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CRISPR/Cas9-based genome-editing in the filamentous fungus Fusarium fujikuroi and its application in strain engineering for gibberellic acid production Tian-Qiong Shi, Jian Gao, Wei-Jian Wang, Kai-Feng Wang, Guo-Qin Xu, He Huang, and Xiao-Jun Ji ACS Synth. Biol., Just Accepted Manuscript • Publication Date (Web): 07 Jan 2019 Downloaded from http://pubs.acs.org on January 8, 2019

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ACS Synthetic Biology

CRISPR/Cas9-based genome-editing in the filamentous fungus Fusarium fujikuroi and its application in strain engineering for gibberellic acid production

Tian-Qiong Shi1, Jian Gao5, Wei-Jian Wang1, Kai-Feng Wang1, Guo-Qin Xu1, He Huang 2, 3, 4, Xiao-Jun Ji1, 4

1 College

of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University,

No. 30 South Puzhu Road, Nanjing 211816, People’s Republic of China 2

College of Pharmaceutical Sciences, Nanjing Tech University, No. 30 South Puzhu

Road, Nanjing 211816, People’s Republic of China 3

State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing Tech

University, No. 30 South Puzhu Road, Nanjing 211816, People’s Republic of China 4

Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM),

Nanjing Tech University, No. 5 Xinmofan Road, Nanjing 210009, People’s Republic of China 5

School of Marine and Bioengineering, Yancheng Institute of Technology, Yancheng

224051, People’s Republic of China

Correspondence: [email protected] (X.-J. Ji)

ACS Paragon Plus Environment

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract The filamentous fungus Fusarium fujikuroi is well-known for its production of natural plant growth hormones: a series of gibberellic acids (GAs). Some GAs, including GA1, GA3, GA4, and GA7, are biologically active and have been widely applied in agriculture. However, the low efficiency of traditional genetic tools limits the further research towards making this fungus more efficient and able to produce tailor-made GAs. Here, we established an efficient CRISPR/Cas9-based genome editing tool for F. fujikuroi. First, we compared three different nuclear localization signals (NLS) and selected an efficient NLS from histone H2B (HTBNLS) to enable the import of the Cas9 protein into the fungal nucleus. Then, different sgRNA expression strategies, both in vitro and different promoter-based in vivo strategies, were explored. The promoters of the U6 small nuclear RNA and 5S rRNA, which were identified in F. fujikuroi, had the highest editing efficiency. The 5S rRNA-promoter-driven genome editing efficiency reached up to 79.2%. What’s more, multi-gene editing was also explored and showed good results. Finally, we used the developed genome editing tool to engineer the metabolic pathways responsible for the accumulation of a series GAs in the filamentous fungus F. fujikuroi, and successfully changed its GA product profile, from GA3 to tailor-made GA4 and GA7 mixtures. Since these mixtures are more efficient for agricultural use, especially for fruit growth, the developed strains will greatly improve industrial GA production.

Keywords: CRISPR/Cas9; Fusarium fujikuroi; genome editing; gibberellic acids; metabolic pathway


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Introduction The filamentous fungus Fusarium fujikuroi (formally known as Gibberella fujikuroi) is well-known for its production of many valuable secondary metabolites, such as bikaverin, neurosporaxanthin, fusarubins and gibberellic acids (GAs)

1-4.

Among these, the production of GAs is dominant in F. fujikuroi. GAs are a class of tetracyclic diterpenoids with biological activities in many different organisms, which play an important role in plant growth regulation, and thus have been widely applied in agriculture and the brewing industry, with great economic benefits

5, 6.

To date, 136

different GAs have been identified, but the most biologically active ones are GA1, GA3, GA4, and GA7 (Fig. 1) 7, 8. Compared with the widely used GA3, mixtures of GA4 and GA7 have recently been paid more attention due to their more moderate and diverse effects

9-12.

However, their production is challenging compared to GA3, which limits

their wider use. In recent years, more and more metabolic and regulatory mechanisms of F. fujikuroi have been elucidated, which significantly accelerated the rate of the relevant metabolic research

13-16.

However, because non-homologous end-joining (NHEJ) is

dominant in F. fujikuroi, gene knockout and integration of heterologous fragments requires very long homologous arms, which contributes to the low editing efficiency. Although a number of studies have proved that the disruption of KU70 or KU80 proteins, which are responsible for the NHEJ, can greatly improve the editing efficiency, no corresponding research was reported for F. fujikuroi. Moreover, increasing evidence indicates that KU70/80-disrupted mutants may be more sensitive to factors such as temperature and radiation, and are consequently not suitable for industrial applications 17, 18.

These disadvantages limit further metabolic research in F. fujikuroi, because

multiple gene disruption and integration requires prohibitive labor costs and timeframes.



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Therefore, it is necessary to develop a powerful and versatile genome-editing tool for F. fujikuroi. The CRISPR/Cas9 system, which emerged at an opportune time, avoids many of the limitations of traditional approaches 19. The CRISPR/Cas9 system consists of only two components, the Cas9 protein and the functional sgRNA. When the sgRNA recognizes the target sequence in the genome, the Cas9 protein will cleave the sequence, producing a double-stranded break 20, 21. Then, the cell will repair the genome by nonhomologous end-joining or homologous recombination, whereby the latter can be used for the purpose of gene knockout and integration 22, 23, 50, 51. Due to its high efficiency, versatility, and easy handling, the CRISPR/Cas9 system was quickly applied to many different filamentous fungi 24. Here, we developed an efficient genome editing technology for the filamentous fungus F. fujikuroi using the CRISPR/Cas9 system. The endogenous nuclear localization signal (NLS) from histone H2B (HTBNLS) was selected for the efficient import of the Cas9 protein into the nucleus. Three target sites - Fusarium cyclin C1 (fcc1), orotidine-5'-phosphate decarboxylase (ura3), and 4'-phosphopantetheinyl transferase (ppt1), were selected to verify the efficiency of this CRISPR/Cas9 based system. The disruption of the three target genes directly affects the accumulation of a specific purple pigment, as well as uracil and lysine auxotrophy, respectively

25-27.

Different sgRNA expression strategies, both in vitro and in vivo, were adopted to improve the editing efficiency. Further experiments showed that the established CRISPR/Cas9 system is efficient for multi-gene editing in F. fujikuroi, and can therefore be reasonably expected to be applicable to other filamentous fungi. Finally, we used the developed CRISPR/Cas9 system to rewrite the metabolic pathways to improve the accumulation of GA4 and GA7, which have boarder applicability in certain


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areas compared to the conventional GA3, and thus bring significant economic benefits. Results Modification of the protoplast-based polyethylene glycol and Ca2+ transformation method in F. fujikuroi In filamentous fungi, an efficient transformation method is a sine qua non for any genetic engineering technology. In order to improve the transformation efficiency in F. fujikuroi, we optimized several key factors including the used cell-wall ablation enzymes, the enzyme ratio and digestion time. Firstly, we tested the performance of 5 different enzymes in the protoplast formation process: lysozyme (Sangon Biotech Co., Ltd., China), snailase (Sangon Biotech Co., Ltd., China), cellulase (Sangon Biotech Co., Ltd., China), lysing enzyme (Sigma-Aldrich, USA) and driselase (Sigma-Aldrich, USA). Samples comprising 1.0 g of wet mycelia were collected and suspended in 10 mL of phosphate buffered saline (PBS) which included 15 mg/mL of the indicated enzymes and 1.0 M sorbitol to balance the osmotic pressure. After incubation at 28 °C for 3 h, the mycelia were filtrated through 4 layers of filter paper and the protoplast solution was collected to count using a blood count plate. As shown in Fig. 2A, the lysing enzyme and driselase had higher protoplast formation efficiencies than the other enzymes. Considering that mixed enzymes often display a better performance in cell wall digestion, we next optimized the relative ratio of the lysing enzyme and driselase. At a ratio of lysing enzyme to driselase of 3:2, the number of protoplasts reached the maximum (Fig. 2B). Finally, the digestion time was also optimized. Although 4 h of incubation gave the highest protoplast number, the regeneration frequency of the resulting protoplasts was decreased significantly. Therefore, an incubation time of 3.5 h was chosen (Fig. 2C and 2D). The PBS solution was removed by centrifugation at 3000 g for 5 min twice, and



the protoplasts were resuspended in 1.0 M sorbitol to a concentration of 107 per mL. In order to verify the transformation efficiency, 50 μL of the protoplast suspension was mixed with 25 μL of buffer with 1.0 M sorbitol (SMS: 1.0 M sorbitol, 10 mM Tris-Cl, 50 mM CaCl2, pH=7.5) and 5 μg of pAN7-1 vector DNA which carries the hygromycin resistance marker, followed by incubation on ice for 20 min. Then, 50 μL 25% PEG 6000 was added to the protoplast solution and incubated for 20 min in an ice bath. Finally, 500 μL PEG 6000, and 1 mL SMS were added and mixed thoroughly with the protoplast solution. An aliquot comprising 200 μL of the final transformation solution was spread on a regeneration plate. After 12 h of incubation at 30°C, every plate was covered with 10 mL soft-agar regeneration medium with 100 μg/mL hygromycin. After 3-5 days of incubation at 28°C, the transformants were streaked for a second round of hygromycin screening to remove false-positives. Selection of an efficient nuclear localization signal for establishment of the CRISPR/Cas9 system in F. fujikuroi When applying the CRISPR/Cas9 system to eukaryotic cells, the Cas9 protein must be fused to a nuclear localization signal (NLS) to allow its import into the nucleus in order to catalyze a double-strand break (DSB) after the target DNA sequence is recognized. Generally, the classical SV40 NLS (SV40NLS) can meet most needs. However, in many filamentous fungi, the SV40NLS was reported to be ineffective for the import of the Cas9 protein into the nucleus 28, 29. In order to establish an efficient CRISPR/Cas9 system in F. fujikuroi, we selected three NLSs to individually fuse with the fFuCas9 protein. These included the SV40NLS, the endogenous NLS from histone H2B (HTBNLS) and the endogenous NLS from Velvet (VELNLS). The HTBNLS is conserved in eukaryotes (Fig. S2) and was used in the CRISPR/Cas9 system developed for Fusarium oxysporum29. In addition, the Velvet gene has been proved to be a nuclear protein which is related to


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the secondary metabolism in many filamentous fungi 30. The Fusarium cyclin C1 gene (fcc1) was first chosen as the target gene, because previous research has demonstrated that fcc1-disrupted F. fujikuroi strains exhibited significantly more red pigment accumulation on PDA compared with the control

25.

In order to verify whether this

phenomenon is also present in our strain, we constructed an fcc1-disruption cassette and disrupted the fcc1 gene via homologous recombination (Fig. 3A). Interestingly, when grown on PDA, the fcc1-disrupted F. fujikuroi accumulated more purple pigment rather than red (Fig. 3B). Considering that no RNA polymerase III promoters such as the U6 small nuclear RNA promoter were reported in F. fujikuroi, in-vitro produced sgRNAs and fFuCas9 vectors carrying different NLSs were utilized to establish the CRISPR/Cas9 system (Fig. 4A). According to the fcc1 sequence, we designed three target sites as the protospacers, which were named locus1, locus2, and locus3. All protospacer-adjacent motif (PAM) sequences were chosen as the TGG for the control (Fig. 4B). First, 5 μg and 10 μg of in-vitro produced locus2-sgRNA were introduced into protoplasts with different fFuCas9 fusion protein vectors (Table 1). However, no mutations were found in the Cas9-SV40NLS fusion protein-mediated system, which showed that the SV40NLS was inefficient or non-functional in F. fujikuroi. By contrast, both VELNLS and HTBNLS were functional in the CRISPR/Cas9 system, whereby the mutation efficiency meditated by VELNLS reached to 4%, and that of the HTBNLS even 41.7%. Compared to the SV40NLS and VELNLS, the HTBNLS can apparently mediate the import of fFuCas9 into the nucleus much more efficiently and contribute to the higher editing efficiency. Therefore, we finally chose the HTBNLS for fusion with the fFuCas9 protein to conduct further experiments. Considering that different loci can also influence the editing efficiency, we next verified the efficacy of the CRISPR/Cas9 system mediated by the locus1, locus



2, and locus 3 sgRNAs under the control of the vector pUC-Cas9-HTBNLS-hph. A total of 10 μg in-vitro produced sgRNA was used for transformation. In all tests, the locus2 sgRNAs-meditated CRISPR/Cas9 system had a higher editing efficiency, and we therefore chose locus2 for further experiments (Fig. 4C). At the end, in order to verify whether expressing fFuCas9 gene will significantly affect the cell growth ability, the dry cell weight was further measured during the fermentation period. The result displayed that the Cas9 expression will not affect the cell growth (Fig. S1). CRISPR/Cas9-based gene disruption using different promoters to drive sgRNA expression The sgRNA expression strategies can be based on either in vivo or in vitro RNA production. Based on the above results, the in-vitro produced sgRNAs were proved to be functional in the CRISPR/Cas9 system. However, the in-vitro production of sgRNA is generally time-consuming and the isolated product is unstable. Consequently, we attempted to find a method to express the sgRNA in vivo. Different promoters driving sgRNA expression were subsequently tested in F. fujikuroi (Fig. 5A). Owing to the lack of a cap structure and poly A-tail, the synthetic sgRNA cannot be transcribed directly by RNA polymerase II, and therefore typically needs to be transcribed using RNA polymerase III promoters 31. However, due to the complexity of the metabolic networks of filamentous fungi, RNA polymerase III promoters were rarely utilized, with few reports in the literature. In order to overcome this difficulty, Nødvig et al. 31 firstly used the two ribozyme sequences, 5’-end hammerhead (HH) and 3’-end hepatitis delta virus (HDV)-to flank the sgRNA in filamentous fungi. Due to self-processing RNA cleavage and subsequent release of the sgRNA sequence without modifications, the successful sgRNA expression only needs a strong RNA polymerase II promoter. Considering that no RNA polymerase III promoters were reported in F.


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fujikuroi, we first adopted the HH and HDV strategy to target the fcc1 gene. The locus2 sgRNA was selected as the protospacer due to its relatively high editing efficiency. Unfortunately, no mutations were found among 48 tested clones, which proved that this strategy was unsuitable for F. fujikuroi. Therefore, we attempted to identify endogenous RNA polymerase III promoters, which can drive sgRNA expression in F. fujikuroi. First, we paid more attention to the U6 small nuclear RNA (snRNA) which is generally conserved in most species, since its promoter has been used in to transcribe sgRNAs in many filamentous fungi, including Aspergillus fumigatus, Aspergillus oryzae, Myceliophthora thermophila, Penicillium chrysogenum and Ustilago maydis

32-36.

As

shown in Fig. 5B, we compared the U6 snRNA sequence from Solanum lycopersicum and Myceliophthora thermophila with the F. fujikuroi genome, and successfully identified the endogenous U6 snRNA sequence in F. fujikuroi (Ff snRNA), which shared 81.4% sequence similarity. Consequently, we used a 467 bp sequence upstream of the Ff snRNA and a TTTTTT sequence as the promoter (Ff U6) and terminator of the fcc1sgRNA, respectively. Among 24 tested clones, the nine had fcc1 mutations by sequencing, which corresponds to an efficiency of up to 37.5%. What’s more, in a recent research, Zheng et al.

37

demonstrated that the 5SrRNA

gene, which is both highly conserved and efficiently expressed in eukaryotes, can be used as a guide RNA promoter. The genome editing efficiency with this system even reached 100% in A. niger. Therefore, we tested whether 5SrRNA was also effective in F. fujikuroi. Using the 5S rRNA sequence of A. niger, we identified over one hundred copies of endogenous 5S rRNA sequences in the F. fujikuroi genome. However, we found these 5S rRNA sequences were not identical, several nucleotides varied between these sequences and we did not know which one was functional. Therefore, we selected one 5S rRNA which existed more than seven copies in F. fujikuroi, the copy number



was more than other 5S rRNAs. According to this characteristic, we supposed that it was functional in F. fujikuroi. By the sequence alignment, the selected 5S rRNA shared 82.5% similarity with the functional 5S rRNA in A. niger (Fig. 5B). At the beginning, we used the endogenous 5SrRNA (Ff 5SrRNA) to express the sgRNA targeting the fcc1 gene, and the final result demonstrated that the Ff 5srRNA was highly efficient in transcribing the sgRNA in F. fujikuroi. Among the 24 tested clones, 19 showed the accumulation of purple pigment. Hence, the editing efficiency reached up to 79.2% (Fig. 5C). In order to verify the robustness of the Ff5SrRNA-based CRISPR/Cas9 system, the ura3 and ppt1 genes were targeted individually. The editing efficiency reached 58.3% and 66.7%, respectively, which was much higher than what was obtained using other sgRNA expression strategies (Fig. 5D). Multi-gene disruption using the efficient CRISPR/Cas9 system in F. fujikuroi In all sgRNA expression strategies, the 5SrRNA promoter contributed to the highest genome editing efficiency. The highest editing efficiency for single-gene disruptions reached up to 79.2%. Here, we attempted to verify if the 5SrRNA promoter is strong enough to support double- and triple-gene disruption. In the double-gene disruption experiment, fcc1 and ura3 were selected as the target genes. The fcc1-sgRNA and ura3-sgRNA expression cassettes were cloned into the vector pUC-fFuCas9HTBNLS-hph and then introduced into the protoplasts. The results revealed 5 desired double mutations among 24 tested clones, which means that the efficiency of doublegene disruption reached 20.8%. Then, the fcc1-sgRNA, ura3-sgRNA expression and ppt1-sgRNA cassettes were all cloned into the vector pUC-fFuCas9-HTBNLS-hph and then introduced into the protoplasts. One desired mutant was found among 24 tested clones by sequencing. Hence the efficiency of triple gene deletion was 4.2%. Therefore, the CRISPR/Cas9 system developed here for F. fujikuroi was proved to be efficient


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enough for multi-gene disruption, which will greatly speed up the metabolic research in F. fujikuroi. Rewriting the metabolic pathways in F. fujikuroi to change its GA product profile As well-known diterpenoids, the biosynthesis pathway of GAs is similar to the other terpenoids and is mainly divided into three parts. As shown in Fig. 1, the first part is the biosynthesis of geranylgeranyl diphosphate (GGPP). At the beginning, hydroxymethylglutaryl coenzyme A (HMG-CoA) is synthesized through the condensation of two acetyl-CoA molecules and subsequently reduced to mevalonate by HMG-CoA reductase (HmgR) 38. This was long viewed as the rate-limiting step in the isoprenoid pathway and was targeted for strengthening the production of many different terpenoids

39-42.

Then, after further multi-step catalysis, the isopentenyl diphosphate

(IPP), dimethylallyl diphosphate (DMAPP), geranyl diphosphate (GPP) and farnesyl diphosphate (FPP) intermediates are synthesized. The FPP is eventually converted into GGPP in a reaction catalyzed by geranylgeranyl diphosphate synthase (Ggs2) 43. The second part is the synthesis of GA12-aldehyde. GGPP is first converted to ent-kaurene by the bifunctional enzyme copalyldiphosphate synthase/kaurene synthase (Cps/Ks) 44. This pathway regarded as the key step in the synthesis of GAs. Previous research has proved that the overexpression of Cps/Ks has a positive effect on the production of GA4 and GA7 45. In the next steps, the ent-kaurene is converted to GA12-aldehyde by the two-step cytochrome P450 monooxygenases P450-4 and P450-1 46. In this part, the GA biosynthesis pathways of higher plants and fungi are similar. The third part is the biosynthesis of a series of different GAs. The GA12-aldehyde is further converted by P450-1, which contributes to the formation of GA14, which is in turn converted by P450-2 to produce GA4, which is the first bioactive GA

47.

After that, a desaturase

enzyme converts GA4 into GA7, which can be further converted to GA3 by P450-3. A



small amount of GA4 is converted to GA1 by the same P450-3 48. According to what is known, we attempted to rewrite the metabolic pathway of GA biosynthesis in F. fujikuroi to change its GA product profile. In F. fujikuroi NJtech 02, the production of GA3 is dominant (376.92 mg/L), and only small amounts of GA4 and GA7 were detected (GA4 (25.10 mg/L; GA7, 63.28 mg/L). In addition, no GA1 was found, which can be partly ascribed to the quite low GA4 yield (Fig. 6A). Therefore, the P450-3 gene was first disrupted to block the synthesis of GA3. As shown in Fig. 6B, the production of GA4 and GA7 in the △P450-3 disruption mutant (410.27 mg/L) was obviously improved compared to the reference strain (88.38 mg/L). Then, using the △P450-3 disruption mutant as the basis, the two key genes Cps/Ks and the truncated HmgR (tHmgR) were overexpressed separately. The truncated HmgR avoids the selfdegradation mediated by its N-terminal domain and is thus stabilized in the cytoplasm. Therefore, the cytosolic domain of HmgR was generally overexpressed to strengthen the supply of precursors in the mevalonate pathway. The fermentation performance of the corresponding strain indicated that overexpression of the Cps/Ks can contribute to an enhanced GA4 and GA7 content (24.23%, 509.68 mg/L). The overexpression of tHmgR had a more significant effect on GA4 and GA7 accumulation (170.14%, 698.03 mg/L) compared to the △P450-3 disruption mutant. To further improve the content, the two genes were simultaneously integrated into the genome. The production of the GA4/GA7 mixture reached 716.37 mg/L, a small improvement compared to the parent strain, whereby GA4 production reached 292.46 mg/L and GA7 production reached 423.91 mg/L, corresponding to a ratio of 1:1.47. In summary, this experiment demonstrated that rewriting the metabolic pathway in F. fujikuroi by combined use of the developed CRISPR/Cas9 tools and overexpression methods, is quite effective for increasing the production of GA4/GA7 mixtures, the content of which was improved


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8.1-fold compared with the control. Discussion Due to the high efficiency, versatility, and easy handling, the CRISPR/Cas9 system has been applied in many different species, including bacteria, fungi, plants, animals and human cells. Due to the complexity of the genetic background of filamentous fungi compared to other organisms, the CRISPR/Cas9 system is still in its infancy. For example, the NLS plays a vital role in the CRISPR/Cas9 system. However, in many filamentous fungi, the general SV40 NLS is not functional and cannot guide the import of Cas9 into the nucleus. Thus, additional time and work is required to identify an efficient NLS in filamentous fungi. What’s more, sgRNA expression is still a problem. The in-vitro produced sgRNA is unstable and is not suitable for those filamentous fungi that must be transformed using Agrobacterium tumefaciens. The invivo expression of sgRNA is usually driven by the Pol III promoter. Both aspects hamper the broader application of the CRISPR/Cas9 system in filamentous fungi. In the present study, we proved that HTBNLS from histone H2B is efficient enough for use in the CRISPR/Cas9 system in F. fujikuroi. Considering that this protein is conserved in most filamentous fungi, and that the HTBNLS has also been applied in the CRISPR/Cas9 system in F. oxysporum, we hypothesize that the HTBNLS is suitable for other filamentous fungi, which can solve the problem of the nuclear import of Cas9. In order to solve the problem of the instability of in-vitro produced sgRNAs, different in vivo expression strategies were explored. Due to a lack of wellcharacterized Pol III promoters in F. fujikuroi, the universal Pol II promoter-meditated HH-sgRNA-HDV expression cassette was adopted. Unfortunately, no mutations were found among 48 tested clones, which indicated that this strategy was unsuitable in F. fujikuroi. Subsequently, considering that the promoter of U6 snRNA was generally used



successfully in filamentous fungi, we identified the homologous promoter of F. fujikuroi and used it for sgRNA expression, after which the editing efficiency reached 37.5%. Another major finding of this study is that the 5S rRNA promoter can also be used for the efficient expression of sgRNAs, which was similar to the results reported for A. niger 25. Different from the U6 promoter, which is only present in 1-2 copies in filamentous fungi, the 5S rRNA is present in several hundred copies, and therefore can offer higher sgRNA transcription levels. The highest efficiency we observed in F. fujikuroi reached 80%. What’s more, 5S rRNA-meditated multi-gene disruption also showed good results. Considering that the 5S rRNA is highly conserved among filamentous fungi, the strategy can also be expanded to other species to solve the problem of sgRNA expression. In summary, we proved that the CRISPR/Cas9 system is stable enough for application in F. fujikuroi, and we expect it to be expanded to other filamentous fungi as well. In addition, with the help of the established CRISPR/Cas9 technology in F. fujikuroi, we successfully rewrote the metabolic pathways to change its product profile from predominantly GA3 to a tailor-made mixture of GA4 and GA7. The final production of the GA4/GA7 mixture was improved 8.1-fold compared with the initial strain. Materials and methods Strains and culture media The F. fujikuroi strains used in this study are listed in Table S2. F. fujikuroi NJtech 02 (CCTCC M2015614), preserved in the China Centre for Type Culture Collection, was used for CRISPR/Cas9 genome editing. Escherichia coli DH5α (Vazyme Biotech Co., Ltd., China) was used for plasmid construction and transformation, and was cultured at 37 °C in Luria-Bertani (LB) medium containing 100 μg/mL ampicillin. F. fujikuroi protoplasts were cultivated on regeneration solid


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medium (2% glucose, 0.3% yeast extract, 1.5% agar, 0.6 M sucrose). After 12 h of incubation, each plate was covered with 10 mL of soft-agar regeneration medium (2% glucose, 0.3% yeast extract, 0.8% agar, 0.6 M sucrose, 100 μg/mL hygromycin). F. fujikuroi clones were cultivated on basic rich medium (1% yeast extract (2% peptone (2% glucose) for genomic DNA extraction and PCR. The seed medium was composed of (g/L): glucose, 60; yeast extract, 5.5; MgSO4·7H2O, 0.2 and KH2PO4, 1.5. The fermentation medium was composed of (g/L): glucose, 90; defatted soybean meal, 12; MgSO4·7H2O, 0.1; KH2PO4, 1.5, and the pH was set to 6.5 using NaOH and HCl. For seed cultures, F. fujikuroi NJtech 02 from a fresh slant tube was used to inoculate 500 mL flasks containing 100 mL of fresh seed medium and cultivated at 200 rpm and 30°C for 24 h. The resulting seed cultures (5%, v/v) were used to inoculate the fermentation medium. Plasmid construction The primers used in this research are listed in Table S1. Gene sequences and sources are listed in Data S1. Plasmids used in this research are listed in Table S2. The plasmids were constructed through the ClonExpress® MultiS One Step Cloning Kit (Vazyme Biotech Co., Ltd., China). The Cas9 gene from Streptococcus pyogenes was codon-optimized for efficient expression in F. fujikuroi (fFuCas9, Synbio-tech Co., Ltd., China). The A. nidulans gpdA promoter (PgpdA) and trpC terminator (TtrpC) were amplified from vector pAN7-1 49. The hygromycin resistance gene expression cassette (hph) was amplified from vector pFC332 31. The nuclear localization signals HTBNLS and VELNLS were amplified from the genomic DNA of F. fujikuroi. The fragments encoding the gpdA promoter, fFuCas9, individual NLS, trpC terminator and hph were assembled simultaneously and ligated into the HindIII site of the vector pUC57. The clones were selected using colony PCR and confirmed by sequencing. The final vectors



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were named as pUC-fFuCas9-SV40NLS-hph, pUC-fFuCas9-HTBNLS-hph (Saved in Addgene,

plasmid#121092)

and

pUC-fFuCas9-VELNLS-hph.

The

fcc1

upstream/downstream homologous arms and hph were amplified using the primer pairs Fcc1-U-F/R, Hph-F/R and Fcc1-D-F/R. Three fragments were cloned into the HindIII site of the vector pcDNA3.1- (provided by Synbio-tech Co., Ltd., China), yielding the final fcc1-disruption cassette, which was named pcDNA-△fcc1. The PgpdA-HH-sgRNA-HDV-TtrpC expression cassette and the Ff5SrRNAbased fcc1, ura3 and ppt1 sgRNA expression cassettes were synthesized by Synbiotech Co., Ltd., China. The Ff5SrRNA-fcc1 sgRNA cassette was amplified using the primer pair 5SrRNA1-F/R and ligated into the EcoRI site of the vector pUC-fFuCas9HTBNLS-hph for fcc1-disruption, yielding pUC-fFuCas9-HTBNLS-hph-fcc1. The Ff5SrRNA-ura3 and Ff5SrRNA-ppt1 sgRNA cassettes were amplified using the primer pair 5SrRNA-U-F/R, and ligated into the EcoRI site of the vectors pUC-fFuCas9HTBNLS-hph and pUC-fFuCas9-HTBNLS-hph-fcc1 for double- and triple-gene disruption, yielding the vector pUC-fFuCas9-HTBNLS-hph-fcc1-ura3/fcc1-ura3-ppt1. The U6 promoter was amplified from the F. fujikuroi genome using the primers U6-1EcoRI-F and U6-1-R, and the fcc1-sgRNA cassette was amplified using the primers U6-1-F and U6-1-EcoRI-R. The two fragments were ligated into the EcoRI site of the vector pUC-fFuCas9-HTBNLS-hph for fcc1 disruption, yielding pUC-fFuCas9HTBNLS-hph-U6-fcc1. The PgpdA-HH-sgRNA-HDV-TtrpC cassette was amplified using the primer pair HH-sgRNA-HDV-F/R and ligated into the EcoRI site of the vector pUC-fFuCas9-HTBNLS-hph for fcc1 disruption, yielding pUC-fFuCas9-HTBNLS-hphHH-HDV-fcc1. For the disruption of the gene encoding P450-3, the Ff 5SrRNA-P450-3 sgRNA cassette was synthesized and ligated to the EcoRI site of pUC-fFuCas9-HTBNLS-hph,


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yielding pUC-fFuCas9-HTBNLS-hph-P450-3. For the integration of Cps/Ks and tHmgR, a gene located on chromosome II (chII, XM_023572143.1), which does not affect the normal growth of F. fujikuroi, was selected as the integration site. The donor vector carrying the upstream/downstream homologous arms of the chII target site and G418 resistance cassette was constructed first (pUC-△chII). Subsequently, the Cps/Ks and tHmgR expression cassettes controlled by the strong PgpdA promoter were ligated into the vector, both separately and simultaneously. The fFuCas9 vector targeting the chII site was introduced via protoplast transformation with the donor vectors for gene overexpression. sgRNA in vitro transformation The sgRNA cassettes targeting the three different sites in fcc1 (locus 1, locus 2 and locus 3) were controlled by the T7 promoter (Data S1). The fragments were ligated into pUC57 for subsequent transcription. The HiScribe T7 High Yield RNA Synthesis Kit (NEB, USA) was used for in vitro transcription of RNA using T7 RNA polymerase. A total of 1 μg of sgRNA-template was mixed with the sgRNA synthesis reagents and incubated at 37 °C overnight. After purification, the sgRNA content was measured using the NanoDrop instrument (Thermo Fisher Scientific, USA). The in vitro produced sgRNA and the fFuCas9 vector were introduced via protoplast transformation using the modified method 25. The positive clones were selected on solid regeneration medium plates containing 100 μg/mL hygromycin, and the genome editing efficiency was verified by observing the production of the purple pigment. Dry cell weight measurement The dry cell weight was measured every 24 h. The 50 mL samples from the fermentation medium are vacuum filtered through a glass-fiber filter and then washed twice with distilled water. The wet mycelia were dried at 60 °C to a constant weight.



Nuclear localization signal prediction In this research, we compared three different nuclear localization signals including the SV40NLS, HTBNLS and VELNLS to improve the editing efficiency. Apart from the known the SV40NLS (PKKKRKV), the other two nuclear localization signal sequences from histone H2B and the Velvet are unknown. Therefore, the NLStradamus website (http://www.moseslab.csb.utoronto.ca/NLStradamus/) was used to predict the nuclear localization signal. Bioinformatic analysis revealed the histone H2B contained a NLS at its N-terminus as described previously 29 (aa: 6-45, Fig. S3A). In order to confirm the predicted HTBNLS was sufficient, the first 54 amino acids including the entire NLS sequence was fused with fFuCas9. Similar method was applied to evaluate the VELNLS. Compared with the constant HTBNLS sequence, VELNLS varied apparently when different prediction cutoffs were set (aa: 208-259, 408-428, 465-480, Fig. S3B). In order to confirm the VELNLS is functional, the amino acids from 200 to 530 was fused with fFuCas9. Analysis of gibberellic acids After 5 days of cultivation in fermentation medium, the culture was filtered, and then the supernatant fraction, which contains GA1, GA3, GA4, GA7 and other GAs, were analyzed using high performance liquid chromatography (HPLC, Dionex U3000, Dionex, USA), equipped with a Venusil MPC18 column (Agela Technologies, China). The pretreated samples were separated using a mobile phase comprising methanol/water/phosphoric acid (68:32:0.05) at a flow rate of 0.7 mL/min. The detection wavelength was 210 nm, and the injection volume was 10 μL. The retention time GA7, GA4 and GA3 is 10.791, 12.446 and 17.565min (Fig. S4A). The standard curves of GA3, GA4 and GA7 concentration were listed such as y3, y4 and y7 in Fig. S4B.


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Acknowledgements We are grateful to Prof. Uffe H. Mortensen from Technical University of Denmark for providing the pFC332 plasmid. This work was financially supported by the National Natural Science Foundation of China (Nos. 21776131, 21476111, 21376203), the Six Talent Peaks Project in Jiangsu Province of China (No. 2018-SWYY-047), the Program for Innovative Research Teams in Universities of Jiangsu Province, and the Jiangsu Synergetic Innovation Center for Advanced Bio-Manufacture (No. XTD1814). Supporting Information Strains, plasmids and primers used in this study; sequences of the synthetic genes and the constructed cassettes. AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]. ORCID He Huang: 0000-0003-2192-9620 Xiao-Jun Ji: 0000-0002-6450-0229 Author Contributions Tian-Qiong Shi, Wei-Jian Wang, Kai-Feng Wang, and Xiao-Jun Ji developed the research plan. Tian-Qiong Shi, Wei-Jian Wang, Kai-Feng Wang performed the experiments. Tian-Qiong Shi, Guo-Qin Xu collected and analyzed data. Tian-Qiong Shi, Jian Gao, He Huang, and Xiao-Jun Ji wrote the manuscript. All authors commented on and revised the manuscript. Notes The authors declare no competing financial interest.



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Table 1 Editing efficiency of fcc1 with different nuclear localization signals used for Cas9 Type of Cas9

fcc1-sgRNA

fusion protein

in vitro (μg)

Cas9-SV40NLS

5

24

0

0

10

24

0

0

5

26

0

0

10

23

1

4

5

24

7

29.2

10

24

10

41.7

Cas9-VELNLS

Cas9-HTBNLS

Clones tested

Mutant clones

Mutation rate (%)



Page 28 of 36

Figure captions: Fig. 1 Metabolic pathways for the synthesis of a series of gibberellic acids in Fusarium fujikuroi Hydroxymethylglutaryl coenzyme A, HMG-CoA; truncated HMG-CoA reductase, tHmgR; isopentenyl diphosphate, IPP; dimethylallyl diphosphate, DMAPP; geranyl diphosphate, GPP; farnesyl diphosphate, FPP; farnesyl diphosphate synthase, FppS; geranylgeranyl diphosphate synthase 2, Ggs2; geranylgeranyl diphosphate, GGPP; copalyldiphosphate

synthase/kaurene

synthase,

Cps/Ks;

cytochrome

P450

monooxygenase, P450-1, P450-2, P450-3, P450-4.

Fig. 2 Modified protoplast-based polyethylene glycol and Ca2+ transformation method for F. fujikuroi. (A) The number of protoplasts produced by the treatment of the mycelia of F. fujikuroi with 5 different enzymes (15 mg/mL). (B) The number of protoplasts produced with different enzyme ratios of the lysing enzyme and driselase. (C) The number of protoplasts produced after different digestion times. (D) Regeneration frequency of the protoplasts produced after different digestion times.

Fig. 3 The fcc1-diruption cassette designed for verification of successful mutagenesis. (A) The fcc1-diruption cassette was constructed using upstream/downstream homologous arms of fcc1 and the hygromycin resistance fragment (hph), and subsequently introduced into the protoplasts for gene knockout via homologous recombination. (B) The fcc1-disrupted strain accumulated more purple pigment than the control.


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Fig. 4 Selection of nuclear localization signals (NLS) for establishment of the CRISPR/Cas9 system in F. fujikuroi. (A) fFuCas9 expression cassettes carrying different NLSs including SV40NLS, VELNLS and HTBNLS were constructed. (B) Three different sites (locus1, locus2 and locus3) in the fcc1 gene were selected. All protospacer adjacent motif (PAM) sequences were chosen as the TGG for the control. (C) The vector pUC-fFuCas9-HTBNLS-hph was used to verify the editing efficiency of three in-vitro produced sgRNAs .

Fig. 5 The in vivo sgRNA expression strategies. (A) Three methods were explored to express the sgRNA in vivo: Pol II promoter driving sgRNA expression, as well as the Pol III Ff U6 and Ff 5S rRNA promoters driving sgRNA expression. (B) Nucleotide blast of the F. fujikuroi U6 snRNA and 5S rRNA promoter sequences against those from other filamentous fungi. (C) Editing efficiency of systems with different promoters driving sgRNA expression. (D) The disruption efficiency of fcc1, ura3 and ppt1 as the targets, using the 5S rRNA promoter.

Fig. 6 Rewriting the metabolic pathways of F. fujikuroi to change its gibberellic acid (GA) product profile.

(A) The GA product profile of the wild-type control strain,

including GA1, GA3, GA4 and GA7. (B) Improving the accumulation of GA4 and GA7 by deleting the P450-3 gene and overexpressing the genes Cps/Ks and tHmgR, which encode rate-limiting enzymes.



Graphic abstract 203x101mm (300 x 300 DPI)


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Fig. 1 Metabolic pathways for the synthesis of a series of gibberellic acids in Fusarium fujikuroi Hydroxymethylglutaryl coenzyme A, HMG-CoA; truncated HMG-CoA reductase, tHmgR; isopentenyl diphosphate, IPP; dimethylallyl diphosphate, DMAPP; geranyl diphosphate, GPP; farnesyl diphosphate, FPP; farnesyl diphosphate synthase, FppS; geranylgeranyl diphosphate synthase 2, Ggs2; geranylgeranyl diphosphate, GGPP; copalyldiphosphate synthase/kaurene synthase, Cps/Ks; cytochrome P450 monooxygenase, P450-1, P450-2, P450-3, P450-4. 191x121mm (300 x 300 DPI)



Fig. 2 Modified protoplast-based polyethylene glycol and Ca2+ transformation method for F. fujikuroi. (A) The number of protoplasts produced by the treatment of the mycelia of F. fujikuroi with 5 different enzymes (15 mg/mL). (B) The number of protoplasts produced with different enzyme ratios of the lysing enzyme and driselase. (C) The number of protoplasts produced after different digestion times. (D) Regeneration frequency of the protoplasts produced after different digestion times. 284x200mm (300 x 300 DPI)


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Fig. 3 The fcc1-diruption cassette designed for verification of successful mutagenesis. (A) The fcc1-diruption cassette was constructed using upstream/downstream homologous arms of fcc1 and the hygromycin resistance fragment (hph), and subsequently introduced into the protoplasts for gene knockout via homologous recombination. (B) The fcc1-disrupted strain accumulated more purple pigment than the control. 227x186mm (300 x 300 DPI)



Fig. 4 Selection of nuclear localization signals (NLS) for establishment of the CRISPR/Cas9 system in F. fujikuroi. (A) fFuCas9 expression cassettes carrying different NLSs including SV40NLS, VELNLS and HTBNLS were constructed. (B) Three different sites (locus1, locus2 and locus3) in the fcc1 gene were selected. All protospacer adjacent motif (PAM) sequences were chosen as the TGG for the control. (C) The vector pUCfFuCas9-HTBNLS-hph was used to verify the editing efficiency of three in-vitro produced sgRNAs . 366x196mm (300 x 300 DPI)


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Fig. 5 The in vivo sgRNA expression strategies. (A) Three methods were explored to express the sgRNA in vivo: Pol II promoter driving sgRNA expression, as well as the Pol III Ff U6 and Ff 5S rRNA promoters driving sgRNA expression. (B) Nucleotide blast of the F. fujikuroi U6 snRNA and 5S rRNA promoter sequences against those from other filamentous fungi. (C) Editing efficiency of systems with different promoters driving sgRNA expression. (D) The disruption efficiency of fcc1, ura3 and ppt1 as the targets, using the 5S rRNA promoter. 210x276mm (300 x 300 DPI)



Fig. 6 Rewriting the metabolic pathways of F. fujikuroi to change its gibberellic acid (GA) product profile. (A) The GA product profile of the wild-type control strain, including GA1, GA3, GA4 and GA7. (B) Improving the accumulation of GA4 and GA7 by deleting the P450-3 gene and overexpressing the genes Cps/Ks and tHmgR, which encode rate-limiting enzymes. 278x106mm (96 x 96 DPI)


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Cas9-Based Genome Editing in the ... - ACS Publications

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