Cordyceps sinensis strain not producing monosporic rice fungus and its creation method

Abstract

This invention belongs to the field of edible fungi genetic breeding technology, and relates to a Cordyceps militaris strain that does not produce aspergillin, with the preservation number CGMCCNo.40266. This strain does not contain the CCM_02059 gene, CCM_02060 gene, or any of the CCM_02059 to CCM_02060 genes. It also relates to a method for creating this strain through CRISPR / Cas9 gene editing, comprising the following steps: S1, determining and synthesizing the sgRNA nucleotide sequence of the target gene; S2, determining and synthesizing the nucleotide sequences of the upstream and downstream homologous arms of the target gene; S3, ligating the sgRNA nucleotide sequence described in S1 and the homologous arm nucleotide sequences described in S2 into the pAMA1-Cas9-hyg vector to construct a knockout vector; and S4, transforming the knockout vector described in S3 into Cordyceps militaris protoplasts via PEG-mediated transformation to obtain a Cordyceps militaris strain with the target gene knocked out. This invention utilizes CRISPR / Cas9 editing technology to edit the gene cluster of inoculin synthesis in Cordyceps militaris, disrupting the inoculin synthesis pathway and breeding Cordyceps militaris strains that do not produce inoculin and have no foreign gene insertions, which is of great significance to the development of the Cordyceps militaris industry.

Description

Technical Field

[0001] This invention belongs to the field of edible fungi genetic breeding technology, specifically relating to a Cordyceps militaris strain that does not produce aspergillin, and a method for creating this strain through CRISPR / Cas9 gene editing. Background Technology

[0002] Cordyceps militaris (L.) Fr. is the type species of the genus Cordyceps. It is rich in various active ingredients such as cordycepin, cordyceps polysaccharides, cordycepic acid, and carotenoids, possessing diverse pharmacological activities including antibacterial, anti-fatigue, anti-tumor, and immunomodulatory effects. It is a valuable resource for food and pharmaceutical development. Currently, besides being sold directly as a fruiting body, Cordyceps militaris is also being developed into various foods, health products, and cosmetics, forming a large-scale industrial chain in China with an annual output value of tens of billions of yuan.

[0003] Secondary metabolites produced by fungi possess diverse structures and biological activities. Our research group previously confirmed the production of aspergillin B by Cordyceps militaris using high-performance liquid chromatography-high-resolution mass spectrometry. The content of aspergillin B in a mixture of silkworm pupae and mycelium using silkworm pupae as the cultivation substrate was 106.5 μg / g, while the content in fruiting bodies cultivated on wheat culture medium was 66.9 μg / g. Furthermore, aspergillin B was detected in artificially cultivated Cordyceps militaris fruiting bodies from different origins (Wang Anning, Study on the Synthetic Gene Clusters of Two Secondary Metabolites of Cordyceps militaris under Photoregulation, Master's Thesis, University of Chinese Academy of Sciences, 2022).

[0004] Inoaricin, a fungal toxin produced by *Aspergillus oryzae*, can contaminate water sources and rice. Currently, no toxicological studies have been reported on inoaricin B. However, mouse toxicology experiments with inoaricin A showed that continuous intraperitoneal injection of inoaricin A at a dose of 400 μg / kg body weight into mice resulted in liver and kidney lesions observed after 10-12 days. Based on the dosage used in current mouse toxicology experiments with inoaricin A, within the prescribed intake range for *Cordyceps militaris* (the former Ministry of Health stipulated that the consumption intake of *Cordyceps militaris* should be ≤2g / day), the inoaricin content should not pose a safety concern for the consumption of *Cordyceps militaris*. However, since the toxicology of inoaricin B is currently unknown and toxicological studies of inoaricin in healthy and sub-healthy individuals are scarce, the selection and breeding of *Cordyceps militaris* strains that do not produce inoaricin is of great significance. Summary of the Invention

[0005] In view of this, the purpose of this invention is to create a Cordyceps militaris strain that does not produce aspergillin through CRISPR / Cas9 (Clustered regularly interspaced short palindromic repeats / Cas9) gene editing, thereby providing a new strain for the safe production of Cordyceps militaris and providing genetic material for the targeted improvement of its new varieties.

[0006] On the one hand, the present invention provides a Cordyceps militaris strain that does not produce aspergillin. The taxonomic name of this strain is Cordyceps militaris, and it is currently deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China, with accession number CGMCC No. 40266 and deposit date of August 3, 2022.

[0007] On the other hand, the present invention provides a method for creating a Cordyceps militaris strain that does not produce aspergillin through CRISPR / Cas9 gene editing, the method comprising the following steps:

[0008] S1. Determine and synthesize the sgRNA nucleotide sequence of the target gene;

[0009] S2. Determine and synthesize the nucleotide sequences of the upstream and downstream homologous arms of the target gene;

[0010] S3. Ligate the sgRNA nucleotide sequence described in S1 and the homologous arm nucleotide sequence described in S2 to the pAMA1-Cas9-hyg vector to construct a knockout vector; and

[0011] S4. The knockout vector described in S3 is transformed into the pupae grassland bioplast via PEG-mediated transformation to obtain a transformant containing the knockout vector.

[0012] In an embodiment of the present invention, the target gene is the CCM_02059 gene, CCM_02060 gene, or CCM_02059 to CCM_02060 gene of Cordyceps militaris, and the nucleotide sequences of the CCM_02059 gene and the CCM_02060 gene are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively.

[0013] In an embodiment of the method of the present invention, the method for creating Cordyceps militaris strains that do not produce aspergillin via CRISPR / Cas9 gene editing may further include:

[0014] S5. At the genomic DNA level, using PCR, identify the transformants that knock out the target gene from the transformants obtained in S4, i.e., positive transformants.

[0015] S6. At the cDNA level, the positive transformants obtained in S5 were verified using semi-quantitative RT-PCR (Reverse transcription PCR) to confirm that the target genes of the positive transformants were not expressed at the transcriptional level; and

[0016] The positive transformants obtained in S7 and S6 were passaged to obtain Cordyceps militaris strains without exogenous gene insertion and without vector loss; and

[0017] S8. Detect the aspergillin content in the cultivated fruiting bodies of the Cordyceps militaris strain confirmed in S7.

[0018] In a third aspect, the present invention provides a CRISPR / Cas9 gene editing vector, which uses pAMA1-Cas9-hyg as a backbone vector and further contains the sgRNA nucleotide sequence of the target gene and the nucleotide sequences of the upstream and downstream homologous arms of the target gene, wherein the target gene is the CCM_02059 gene (SEQ ID NO:1), the CCM_02060 gene (SEQ ID NO:2), or the CCM_02059 to CCM_02060 genes.

[0019] Fourthly, the present invention provides a nucleotide sequence involved in regulating the synthesis of cordyceps militaris aspergillin, as shown in SEQ ID NO:1 or SEQ ID NO:2.

[0020] Compared with existing factory-produced Cordyceps militaris strains, this invention has significant beneficial effects. This invention creates a Cordyceps militaris strain that does not produce aspergillin by knocking out the CCM_02059, CCM_02060, or CCM_02059 to CCM_02060 genes in the Cordyceps militaris genome using CRISPR / Cas9 gene editing technology. This does not affect the growth of the fruiting body, thus avoiding the potential health hazards of consuming large quantities of Cordyceps militaris and preventing food safety issues caused by the integration of transgenic elements into the genome. This provides a framework and experimental basis for targeted breeding of Cordyceps militaris. Attached Figure Description

[0021] Figure 1 A structural diagram of the pAMA1-Cas9-hyg vector of the present invention is shown.

[0022] Figure 2 A diagram illustrating the gene knockout strategy of an embodiment of the present invention is shown.

[0023] Figure 3The agarose gel electrophoresis images of PCR verification of genomic DNA and cDNA of the transformants are shown. A. Genomic DNA PCR verification: 33 is a positive transformant, 34 is a negative control, and WT is wild type; B. Semi-quantitative RT-PCR verification: CCM_02059 semi-quantitative primer amplification, WTc is cDNA, and WTg is wild type genomic DNA.

[0024] Figure 4 The high-performance liquid chromatography and mass spectra of aspergillin are shown.

[0025] Figure 5 The images show the growth of fruiting bodies in silkworm pupae and wheat culture media. A: Wheat culture medium, B: Silkworm pupae culture medium. Detailed Implementation

[0026] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. However, those skilled in the art should understand that the embodiments described below are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] The inventors discovered that a gene cluster (CCM_02054 to CCM_02066) in the *Cordyceps militaris* genome is related to the synthesis of inoaspergillus B. Furthermore, they confirmed that the protein products encoded by the core genes CCM_02059 and CCM_02060 of this inoaspergillus B synthesis gene cluster both contain the same conserved oxidase domain DUF3328, which plays a crucial role in the formation of the initial cyclic peptide backbone of inoaspergillus. Therefore, knocking out the CCM_02059 and CCM_02060 genes in the *Cordyceps militaris* genome, thus disrupting the inoaspergillus synthesis gene cluster, can yield *Cordyceps militaris* strains that do not produce inoaspergillus.

[0028] CRISPR / Cas9 editing technology can improve bacterial strains through targeted genome editing. Compared with traditional breeding methods such as hybridization, mutagenesis, and domestication, this technology has advantages such as high targeting, short operation cycle, simplicity, high efficiency, and no introduction of exogenous DNA fragments. The vector element AMA1 (a self-replicating nucleotide sequence in filamentous fungi) ensures that the vector replicates independently of the chromosome. Therefore, the vector almost never inserts into the fungal genome. Under conditions without antibiotic selection pressure, vector loss in transformants is easily achieved, thus realizing truly traceless editing without exogenous gene insertion and avoiding the safety issues associated with transgenic technology.

[0029] Therefore, if the CCM_02059 and CCM_02060 genes in the Cordyceps militaris genome can be knocked out using CRISPR / Cas9 editing technology, thereby disrupting the rice aspergillin synthesis gene cluster and thus losing the ability to synthesize rice aspergillin, a Cordyceps militaris strain that does not produce rice aspergillin can be obtained, thus obtaining the present invention.

[0030] On the one hand, this invention provides a Cordyceps militaris strain that does not produce aspergillin. This strain's genome lacks the CCM_02059 gene, the CCM_02060 gene, or both of these genes (CCM_02059 to CCM_02060). The mycelial growth rate and light-induced color change (carotenoid accumulation) of this gene-deleted Cordyceps militaris strain are not significantly different from those of the wild-type strain. It can normally develop into fruiting bodies on wheat culture medium and silkworm pupae, and the gene deletion has no negative impact on the cultivation traits of Cordyceps militaris.

[0031] In an embodiment of the present invention, the nucleotide sequence of the CCM_02059 gene is shown in SEQ ID NO:1, and the nucleotide sequence of the CCM_02060 gene is shown in SEQ ID NO:2.

[0032] On the other hand, the present invention provides a method for creating Cordyceps militaris strains that do not produce aspergillin via CRISPR / Cas9 gene editing, the method comprising the following steps:

[0033] S1. Determine and synthesize the sgRNA nucleotide sequence of the target gene;

[0034] S2. Determine and synthesize the nucleotide sequences of the upstream and downstream homologous arms of the target gene;

[0035] S3. Ligate the sgRNA nucleotide sequence described in S1 and the homologous arm nucleotide sequence described in S2 to the pAMA1-Cas9-hyg vector to construct a knockout vector; and

[0036] S4. The knockout vector described in S3 is transformed into the pupae grassland bioplast via PEG-mediated transformation to obtain a transformant containing the knockout vector.

[0037] In embodiments of the present invention, the method for creating Cordyceps militaris strains that do not produce aspergillin via CRISPR / Cas9 gene editing may further include:

[0038] S5. At the genomic DNA level, PCR is used to identify the transformants with the target gene knocked out from the transformants obtained in S4, i.e., the positive transformants.

[0039] S6. At the cDNA level, semi-quantitative RT-PCR was used to verify the positive transformants obtained in S5, confirming that the target genes of the positive transformants were not expressed at the transcriptional level; and

[0040] The positive transformants obtained in S7 and S6 were passaged to obtain Cordyceps militaris strains without exogenous gene insertion and without vector loss; and

[0041] S8. Detect the aspergillin content in the cultivated fruiting bodies of the Cordyceps militaris strain confirmed in S7.

[0042] In an embodiment of the present invention, the target gene is CCM_02059, CCM_02060, or CCM_02059 to CCM_02060 genes in the Cordyceps militaris genome.

[0043] In an embodiment of the present invention, the sgRNA nucleotide sequence is 20 bp upstream of the protospacer-associated motif (PAM) adjacent to the target gene's protospacer-associated nucleotide sequence, i.e., 5'-N20-NGG-3', where NGG represents the PAM nucleotide sequence and N20 represents the 20 bp recognition nucleotide sequence. CRISPR / Cas9 technology is used to create a double-strand break (DSB) site in the target gene. Utilizing the fungal's own DSB repair mechanism, homologous arm nucleotide sequences are introduced at both ends to initiate homologous recombination repair. The upstream homologous arm nucleotide sequence is located upstream of the target gene's coding region, and the downstream homologous arm nucleotide sequence is located downstream of the target gene's coding region. The upstream and downstream homologous arm nucleotide sequences are tightly connected, resulting in complete deletion of the target gene without the insertion of an resistance gene.

[0044] In a specific embodiment of the present invention, for the CCM_02059 gene, that is, when only the CCM_02059 gene is knocked out, the sgRNA nucleotide sequence is Target 1, and its nucleotide sequence is shown in SEQ ID NO:3. The synthesized Target 1 expression cassette sequence is shown in SEQ ID NO:4.

[0045] In a specific embodiment of the present invention, for the CCM_02059 gene, i.e., when only the CCM_02059 gene is knocked out, its upstream homologous arm nucleotide sequence is shown in SEQ ID NO:5, and its downstream homologous arm nucleotide sequence is shown in SEQ ID NO:6. The primers for synthesizing the upstream homologous arm nucleotide sequence are up 1-F (SEQ ID NO:7) / up 1-R (SEQ ID NO:8), and the primers for synthesizing the downstream homologous arm nucleotide sequence are down 1-F (SEQ ID NO:9) / down 1-R (SEQ ID NO:10).

[0046] In a specific embodiment of the present invention, for the CCM_02060 gene, i.e., when only the CCM_02060 gene is knocked out, the sgRNA nucleotide sequence is Target 2, and its nucleotide sequence is shown in SEQ ID NO:11. The synthesized Target 2 expression cassette sequence is shown in SEQ ID NO:12.

[0047] In a specific embodiment of the present invention, for the CCM_02060 gene, i.e., when only the CCM_02060 gene is knocked out, its upstream homologous arm nucleotide sequence is shown in SEQ ID NO:13, and its downstream homologous arm nucleotide sequence is shown in SEQ ID NO:14. The primers for synthesizing the upstream and downstream homologous arm nucleotide sequences are up 2-F (SEQ ID NO:15) / up 2-R (SEQ ID NO:16) and down 1-F (SEQ ID NO:17) / down 1-R (SEQ ID NO:18), respectively.

[0048] Since the CCM_02059 gene and the CCM_02060 gene are adjacent, the CCM_02059 gene and the CCM_02060 gene and their intervening nuclear sequences (i.e., CCM_02059 to CCM_02060 genes) can be simultaneously knocked out by designing the sgRNA within the spacer nucleotide sequence of the two genes, designing the upstream homologous arm nucleotide sequence upstream of the coding region of the CCM_02059 gene, and designing the downstream homologous arm nucleotide sequence downstream of the coding region of the CCM_02060 gene.

[0049] Therefore, in another embodiment of the method of the present invention, the present invention provides a method for creating Cordyceps militaris strains that do not produce aspergillin through CRISPR / Cas9 gene editing, the method comprising the following steps:

[0050] S1. Determine and synthesize the sgRNA nucleotide sequence of the spacer sequence between the CCM_02059 gene and the CCM_02060 gene;

[0051] S2. Determine and synthesize the nucleotide sequences of the upstream and downstream homologous arms of the CCM_02059 to CCM_02060 genes;

[0052] S3. Ligate the sgRNA nucleotide sequence described in S1 and the homologous arm nucleotide sequence described in S2 to the pAMA1-Cas9-hyg vector to construct a knockout vector; and

[0053] S4. The knockout vector described in S3 is transformed into the pupae grassland bioplast via PEG-mediated transformation to obtain a transformant containing the knockout vector.

[0054] In a specific implementation scheme, the spacer sequence between the CCM_02059 gene and the CCM_02060 gene is shown in SEQ ID NO:19.

[0055] In the specific implementation scheme, for the CCM_02059 to CCM_02060 genes, i.e., when both the CCM_02059 and CCM_02060 genes are knocked out simultaneously, the sgRNA nucleotide sequence is Target 3, and its nucleotide sequence is shown in SEQ ID NO:20. The synthesized Target 3 expression cassette sequence is shown in SEQ ID NO:21.

[0056] In the specific implementation scheme, the upstream homologous arm nucleotide sequences of the CCM_02059 to CCM_02060 genes are shown in SEQ ID NO:22, and the downstream homologous arm nucleotide sequences are shown in SEQ ID NO:23. The primers for synthesizing the upstream homologous arm nucleotide sequences are up 3-F (SEQ ID NO:24) / up 3-R (SEQ ID NO:25), and the primers for synthesizing the downstream homologous arm nucleotide sequences are down 3-F (SEQ ID NO:26) / down 3-R (SEQ ID NO:27).

[0057] Therefore, in the implementation plan for obtaining Cordyceps militaris strains with CCM_02059 to CCM_02060 genes knocked out, the method for creating Cordyceps militaris strains that do not produce aspergillin through CRISPR / Cas9 gene editing specifically includes:

[0058] S1. Design the nucleotide sequence of Target 3 (SEQ ID NO:20) and synthesize the Target 3 expression cassette sequence.

[0059] The synthesized Target 3 expression cassette nucleotide sequence is SEQ ID NO:21.

[0060] S2. Design and synthesize primers for the upstream and downstream homologous arm nucleotide sequences of the CCM_02059 to CCM_02060 genes, and amplify the upstream and downstream homologous arm nucleotide sequences by PCR.

[0061] Primers up 3-F / up 3-R and down 3-F and down 3-R were designed based on the upstream and downstream homologous arm nucleotide sequences. Using wild-type Cordyceps militaris strain genomic DNA as a template, the upstream homologous arm nucleotide sequence (SEQ ID NO:22) and the downstream homologous arm nucleotide sequence (SEQ ID NO:23) were amplified, respectively. The PCR amplification products were recovered. The primer nucleotide sequences are as follows:

[0062] up 3-F:

[0063] 5'-ACCCTGATAAATGCTTCAATAATATTgtcgacaagagaggtggtcc-3' (SEQ ID NO: 24);

[0064] up 3-R: 5'-AGATTCGCAGcctggacaatctactccgattca-3' (SEQ ID NO: 25);

[0065] down 3-F: 5'-agattgtccaggCTGCGAATCTGAGTGGTTGG-3' (SEQ ID NO: 26);

[0066] down 3-R:

[0067] 5'-ACTCATACTCTTCCTTTTTCAATATTCAGCGCCCTGTATCCTTTGA-3' (SEQ ID NO: 27);

[0068] S3. Link the nucleotide sequences of Target 3 described in S1 and the homologous arms described in S2 to the pAMA1-Cas9-hyg vector to construct a knockout vector.

[0069] In step S3, the synthesized Target 3 expression cassette sequence described in S1 can be ligated to the vector pAMA1-Cas9-hyg, and the successfully ligated vector can be named pAMA1-Cas9-hyg-Target 3. Successful ligation can be verified, for example, by transforming the ligated vector into E. coli DH5α competent cells, picking E. coli colonies, and using PCR to verify whether the Target 3 described in S1 has been successfully ligated to the pAMA1-Cas9-hyg vector. The nucleotide sequences of the primers used for PCR are shown in SEQ ID NO:28 and SEQ ID NO:29. The colonies correctly identified by PCR are then amplified, the vector is extracted and sequenced, and the sequencing results are compared with the Target 3 expression cassette sequence described in S1.

[0070] Next, the upstream and downstream homologous arm nucleotide sequences described in S2 are ligated to the vector pAMA1-Cas9-hyg-Target 3. The successfully ligated vector is named pAMA1-Cas9-hyg-Target 3-HR, i.e., the knockout vector. The success of the ligation can be verified by, for example, the following method: the ligated vector is transformed into, for example, E. coli DH5α competent cells, and E. coli colonies are picked and PCR is used to verify whether the upstream and downstream homologous arm nucleotide sequences described in S2 are ligated to the vector pAMA1-Cas9-hyg-Target 3. The nucleotide sequences of the primers used for PCR are shown in SEQ ID NO:30 and SEQ ID NO:31. The colonies that have been verified by PCR are amplified, the vector is extracted and sequenced, and the sequencing results are compared with the upstream and downstream homologous arm nucleotide sequences described in S2.

[0071] S4. The knockout vector pAMA1-Cas9-hyg-Target 3-HR described in S3 is transformed into *Pteris vittata* bioplasts via PEG-mediated transformation to obtain transformants containing the knockout vector pAMA1-Cas9-hyg-Target 3-HR.

[0072] Transformers containing the knockout vector pAMA1-Cas9-hyg-Target 3-HR can be obtained, for example, by screening protoplast resuscitation medium plates containing 500 μg / mL hygromycin.

[0073] In a further embodiment of the double gene knockout (i.e., knockout of CCM_02059 to CCM_02060 genes), the method may further include step S5, i.e., at the genomic DNA level, using PCR to identify transformants with knockout target genes from the transformants obtained in S4, i.e., positive transformants, wherein the genomic DNA of the transformants obtained in step S4 is extracted, and using this as a template, PCR is performed on the transformants using primers SEQ ID NO:32 and SEQ ID NO:33 to identify transformants with knockout of CCM_02059 to CCM_02060 genes, i.e., positive transformants.

[0074] In a further embodiment of the knockout of two genes (i.e., knockout of CCM_02059 and CCM_02060 genes), the method may further include step S6, which involves verifying the positive transformants obtained in step S5 at the cDNA level using semi-quantitative RT-PCR to confirm that the positive transformants are not expressed at the transcriptional level. Specifically, RNA is extracted from the positive transformants obtained in step S5 and reversed to cDNA. Using cDNA as a template, semi-quantitative RT-PCR is performed using semi-quantitative primers SEQ ID NO:34 and SEQ ID NO:35 to verify the positive transformants obtained in step S5, confirming that the positive transformants are not expressed at the transcriptional level. This verifies the knockout of CCM_02059 to CCM_02060 genes at the transcriptional level.

[0075] In a further embodiment of the double gene knockout, the method further includes S7, whereby the positive transformant confirmed not to synthesize isoaspergillus is separated and passaged by conidia to achieve the loss of free vector, and cultured on silkworm pupa or wheat culture medium to finally obtain a Cordyceps militaris strain that does not produce isoaspergillus and can develop into a fruiting body normally.

[0076] In a further embodiment of the double gene knockout, the method may further include step S8, namely, detecting the content of rice arsenic obtained in S7, for example, extracting and purifying rice arsenic from the cultivated fruiting bodies of the Cordyceps militaris strain obtained in step S7, and detecting its rice arsenic content using high performance liquid chromatography.

[0077] In a third aspect, the present invention provides a CRISPR / Cas9 gene editing vector, which uses pAMA1-Cas9-hyg as a backbone vector and further contains the sgRNA nucleotide sequence of the target gene and the nucleotide sequences of the upstream and downstream homologous arms of the target gene, wherein the target gene is the CCM_02059 gene (SEQ ID NO:1), the CCM_02060 gene (SEQ ID NO:2), or the CCM_02059 to CCM_02060 genes.

[0078] Example

[0079] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0080] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0081] The wild-type Cordyceps militaris strain used in the following examples is CGMCC 3.16323, and the vector pAMA1-Cas9-hyg was kindly provided by the research group of Professor Liu Gang at the Institute of Microbiology, Chinese Academy of Sciences. The specific implementation method is illustrated using double gene knockout as an example.

[0082] 1.1 sgRNA Design

[0083] The interstitial sequence between the CCM_02059 and CCM_02060 genes in the *Cordyceps militaris* genome was selected as the target nucleotide sequence. The *Cordyceps militaris* CM01 (CGMCC 3.14242) genome (Zheng et al., Genome sequence of the insect pathogenic fungus *Cordyceps militaris*, a valued traditional Chinese medicine. *Genome Biology*, 2012, R116) was used as the reference genome. The sgRNA was designed online using the Eukaryotic Pathogen CRISPRguide RNA / DNA Design Tool website (http: / / grna.ctegd.uga.edu / ), with all parameters set to default.

[0084] 1.2 Construction of pAMA1-Cas9-hyg-Target 3 intermediate vector

[0085] a.Target 3 Expression Box Composition

[0086] The synthesized Target 3 expression cassette nucleotide sequence is SEQ ID NO:21.

[0087] b. Enzyme digestion and fragment ligation of pAMA1-Cas9-hyg vector

[0088] Using pAMA1-Cas9-hyg as the starting vector, the synthesized Target 3 expression cassette and pAMA1-Cas9-hyg (linearized with restriction endonuclease BstEII) were purified by gel extraction using a gel extraction kit (D205-01) from Beijing Kangrun Chengye Biotechnology Co., Ltd. The nucleotide concentrations of the DNA fragments and linearized vector were determined using a Thermo Scientific NanoDrop Lite spectrophotometer (ND-NDL-US-CAN). Ligation reactions were performed using a Vazyme one-step cloning kit (C115-02) according to the manufacturer's instructions. The BstEII restriction enzyme digestion reaction system is as follows:

[0089] Carrier: 2μg

[0090] Restriction endonuclease: 2 μL

[0091] 10×buffer B: 10μL

[0092] Nuclease-free water: to 100μL

[0093] React for 5-12 hours at the optimal operating temperature.

[0094] The structure of the pAMA1-Cas9-hyg vector is as follows: Figure 1 As shown.

[0095] c. Colony PCR Validation and Sequencing

[0096] The ligation product was transformed into E. coli DH5α competent cells using a heat shock freeze-thaw method. E. coli colonies were picked from LB / Amp+ (tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L) plates and identified by PCR using the primer nucleotide sequences of SEQ ID NO:28 and SEQ ID NO:29. Colonies correctly identified by PCR were inoculated into LB / Amp+ liquid medium and cultured at 37℃ and 200 rpm for 12 h for propagation. Using the plasmid extraction kit (D201-04) from Beijing Kangrun Chengye Biotechnology Co., Ltd., the vector was extracted from the propagated bacterial culture according to the manufacturer's instructions. The vector was then sent to Sangon Biotech (Shanghai) Co., Ltd., Beijing Branch for sequencing. Sequence alignment showed that the sequencing results were consistent with the Target 3 expression cassette sequence, confirming the successful construction of the vector pAMA1-Cas9-hyg-Target 3.

[0097] The PCR identification reaction system for E. coli DH5α colonies is as follows:

[0098] 2×RapidTaqMasterMix: 5μL;

[0099] Primer F (10 μmol / L): 0.2 μL;

[0100] Primer R (10 μmol / L): 0.2 μL;

[0101] Bacterial suspension (capillaries from LB / Amp+ plates dipped into 10 μL ddH2O): 4.6 μL.

[0102] The PCR amplification reaction procedure is as follows:

[0103] Step 1: 95℃ for 3 minutes;

[0104] Step 2: 95℃ for 30 seconds;

[0105] Step 3: 58℃ for 30 seconds;

[0106] Step 4: 72℃ for 5 seconds;

[0107] Step 5: Repeat steps 2-4 for 35 cycles;

[0108] Step 6: 72℃ for 10 minutes.

[0109] 1.3 Construction of pAMA1-Cas9-hyg-Target 3-HR vector

[0110] Using primers up 3-F / up 3-R and down 3-F / down 3-R, and with wild-type Cordyceps militaris strain genomic DNA as a template, the upstream and downstream homologous arm nucleotide sequences of the CCM_02059 to CCM_02060 genes were amplified. The nucleotide sequences of the primers are as follows:

[0111] up 3-F:

[0112] 5'-ACCCTGATAAATGCTTCAATAATATTgtcgacaagagaggtggtcc-3' (SEQ ID NO: 24);

[0113] up 3-R: 5'-AGATTCGCAGCCTGGACAATCTACTCCGATTCA-3' (SEQ ID NO: 25);

[0114] down 3-F: 5'-AGATTGTCCAGGCTGCGAATCTGAGTGGTTGG-3' (SEQ ID NO: 26);

[0115] down 3-R:

[0116] 5'-ACTCATACTCTTCCTTTTTCAATATTCAGCGCCCTGTATCCTTTGA-3' (SEQ ID NO: 27).

[0117] The amplified upstream and downstream homologous arm nucleotide sequences and pAMA1-Cas9-hyg-Target 3 (restriction endonuclease SspⅠ linearization) were purified by gel extraction using a gel extraction kit (D205-01) from Beijing Kangrun Chengye Biotechnology Co., Ltd. The SspⅠ linearization reaction system followed the method described in section 1.2. The nucleotide concentrations of the DNA fragments and linearized vectors were determined using a Thermo Scientific NanoDrop Lite spectrophotometer (ND-NDL-US-CAN). Ligation was performed using a Vazyme one-step cloning kit (C115-02) according to the manufacturer's instructions. The ligation products were transformed into E. coli DH5α competent cells using a heat shock freeze-thaw method. E. coli colonies were picked from LB / Amp+ plates and identified by PCR using the primer nucleotide sequences of SEQ ID NO:30 and SEQ ID NO:31. Colonies correctly identified by PCR were inoculated into LB / Amp+ liquid medium for propagation. Using the plasmid extraction kit (D201-04) from Beijing Kangrun Chengye Biotechnology Co., Ltd., and following the manufacturer's instructions, the amplified bacterial culture was used to extract the vector. The vector was then sent to Sangon Biotech (Shanghai) Co., Ltd., Beijing Branch for sequencing. Sequence alignment confirmed that the sequencing results were consistent with the homologous arm nucleotide sequences described above, thus proving the successful construction of the vector pAMA1-Cas9-hyg-Target3-HR.

[0118] 1.4. pAMA1-Cas9-hyg-Target 3-HR transformation of wild-type pupae grassland bioplasts

[0119] Wild-type Cordyceps militaris mycelia were collected, and 1 mL of 2% lysozyme (developed by Guangdong Institute of Microbiology, lysozyme powder dissolved in 0.8 mol / L KCl, pH = 6.5) was added. The mixture was incubated at 32℃ and 90 rpm for 3 h. The mycelia were filtered through four layers of gauze. 1 mL of the lower filtrate was transferred to a 1.5 mL centrifuge tube and centrifuged at 3000 rpm for 10 min at room temperature. The supernatant was discarded, and then 100-200 μL of STC buffer (sorbitol: 18.2 g; Tris: 0.121 g; CaCl2: 0.368 g; ddH2O) was added to bring the volume to 100 mL. The mixture was gently resuspended using a pipette until the protoplast concentration was 1 × 10⁻⁶. 7 per mL.

[0120] Add 2 μg of pAMA1-Cas9-hyg-Target 3-HR vector to 100 μL of *Pteris vittata* bioplasts and incubate on ice for 5 min. Next, add 50 μL of PEG buffer (PEG 4000: 25 g; Tris: 0.121 g; CaCl2: 0.368 g; ddH2O to a final volume of 100 mL) to the protoplast-vector mixture and incubate on ice for 30 min. Then, add 0.5 mL of PEG buffer to the mixture, gently mix by pipetting, and incubate in a 28°C metal bath for 20 min. Finally, add 1 mL of PEG buffer… Mix the STC buffer solution gently by pipetting, centrifuge at 3000 rpm for 10 min and discard the supernatant. Resuspend the protoplast pellet at the bottom of the centrifuge tube in STC buffer and spread it onto a plate containing 500 μg / mL hygromycin in protoplast resuscitation medium (filtered juice from 200g peeled potatoes, 20g glucose, 10g tryptone, 0.6M mannitol, and ddH2O to a final volume of 1L). Incubate in the dark at 25°C for 5-7 days until the transformant mycelia germinate and form colonies. Extract genomic DNA from the transformants for PCR identification.

[0121] 1.5 PCR Validation of Transformants

[0122] Genomic DNA was extracted from the above transformants using the CTAB method (Yan Qingxiang et al., Extraction of cassava genomic DNA using a modified CTAB method, Chinese Agricultural Science Bulletin, 2010, (4): 30-32). Primers SEQ ID NO:32 and SEQ ID NO:33 were used for initial PCR screening of the transformants. Figure 3 As shown in Figure A, the amplification product of transformant 33 was 1197 bp, while the amplification product of the wild-type Cordyceps militaris strain was 3544 bp, indicating that gene knockout was successfully achieved in transformant 33, making it a positive transformant (M33). To verify gene knockout at the transcriptional level, RNA was extracted from transformant 33 and the wild-type Cordyceps militaris strain using the OMEGA RNAPlant Kit (R6827-02). The RNA was then analyzed using Vazyme HiScript. II. Using the QRT SuperMix for qPCR (+gDNA wiper) kit (R223-01), following the instructions, RNA was reverse transcribed into cDNA. Using cDNA as a template, semi-quantitative primers SEQ ID NO:34, SEQ ID NO:35, and rpb1-F / R (Lian et al., Variations of SSU rDNA group I introns in different isolates of Cordyceps militaris and the loss of an intron during cross-mating. Journal of Microbiology. 201452(8):659-666) were used to further verify at the gene transcription level whether the CCM_02059 to CCM_02060 genes had been knocked out. Figure 3 As shown in Figure B, the CCM_02059 gene was not expressed at the transcriptional level in transformant M33, indicating that transformant M33 was a positive transformation (CGMCC No. 40266), meaning that the deletion of the CCM_02059 to CCM_02060 genes was successfully achieved.

[0123] Semi-quantitative RT-PCR reaction system:

[0124] 2×RapidTaqMasterMix: 5μL;

[0125] Primer F (10 μmol / L): 0.2 μL;

[0126] Primer R (10 μmol / L): 0.2 μL;

[0127] ddH2O: 3.6 μL;

[0128] Template: 1μL.

[0129] The PCR amplification reaction procedure is the same as above.

[0130] 1.6 Extraction, purification and detection of inoculin

[0131] The extraction and purification steps for astiloxins were based on existing literature (Cao et al., Analysis of astiloxins in rice using polymer cation exchange cleanup followed by liquid chromatography-tandem mass spectrometry. Journal of Chromatography A, 2016, 1476:46-52), with appropriate adjustments. The specific steps are as follows:

[0132] Wild-type Cordyceps militaris mycelium and the above-mentioned transformant M33 (CGMCC No. 40266) mycelium were freeze-dried in a freeze dryer. Three biological replicates were taken for each sample. The freeze-dried samples were thoroughly ground in a mortar and pestle for later use. 100 mg of freeze-dried sample powder was weighed into a 2 mL centrifuge tube, 1 mL of ddH2O and ceramic beads were added, and the mixture was thoroughly ground using a tissue homogenizer. The homogenate obtained by homogenization was transferred into a 4.5 mL centrifuge tube, and the volume was adjusted to 3 mL with ddH2O. The mixture was then treated in an ultrasonic extractor for 1 h, followed by centrifugation at 12,000 rpm for 6 min at room temperature. The resulting supernatant was transferred into a 10 mL centrifuge tube. Next, 2 mL of dichloromethane was added to the supernatant, and after thorough mixing, the mixture was centrifuged at 12,000 rpm for 6 min. The supernatant was collected to prepare a purification solution with a formic acid content of 5%. This solution was filtered through a 0.22 μm aqueous filter and pretreated with 3 mL of methanol (chromatographic grade) to equilibrate the PCX solid-phase extraction column with 3 mL of 5% formic acid. When the liquid level reached the surface of the column adsorption layer, the acidified sample was immediately added, and the column was eluted successively with 3 mL of 5% formic acid and 3 mL of methanol. The target substance was eluted with 3 mL of methanol containing 5% ammonia (NH3·H2O 25%-28%). The eluent was concentrated to near dryness under reduced pressure at 45 °C, and then 1 mL of 15% methanol was added. The solution was reconstituted by sonication for 15 min and filtered through a 0.22 μm aqueous filter.

[0133] The detection of inocrican was performed using a Shimadzu LC-20A HPLC system. The chromatographic column was a Luna Omega Polar C18 column (4.6 mm × 250 mm, 5 μm), the mobile phase was 0.01% trifluoroacetic acid aqueous solution (A)-methanol (B), and the detection wavelength was 190-400 nm.

[0134] HPLC detection results are as follows Figure 4 As shown. By Figure 4It can be seen that the retention time of inoakisin in the wild-type sample is 22-23 min. In contrast, the corresponding compound of the transformant M33 (CGMCC No.40266) was not detected at the corresponding retention time, indicating that the Cordyceps militaris strain that does not produce inoakisin has been successfully obtained.

[0135] 1.7, pAMA1-Cas9-hyg-Target 3-HR vector loss

[0136] Using a sterile toothpick, collect the conidia of transformant M33 (CGMCC No. 40266) and inoculate them onto the above-mentioned resuscitation medium plate without hygromycin for subculturing until the hygromycin-resistant vector pAMA1-Cas9-hyg-Target3-HR is lost, thus obtaining a traceless knockout strain without the resistance selection tag.

[0137] 1.8. Experiment on fruiting body formation on silkworm pupae and wheat culture media

[0138] Wild-type strain and transformant M33 (CGMCC No. 40266) were inoculated onto PDA plates and cultured at 20℃ for 16 days. The mycelium on the plates was washed with sterile water and filtered through four layers of gauze. 1 mL of the filtrate was transferred to a 1.5 mL centrifuge tube and centrifuged at 8,000 rpm for 6 min. Conidia of the wild-type strain and transformant M33 (CGMCC No. 40266) were collected and prepared to a concentration of 1×10⁻⁶. 6 A spore / mL suspension was injected into each silkworm pupa using a sterile syringe. The silkworm pupae were then placed in a 20℃ intelligent mushroom cultivation box (Beijing Zhitai Kangxing Biotechnology Co., Ltd.) for cultivation, and the development of the fruiting bodies was observed.

[0139] Wild-type strains and transformant M33 (CGMCC No. 40266) were inoculated onto PDA plates in seed culture medium (200g peeled potato slices boiled in juice, 20g glucose, 3g peptone, 1g KH2PO4, and 0.5g MgSO4·7H2O, with tap water to a final volume of 1L) and cultured at 150rpm for 3 days to obtain liquid spawn. 5mL of this liquid spawn was inoculated onto wheat culture medium (wheat: 20g; nutrient solution: glucose 20g, peptone 10g, KH2PO4 2g, MgSO4·7H2O 1g, ammonium citrate 1g, vitamin B1 20mg, tap water to a final volume of 1L) and placed in a 20℃ intelligent mushroom cultivation box (Beijing Zhitai Kangxing Biotechnology Co., Ltd.) to observe the development of fruiting bodies in the wheat culture medium.

[0140] The results showed that transformant M33 (CGMCC No. 40266) could develop into fruiting bodies normally on both silkworm pupa and wheat culture media. The fruiting bodies were uniform, upright, and brightly colored, and their growth rate was faster than that of the wild-type strain on wheat culture media. Figure 5 As shown.

Claims

1. Cordyceps militaris that does not produce aspergillin B ( Cordyceps militaris ) strain, characterized in The Cordyceps militaris strain has the preservation number CGMCC No. 40266.

2. A method for creating Cordyceps militaris strains that do not produce aspergillin via CRISPR / Cas9 gene editing, comprising the following steps: S1. Determine and synthesize the sgRNA nucleotide sequence of the target gene; S2. Determine and synthesize the nucleotide sequences of the upstream and downstream homologous arms of the target gene; S3. Link the sgRNA nucleotide sequence described in S1 and the homologous arm nucleotide sequence described in S2 to pAMA1-Cas9- hyg On the vector, construct a knockout vector; and S4. The knockout vector described in S3 is transformed into pupae grassland bioplasts via PEG-mediated transformation to obtain transformants containing the knockout vector. in, The target gene is CCM_02059 gene (SEQ ID NO: 1), CCM_02060 gene (SEQ ID NO: 2), or CCM_02059 to CCM_02060 genes.

3. The method according to claim 2, characterized in that, The sgRNA nucleotide sequence is Target 1, 2 or 3, and its nucleotide sequence is shown as SEQ ID NO: 3, SEQ ID NO: 11 or SEQ ID NO: 20, respectively.

4. The method according to claim 3, characterized in that, The nucleotide sequences of the synthesized Target 1, 2 or 3 expression frames are shown in SEQ ID NO: 4, SEQ ID NO: 12 or SEQ ID NO:

21.

5. The method according to claim 2, characterized in that, For the CCM_02059 gene, the nucleotide sequence of the upstream homologous arm is SEQ ID NO: 5, and the nucleotide sequence of the downstream homologous arm is SEQ ID NO: 6; for the CCM_02060 gene, the nucleotide sequence of the upstream homologous arm is SEQ ID NO: 13, and the nucleotide sequence of the downstream homologous arm is SEQ ID NO: 14; for the CCM_02059 to CCM_02060 genes, the nucleotide sequence of the upstream homologous arm is SEQ ID NO: 22; and the nucleotide sequence of the downstream homologous arm is SEQ ID NO:

23.

6. The method according to claim 5, characterized in that, For the CCM_02059 gene, the primer for synthesizing the upstream homologous arm nucleotide sequence is up 1-F / up 1-R, and the primer for synthesizing the downstream homologous arm nucleotide sequence is down 1-F / down 1-R; for the CCM_02060 gene, the primer for synthesizing the upstream homologous arm nucleotide sequence is up 2-F / up 2-R, and the primer for synthesizing the downstream homologous arm nucleotide sequence is down 2-F / down 2-R; for the CCM_02059 to CCM_02060 genes, the primer for synthesizing the upstream homologous arm nucleotide sequence is up 3-F / up 3-R, and the primer for synthesizing the downstream homologous arm nucleotide sequence is down 3-F / down 3-R. The nucleotide sequences of the primers are as follows: up 1-F: 5'-ACCCTGATAAATGCTTCAATAATATTcaagagcagattcgtcaag-3' (SEQ ID NO: 7); up 1-R: 5'-GCCAACCTCGTCAACGGCCagctagttggcaataccgcta-3' (SEQ ID NO: 8); down 1-F: 5'-caactagctGGCCGTTGACGAGGTTGGCAAAGAAGCAG-3' (SEQ ID NO: 9); down 1-R: 5'-ACTCATACTCTTCCTTTTTCAATATTAAGACGGAGCACACCACGG-3' (SEQ ID NO: 10); up 2-F: 5'-ACCCTGATAAATGCTTCAATAATATTgtcgacaagagaggtgg-3' (SEQ ID NO: 15); up 2-R: 5'-CCCTGGGTTGATCAGCGTCcctggacaatctactccgattc-3' (SEQ ID NO: 16) down 2-F: 5'-aggGACGCTGATCAACCAGGGCGCGGTCAATCGGAAG-3' (SEQ ID NO: 17); down 2-R: 5'-ACTCATACTCTTCCTTTTTCAATATTATCACCATGTGGACCACTGT-3' (SEQ ID NO: 18); up 3-F: 5'-ACCCTGATAAATGCTTCAATAATATTgtcgacaagagaggtggtcc-3' (SEQ ID NO: 24); up 3-R: 5'-AGATTCGCAGCCTGGACAATCTACTCCGATTCA-3' (SEQ ID NO: 25); down 3-F: 5'-AGATTGTCCAGGCTGCGAATCTGAGTGGTTGG-3' (SEQ ID NO: 26); down 3-R: 5'-ACTCATACTCTTCCTTTTTCAATATTCAGCGCCCTGTATCCTTTGA-3' (SEQ ID NO: 27).

7. The method according to claim 2, characterized in that, The method further includes: S5. At the genomic DNA level, using PCR, identify the transformants that knock out the target gene from the transformants obtained in S4, i.e., positive transformants. S6. At the cDNA level, the positive transformants obtained in S5 were verified by semi-quantitative RT-PCR to confirm that the target genes of the positive transformants were not expressed at the transcriptional level. The positive transformants obtained in S7 and S6 were passaged to obtain Cordyceps militaris strains without exogenous gene insertion and without vector loss; and S8. Detect the aspergillin content of the cultivated fruiting bodies of the Cordyceps militaris strain obtained in S7.

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