, Zhao J, Sun W, Tian C. 2017. Development of a genome-editing CRISPR/Cas9 system in thermophilic fungal Myceliophthora species and its application to hyper-cellulase production strain engineering. Biotechnol Biofuels 10:1. doi: 10.1186/s13068-016-0693-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Schwartz CM, Hussain MS, Blenner M, Wheeldon I. 2016. Synthetic RNA polymerase III promoters facilitate high-efficiency CRISPR-Cas9-mediated genome editing in Yarrowia lipolytica. ACS Synth Biol 5:356–359. doi: 10.1021/acssynbio.5b00162. [DOI] [PubMed] [Google Scholar]
25.Xie K, Minkenberg B, Yang Y. 2015. Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system. Proc Natl Acad Sci U S A 112:3570–3575. doi: 10.1073/pnas.1420294112. [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Liu R, Chen L, Jiang Y, Zhou Z, Zou G. 2015. Efficient genome editing in filamentous fungus Trichoderma reesei using the CRISPR/Cas9 system. Cell Discov 1:15077. doi: 10.1038/celldisc.2015.7. [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Nielsen ML, Isbrandt T, Rasmussen KB, Thrane U, Hoof JB, Larsen TO, Mortensen UH. 2017. Genes linked to production of secondary metabolites in Talaromyces atroroseus revealed using CRISPR/Cas9. PLoS One 12:e0169712. doi: 10.1371/journal.pone.0169712. [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Weber J, Valiante V, Nodvig CS, Mattern DJ, Slotkowski RA, Mortensen UH, Brakhage AA. 2017. Functional reconstitution of a fungal natural product gene cluster by advanced genome editing. ACS Synth Biol 6:62–68. doi: 10.1021/acssynbio.6b00203. [DOI] [PubMed] [Google Scholar]
29.Li J, Zhang Y, Zhang Y, Yu PL, Pan H, Rollins JA. 2018. Introduction of large sequence inserts by CRISPR-Cas9 to create pathogenicity mutants in the multinucleate filamentous pathogen Sclerotinia sclerotiorum. mBio 9:e00567-18. doi: 10.1128/mBio.00567-18. [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Cernak P, Estrela R, Poddar S, Skerker JM, Cheng YF, Carlson AK, Chen B, Glynn VM, Furlan M, Ryan OW, Donnelly MK, Arkin AP, Taylor JW, Cate JHD. 2018. Engineering Kluyveromyces marxianus as a robust synthetic biology platform host. mBio 9:e01410-18. doi: 10.1128/mBio.01410-18. [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X, Shalem O, Cradick TJ, Marraffini LA, Bao G, Zhang F. 2013. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 31:827–832. doi: 10.1038/nbt.2647. [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Harrington LB, Paez-Espino D, Staahl BT, Chen JS, Ma E, Kyrpides NC, Doudna JA. 2017. A thermostable Cas9 with increased lifetime in human plasma. Nat Commun 8:1424. doi: 10.1038/s41467-017-01408-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Mougiakos I, Mohanraju P, Bosma EF, Vrouwe V, Finger Bou M, Naduthodi MIS, Gussak A, Brinkman RBL, van Kranenburg R, van der Oost J. 2017. Characterizing a thermostable Cas9 for bacterial genome editing and silencing. Nat Commun 8:1647. doi: 10.1038/s41467-017-01591-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
34.LeBlanc C, Zhang F, Mendez J, Lozano Y, Chatpar K, Irish VF, Jacob Y. 2018. Increased efficiency of targeted mutagenesis by CRISPR/Cas9 in plants using heat stress. Plant J 93:377–386. doi: 10.1111/tpj.13782. [DOI] [PubMed] [Google Scholar]
35.Mougiakos I, Bosma EF, Weenink K, Vossen E, Goijvaerts K, van der Oost J, van Kranenburg R. 2017. Efficient genome editing of a facultative thermophile using mesophilic spCas9. ACS Synth Biol 6:849–861. doi: 10.1021/acssynbio.6b00339. [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Toledo-Ortiz G, Huq E, Quail PH. 2003. The Arabidopsis basic/helix-loop-helix transcription factor family. Plant Cell 15:1749–1770. doi: 10.1105/tpc.013839. [DOI] [PMC free article] [PubMed] [Google Scholar]
37.Jacobs JZ, Ciccaglione KM, Tournier V, Zaratiegui M. 2014. Implementation of the CRISPR-Cas9 system in fission yeast. Nat Commun 5:5344. doi: 10.1038/ncomms6344. [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Niu Y, Shen B, Cui Y, Chen Y, Wang J, Wang L, Kang Y, Zhao X, Si W, Li W, Xiang AP, Zhou J, Guo X, Bi Y, Si C, Hu B, Dong G, Wang H, Zhou Z, Li T, Tan T, Pu X, Wang F, Ji S, Zhou Q, Huang X, Ji W, Sha J. 2014. Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell 156:836–843. doi: 10.1016/j.cell.2014.01.027. [DOI] [PubMed] [Google Scholar]
39.Hendel A, Bak RO, Clark JT, Kennedy AB, Ryan DE, Roy S, Steinfeld I, Lunstad BD, Kaiser RJ, Wilkens AB, Bacchetta R, Tsalenko A, Dellinger D, Bruhn L, Porteus MH. 2015. Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat Biotechnol 33:985–989. doi: 10.1038/nbt.3290. [DOI] [PMC free article] [PubMed] [Google Scholar]
40.Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li H, Lin Y, Xie Y, Shen R, Chen S, Wang Z, Chen Y, Guo J, Chen L, Zhao X, Dong Z, Liu YG. 2015. A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol Plant 8:1274–1284. doi: 10.1016/j.molp.2015.04.007. [DOI] [PubMed] [Google Scholar]
41.Hammond A, Galizi R, Kyrou K, Simoni A, Siniscalchi C, Katsanos D, Gribble M, Baker D, Marois E, Russell S, Burt A, Windbichler N, Crisanti A, Nolan T. 2016. A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae. Nat Biotechnol 34:78–83. doi: 10.1038/nbt.3439. [DOI] [PMC free article] [PubMed] [Google Scholar]
42.Port F, Bullock SL. 2016. Augmenting CRISPR applications in Drosophila with tRNA-flanked sgRNAs. Nat Methods 13:852–854. doi: 10.1038/nmeth.3972. [DOI] [PMC free article] [PubMed] [Google Scholar]
43.Zhang C, Meng X, Wei X, Lu L. 2016. Highly efficient CRISPR mutagenesis by microhomology-mediated end joining in Aspergillus fumigatus. Fungal Genet Biol 86:47–57. doi: 10.1016/j.fgb.2015.12.007. [DOI] [PubMed] [Google Scholar]
44.Nissim L, Perli SD, Fridkin A, Perez-Pinera P, Lu TK. 2014. Multiplexed and programmable regulation of gene networks with an integrated RNA and CRISPR/Cas toolkit in human cells. Mol Cell 54:698–710. doi: 10.1016/j.molcel.2014.04.022. [DOI] [PMC free article] [PubMed] [Google Scholar]
45.Shi TQ, Liu GN, Ji RY, Shi K, Song P, Ren LJ, Huang H, Ji XJ. 2017. CRISPR/Cas9-based genome editing of the filamentous fungi: the state of the art. Appl Microbiol Biotechnol 101:7435–7443. doi: 10.1007/s00253-017-8497-9. [DOI] [PubMed] [Google Scholar]
46.Rutledge PJ, Challis GL. 2015. Discovery of microbial natural products by activation of silent biosynthetic gene clusters. Nat Rev Microbiol 13:509–523. doi: 10.1038/nrmicro3496. [DOI] [PubMed] [Google Scholar]
47.Pohl C, Kiel JA, Driessen AJ, Bovenberg RA, Nygard Y. 2016. CRISPR/Cas9-based genome editing of Penicillium chrysogenum. ACS Synth Biol 5:754–764. doi: 10.1021/acssynbio.6b00082. [DOI] [PubMed] [Google Scholar]
48.Zhang JJ, Tang X, Moore BS. 2019. Genetic platforms for heterologous expression of microbial natural products. Nat Prod Rep 36:1313–1332. doi: 10.1039/c9np00025a. [DOI] [PMC free article] [PubMed] [Google Scholar]
49.Wasil Z, Pahirulzaman KAK, Butts C, Simpson TJ, Lazarus CM, Cox RJ. 2013. One pathway, many compounds: heterologous expression of a fungal biosynthetic pathway reveals its intrinsic potential for diversity. Chem Sci 4:3845–3856. doi: 10.1039/c3sc51785c. [DOI] [Google Scholar]
50.Skellam E. 2019. Strategies for engineering natural product biosynthesis in fungi. Trends Biotechnol 37:416–427. doi: 10.1016/j.tibtech.2018.09.003. [DOI] [PubMed] [Google Scholar]
51.Keller NP. 2019. Fungal secondary metabolism: regulation, function and drug discovery. Nat Rev Microbiol 17:167–180. doi: 10.1038/s41579-018-0121-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
52.Fox EM, Howlett BJ. 2008. Secondary metabolism: regulation and role in fungal biology. Curr Opin Microbiol 11:481–487. doi: 10.1016/j.mib.2008.10.007. [DOI] [PubMed] [Google Scholar]
53.Xie M, Wang Y, Tang L, Yang L, Zhou D, Yang L, Zhou D, Li Q, Niu X, Zhang K, Yang J. 2019. AoStuA, an APSES transcription factor, regulates the conidiation, trap formation, stress resistance and pathogenicity of the nematode‐trapping fungus Arthrobotrys oligospora. Environ Microbiol 21:4648–4661. doi: 10.1111/1462-2920.14785. [DOI] [PubMed] [Google Scholar]
54.Xie M, Bai N, Yang J, Jiang K, Zhou D, Zhao Y, Li D, Niu X, Zhang K, Yang J. 2019. Protein kinase Ime2 is required for mycelial growth, conidiation, osmoregulation, and pathogenicity in nematode-trapping fungus Arthrobotrys oligospora. Front Microbiol 10:3065. doi: 10.3389/fmicb.2019.03065. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental file 1

AEM.01486-20-s0001.pdf^{(2.1MB, pdf)}

Data Availability Statement

[B1] 1.Maheshwari R, Bharadwaj G, Bhat MK. 2000. Thermophilic fungi: their physiology and enzymes. Microbiol Mol Biol Rev 64:461–488. doi: 10.1128/mmbr.64.3.461-488.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B2] 2.de Oliveira TB, Gomes E, Rodrigues A. 2015. Thermophilic fungi in the new age of fungal taxonomy. Extremophiles 19:31–37. doi: 10.1007/s00792-014-0707-0. [DOI] [PubMed] [Google Scholar]

[B3] 3.Niu X, Chen L, Yue Q, Wang B, Zhang J, Zhu C, Zhang K, Bills GF, An Z. 2014. Characterization of thermolide biosynthetic genes and a new thermolide from sister thermophilic fungi. Org Lett 16:3744–3747. doi: 10.1021/ol501595z. [DOI] [PubMed] [Google Scholar]

[B4] 4.Zhou P, Zhang G, Chen S, Jiang Z, Tang Y, Henrissat B, Yan Q, Yang S, Chen C, Zhang B, Du Z. 2014. Genome sequence and transcriptome analyses of the thermophilic zygomycete fungus Rhizomucor miehei. BMC Genomics 15:294. doi: 10.1186/1471-2164-15-294. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B5] 5.Huttner S, Nguyen TT, Granchi Z, Chin AWT, Ahren D, Larsbrink J, Thanh VN, Olsson L. 2017. Combined genome and transcriptome sequencing to investigate the plant cell wall degrading enzyme system in the thermophilic fungus Malbranchea cinnamomea. Biotechnol Biofuels 10:265. doi: 10.1186/s13068-017-0956-0. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B6] 6.Berka RM, Grigoriev IV, Otillar R, Salamov A, Grimwood J, Reid I, Ishmael N, John T, Darmond C, Moisan M-C, Henrissat B, Coutinho PM, Lombard V, Natvig DO, Lindquist E, Schmutz J, Lucas S, Harris P, Powlowski J, Bellemare A, Taylor D, Butler G, de Vries RP, Allijn IE, van den Brink J, Ushinsky S, Storms R, Powell AJ, Paulsen IT, Elbourne LDH, Baker SE, Magnuson J, Laboissiere S, Clutterbuck AJ, Martinez D, Wogulis M, de Leon AL, Rey MW, Tsang A. 2011. Comparative genomic analysis of the thermophilic biomass-degrading fungi Myceliophthora thermophila and Thielavia terrestris. Nat Biotechnol 29:922–927. doi: 10.1038/nbt.1976. [DOI] [PubMed] [Google Scholar]

[B7] 7.Morgenstern I, Powlowski J, Ishmael N, Darmond C, Marqueteau S, Moisan MC, Quenneville G, Tsang A. 2012. A molecular phylogeny of thermophilic fungi. Fungal Biol 116:489–502. doi: 10.1016/j.funbio.2012.01.010. [DOI] [PubMed] [Google Scholar]

[B8] 8.Machida M, Asai K, Sano M, Tanaka T, Kumagai T, Terai G, Kusumoto K-I, Arima T, Akita O, Kashiwagi Y, Abe K, Gomi K, Horiuchi H, Kitamoto K, Kobayashi T, Takeuchi M, Denning DW, Galagan JE, Nierman WC, Yu J, Archer DB, Bennett JW, Bhatnagar D, Cleveland TE, Fedorova ND, Gotoh O, Horikawa H, Hosoyama A, Ichinomiya M, Igarashi R, Iwashita K, Juvvadi PR, Kato M, Kato Y, Kin T, Kokubun A, Maeda H, Maeyama N, Maruyama J-I, Nagasaki H, Nakajima T, Oda K, Okada K, Paulsen I, Sakamoto K, Sawano T, Takahashi M, Takase K, Terabayashi Y, Wortman JR, Yamada O, Yamagata Y, Anazawa H, Hata Y, Koide Y, Komori T, Koyama Y, Minetoki T, Suharnan S, Tanaka A, Isono K, Kuhara S, Ogasawara N, Kikuchi H. 2005. Genome sequencing and analysis of Aspergillus oryzae. Nature 438:1157–1161. doi: 10.1038/nature04300. [DOI] [PubMed] [Google Scholar]

[B9] 9.van den Berg MA, Albang R, Albermann K, Badger JH, Daran J-M, Driessen AJM, Garcia-Estrada C, Fedorova ND, Harris DM, Heijne WHM, Joardar V, Kiel JAKW, Kovalchuk A, Martín JF, Nierman WC, Nijland JG, Pronk JT, Roubos JA, van der Klei IJ, van Peij NNME, Veenhuis M, von Döhren H, Wagner C, Wortman J, Bovenberg RAL. 2008. Genome sequencing and analysis of the filamentous fungus Penicillium chrysogenum. Nat Biotechnol 26:1161–1168. doi: 10.1038/nbt.1498. [DOI] [PubMed] [Google Scholar]

[B10] 10.Guo J-P, Zhu C-Y, Zhang C-P, Chu Y-S, Wang Y-L, Zhang J-X, Wu D-K, Zhang K-Q, Niu X-M. 2012. Thermolides, potent nematocidal PKS-NRPS hybrid metabolites from thermophilic fungus Talaromyces thermophilus. J Am Chem Soc 134:20306–20309. doi: 10.1021/ja3104044. [DOI] [PubMed] [Google Scholar]

[B11] 11.He J, Niu X. 2019. An efficient system for targeted gene replacement in the thermophilus fungus Thermomyces dupontii. Mycosystema 38:230–241. [Google Scholar]

[B12] 12.Zhang J-M, Wang H-H, Liu X, Hu C-H, Zou Y. 2020. Heterologous and engineered biosynthesis of nematocidal polyketidenonribosomal peptide hybrid macrolactone from extreme thermophilic fungi. J Am Chem Soc 142:1957–1965. doi: 10.1021/jacs.9b11410. [DOI] [PubMed] [Google Scholar]

[B13] 13.Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. 2013. Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823. doi: 10.1126/science.1231143. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B14] 14.Tapscott T, Guarnieri MT, Henard CA. 2019. Development of a CRISPR/Cas9 system for Methylococcus capsulatus in vivo gene editing. Appl Environ Microbiol 85:e00340-19. doi: 10.1128/AEM.00340-19. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B15] 15.Zhou D, Jiang Z, Pang Q, Zhu Y, Wang Q, Qi Q. 2019. CRISPR/Cas9-assisted seamless genome editing in Lactobacillus plantarum and its application in N-acetylglucosamine production. Appl Environ Microbiol 85:e01367-19. doi: 10.1128/AEM.01367-19. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B16] 16.Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S. 2015. Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol 81:2506–2514. doi: 10.1128/AEM.04023-14. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B17] 17.Wang Y, Wang S, Chen W, Song L, Zhang Y, Shen Z, Yu F, Li M, Ji Q. 2018. CRISPR-Cas9 and CRISPR-assisted cytidine deaminase enable precise and efficient genome editing in Klebsiella pneumoniae. Appl Environ Microbiol 84:e01834-18. doi: 10.1128/AEM.01834-18. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B18] 18.Wasels F, Chartier G, Hocq R, Ferreira NL. 2020. A CRISPR/anti-CRISPR genome editing approach underlines the synergy of butanol dehydrogenases in Clostridium acetobutylicum DSM 792. Appl Environ Microbiol 86:AEM.00408-20. doi: 10.1128/AEM.00408-20. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B19] 19.Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. 2012. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821. doi: 10.1126/science.1225829. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B20] 20.Fang YF, Tyler BM. 2016. Efficient disruption and replacement of an effector gene in the oomycete Phytophthora sojae using CRISPR/Cas9. Mol Plant Pathol 17:127–139. doi: 10.1111/mpp.12318. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B21] 21.Kujoth GC, Sullivan TD, Merkhofer R, Lee TJ, Wang H, Brandhorst T, Wuthrich M, Klein BS. 2018. CRISPR/Cas9-Mediated gene disruption reveals the importance of zinc metabolism for fitness of the dimorphic fungal pathogen Blastomyces dermatitidis. mBio 9:e00412-18. doi: 10.1128/mBio.00412-18. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B22] 22.Fuller KK, Chen S, Loros JJ, Dunlap JC. 2015. Development of the CRISPR/Cas9 system for targeted gene disruption in Aspergillus fumigatus. Eukaryot Cell 14:1073–1080. doi: 10.1128/EC.00107-15. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B23] 23.Liu Q, Gao R, Li J, Lin L, Zhao J, Sun W, Tian C. 2017. Development of a genome-editing CRISPR/Cas9 system in thermophilic fungal Myceliophthora species and its application to hyper-cellulase production strain engineering. Biotechnol Biofuels 10:1. doi: 10.1186/s13068-016-0693-9. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B24] 24.Schwartz CM, Hussain MS, Blenner M, Wheeldon I. 2016. Synthetic RNA polymerase III promoters facilitate high-efficiency CRISPR-Cas9-mediated genome editing in Yarrowia lipolytica. ACS Synth Biol 5:356–359. doi: 10.1021/acssynbio.5b00162. [DOI] [PubMed] [Google Scholar]

[B25] 25.Xie K, Minkenberg B, Yang Y. 2015. Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system. Proc Natl Acad Sci U S A 112:3570–3575. doi: 10.1073/pnas.1420294112. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B26] 26.Liu R, Chen L, Jiang Y, Zhou Z, Zou G. 2015. Efficient genome editing in filamentous fungus Trichoderma reesei using the CRISPR/Cas9 system. Cell Discov 1:15077. doi: 10.1038/celldisc.2015.7. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B27] 27.Nielsen ML, Isbrandt T, Rasmussen KB, Thrane U, Hoof JB, Larsen TO, Mortensen UH. 2017. Genes linked to production of secondary metabolites in Talaromyces atroroseus revealed using CRISPR/Cas9. PLoS One 12:e0169712. doi: 10.1371/journal.pone.0169712. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B28] 28.Weber J, Valiante V, Nodvig CS, Mattern DJ, Slotkowski RA, Mortensen UH, Brakhage AA. 2017. Functional reconstitution of a fungal natural product gene cluster by advanced genome editing. ACS Synth Biol 6:62–68. doi: 10.1021/acssynbio.6b00203. [DOI] [PubMed] [Google Scholar]

[B29] 29.Li J, Zhang Y, Zhang Y, Yu PL, Pan H, Rollins JA. 2018. Introduction of large sequence inserts by CRISPR-Cas9 to create pathogenicity mutants in the multinucleate filamentous pathogen Sclerotinia sclerotiorum. mBio 9:e00567-18. doi: 10.1128/mBio.00567-18. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B30] 30.Cernak P, Estrela R, Poddar S, Skerker JM, Cheng YF, Carlson AK, Chen B, Glynn VM, Furlan M, Ryan OW, Donnelly MK, Arkin AP, Taylor JW, Cate JHD. 2018. Engineering Kluyveromyces marxianus as a robust synthetic biology platform host. mBio 9:e01410-18. doi: 10.1128/mBio.01410-18. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B31] 31.Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X, Shalem O, Cradick TJ, Marraffini LA, Bao G, Zhang F. 2013. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 31:827–832. doi: 10.1038/nbt.2647. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B32] 32.Harrington LB, Paez-Espino D, Staahl BT, Chen JS, Ma E, Kyrpides NC, Doudna JA. 2017. A thermostable Cas9 with increased lifetime in human plasma. Nat Commun 8:1424. doi: 10.1038/s41467-017-01408-4. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B33] 33.Mougiakos I, Mohanraju P, Bosma EF, Vrouwe V, Finger Bou M, Naduthodi MIS, Gussak A, Brinkman RBL, van Kranenburg R, van der Oost J. 2017. Characterizing a thermostable Cas9 for bacterial genome editing and silencing. Nat Commun 8:1647. doi: 10.1038/s41467-017-01591-4. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B34] 34.LeBlanc C, Zhang F, Mendez J, Lozano Y, Chatpar K, Irish VF, Jacob Y. 2018. Increased efficiency of targeted mutagenesis by CRISPR/Cas9 in plants using heat stress. Plant J 93:377–386. doi: 10.1111/tpj.13782. [DOI] [PubMed] [Google Scholar]

[B35] 35.Mougiakos I, Bosma EF, Weenink K, Vossen E, Goijvaerts K, van der Oost J, van Kranenburg R. 2017. Efficient genome editing of a facultative thermophile using mesophilic spCas9. ACS Synth Biol 6:849–861. doi: 10.1021/acssynbio.6b00339. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B36] 36.Toledo-Ortiz G, Huq E, Quail PH. 2003. The Arabidopsis basic/helix-loop-helix transcription factor family. Plant Cell 15:1749–1770. doi: 10.1105/tpc.013839. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B37] 37.Jacobs JZ, Ciccaglione KM, Tournier V, Zaratiegui M. 2014. Implementation of the CRISPR-Cas9 system in fission yeast. Nat Commun 5:5344. doi: 10.1038/ncomms6344. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B38] 38.Niu Y, Shen B, Cui Y, Chen Y, Wang J, Wang L, Kang Y, Zhao X, Si W, Li W, Xiang AP, Zhou J, Guo X, Bi Y, Si C, Hu B, Dong G, Wang H, Zhou Z, Li T, Tan T, Pu X, Wang F, Ji S, Zhou Q, Huang X, Ji W, Sha J. 2014. Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell 156:836–843. doi: 10.1016/j.cell.2014.01.027. [DOI] [PubMed] [Google Scholar]

[B39] 39.Hendel A, Bak RO, Clark JT, Kennedy AB, Ryan DE, Roy S, Steinfeld I, Lunstad BD, Kaiser RJ, Wilkens AB, Bacchetta R, Tsalenko A, Dellinger D, Bruhn L, Porteus MH. 2015. Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat Biotechnol 33:985–989. doi: 10.1038/nbt.3290. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B40] 40.Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li H, Lin Y, Xie Y, Shen R, Chen S, Wang Z, Chen Y, Guo J, Chen L, Zhao X, Dong Z, Liu YG. 2015. A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol Plant 8:1274–1284. doi: 10.1016/j.molp.2015.04.007. [DOI] [PubMed] [Google Scholar]

[B41] 41.Hammond A, Galizi R, Kyrou K, Simoni A, Siniscalchi C, Katsanos D, Gribble M, Baker D, Marois E, Russell S, Burt A, Windbichler N, Crisanti A, Nolan T. 2016. A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae. Nat Biotechnol 34:78–83. doi: 10.1038/nbt.3439. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B42] 42.Port F, Bullock SL. 2016. Augmenting CRISPR applications in Drosophila with tRNA-flanked sgRNAs. Nat Methods 13:852–854. doi: 10.1038/nmeth.3972. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B43] 43.Zhang C, Meng X, Wei X, Lu L. 2016. Highly efficient CRISPR mutagenesis by microhomology-mediated end joining in Aspergillus fumigatus. Fungal Genet Biol 86:47–57. doi: 10.1016/j.fgb.2015.12.007. [DOI] [PubMed] [Google Scholar]

[B44] 44.Nissim L, Perli SD, Fridkin A, Perez-Pinera P, Lu TK. 2014. Multiplexed and programmable regulation of gene networks with an integrated RNA and CRISPR/Cas toolkit in human cells. Mol Cell 54:698–710. doi: 10.1016/j.molcel.2014.04.022. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B45] 45.Shi TQ, Liu GN, Ji RY, Shi K, Song P, Ren LJ, Huang H, Ji XJ. 2017. CRISPR/Cas9-based genome editing of the filamentous fungi: the state of the art. Appl Microbiol Biotechnol 101:7435–7443. doi: 10.1007/s00253-017-8497-9. [DOI] [PubMed] [Google Scholar]

[B46] 46.Rutledge PJ, Challis GL. 2015. Discovery of microbial natural products by activation of silent biosynthetic gene clusters. Nat Rev Microbiol 13:509–523. doi: 10.1038/nrmicro3496. [DOI] [PubMed] [Google Scholar]

[B47] 47.Pohl C, Kiel JA, Driessen AJ, Bovenberg RA, Nygard Y. 2016. CRISPR/Cas9-based genome editing of Penicillium chrysogenum. ACS Synth Biol 5:754–764. doi: 10.1021/acssynbio.6b00082. [DOI] [PubMed] [Google Scholar]

[B48] 48.Zhang JJ, Tang X, Moore BS. 2019. Genetic platforms for heterologous expression of microbial natural products. Nat Prod Rep 36:1313–1332. doi: 10.1039/c9np00025a. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B49] 49.Wasil Z, Pahirulzaman KAK, Butts C, Simpson TJ, Lazarus CM, Cox RJ. 2013. One pathway, many compounds: heterologous expression of a fungal biosynthetic pathway reveals its intrinsic potential for diversity. Chem Sci 4:3845–3856. doi: 10.1039/c3sc51785c. [DOI] [Google Scholar]

[B50] 50.Skellam E. 2019. Strategies for engineering natural product biosynthesis in fungi. Trends Biotechnol 37:416–427. doi: 10.1016/j.tibtech.2018.09.003. [DOI] [PubMed] [Google Scholar]

[B51] 51.Keller NP. 2019. Fungal secondary metabolism: regulation, function and drug discovery. Nat Rev Microbiol 17:167–180. doi: 10.1038/s41579-018-0121-1. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B52] 52.Fox EM, Howlett BJ. 2008. Secondary metabolism: regulation and role in fungal biology. Curr Opin Microbiol 11:481–487. doi: 10.1016/j.mib.2008.10.007. [DOI] [PubMed] [Google Scholar]

[B53] 53.Xie M, Wang Y, Tang L, Yang L, Zhou D, Yang L, Zhou D, Li Q, Niu X, Zhang K, Yang J. 2019. AoStuA, an APSES transcription factor, regulates the conidiation, trap formation, stress resistance and pathogenicity of the nematode‐trapping fungus Arthrobotrys oligospora. Environ Microbiol 21:4648–4661. doi: 10.1111/1462-2920.14785. [DOI] [PubMed] [Google Scholar]

[B54] 54.Xie M, Bai N, Yang J, Jiang K, Zhou D, Zhao Y, Li D, Niu X, Zhang K, Yang J. 2019. Protein kinase Ime2 is required for mycelial growth, conidiation, osmoregulation, and pathogenicity in nematode-trapping fungus Arthrobotrys oligospora. Front Microbiol 10:3065. doi: 10.3389/fmicb.2019.03065. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Two CRISPR/Cas9 Systems Developed in Thermomyces dupontii and Characterization of Key Gene Functions in Thermolide Biosynthesis and Fungal Adaptation

Wei-Ping Huang

Yuan-Jiang Du

Yun Yang

Jia-Ning He

Qian Lei

Xiao-Yu Yang

Ke-Qin Zhang

Xue-Mei Niu

Roles

ABSTRACT

INTRODUCTION

RESULTS

Expression and localization of mesophilic SpCas9 in T. dupontii.

FIG 1.

Expression of sgRNA in T. dupontii.

FIG 2.

CRISPR/SpCas9 gene editing in T. dupontii.

TABLE 1.

FIG 3.

Double-locus editing in T. dupontii via a tandem array in a single architecture.

Activation of a silent gene via tRNAGly/SpCas9-meditated HR in T. dupontii.

FIG 4.

Thermostable CRISPR/Cas9 for gene editing in T. dupontii at 45°C.

Elucidation of the key biosynthetic step for production of potent nematocidal thermolides in T. dupontii.

FIG 5.

Influence of biosynthetic genes on fungal growth, conidiation, and conidial germination.

FIG 6.

Contributions of biosynthetic genes to chemical stress responses.

FIG 7.

DISCUSSION

MATERIALS AND METHODS

Fungal strain and cultural conditions.

Construction of CRISPR/Cas9 system.

Donor plasmid construction.

Protoplasts transformation of T. dupontii.

Subcellular localization of SpCas9 in T. dupontii.

Screening of targeted clones.

RNA preparation and RT-PCR.

Western blotting.

LC-MS analysis.

Fungal morphology, conidial production, and germination.

Stress assays.

Data availability.

Supplementary Material

ACKNOWLEDGMENTS

Footnotes

REFERENCES

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

Activation of a silent gene via tRNA^Gly/SpCas9-meditated HR in T. dupontii.