CN114410635A - Endogenous U6 promoter of Fusarium venezia and its CRISPR/Cas9-based gene editing method - Google Patents

Abstract

The invention discloses a U6 promoter derived from Fusarium venetianus and a gene editing method suitable for Fusarium venetianus. The present invention successfully establishes a CRISPR/Cas9 gene editing system based on the endogenous U6 promoter in Fusarium venetianus, and realizes the site-directed editing of the target gene, thus realizing convenience and speed in Fusarium venetianus with a low homologous recombination rate , Efficient gene editing, with high application value.

Description

Endogenous U6 promoter of Fusarium venezia and its CRISPR/Cas9-based gene editing method

technical field

The invention relates to the field of genetic engineering of Fusarium venetianus, in particular to an endogenous U6 promoter of Fusarium venetianus and a gene editing method based on CRISPR/Cas9.

Background technique

Fusarium venezia is an industrial strain screened from more than 3,000 fungi that can be used for fermentative production of mycelin. Compared with the single-cell protein, the mycelial protein produced by the fermentation of this strain is more delicious, has a meat-like tissue structure, and can provide a good nutritional balance, including low fat content, complete amino acid types, rich in trace elements, vitamins and benefits. Edible crude fiber for human gastrointestinal motility. In addition, Fusarium venezia mycelial protein also has very good safety, and has obtained the marketing license of food raw materials in 18 countries around the world. Therefore, it has the potential to partially or completely replace animal-based and plant-based protein foods.

In the past few years, the CRISPR/Cas9 gene editing system has been widely used in filamentous fungi, greatly improving the ability of researchers to modify filamentous fungi. In the establishment of the CRISPR/Cas9 gene editing system, the efficient type III promoter that regulates the expression of sgRNA plays a key role, among which the U6 promoter is the most commonly studied. There is currently no report on the endogenous efficient U6 promoter in F. venezia. Therefore, mining the efficient endogenous U6 promoter of Fusarium venezia TB01 and establishing a CRISPR/Cas9 gene editing system is expected to greatly improve the efficiency of homologous recombination, reduce the dependence on selection markers, and achieve simultaneous editing of multiple genes. Basic research and development and utilization of this strain.

SUMMARY OF THE INVENTION

In response to the needs of the prior art, the present invention takes the Fusarium venetianus TB01 GFP fluorescent strain as the editing object, and uses the Cas9 protein expression vector that can replicate autonomously in filamentous fungi and contains codon-optimized Aspergillus niger as the Cas9 protein donor, through Comparing the editing efficiency of GFP gene when different endogenous U6 promoters regulate sgRNA _GFP , the U6 promoters U6 ₃₇₄ and U6 ₉₄ were screened, and based on this, a CRISPR capable of realizing accurate and efficient genome editing of Fusarium venezia strain TB01 was established /Cas9 gene editing system.

Therefore, the object of the present invention is to provide a high-efficiency endogenous U6 promoter of F. venetianus and a CRISPR/Cas9-based gene editing method suitable for F. venetianus.

The invention provides an endogenous U6 promoter derived from Fusarium venetianus, which is characterized in that the expression of sgRNA can be induced in Fusarium venetianus, so that Cas9 cuts the target sequence at the correct position, and the endogenous U6 promoter The nucleotide sequence of the sub is as SEQ ID NO:1 or SEQ ID NO:2. More specifically, the promoter is cloned from the filamentous fungus Fusarium venenatum TB01, the strain's deposit number is CGMCC NO. Ordinary Microbiology Center of China Microbial Culture Collection Management Committee (the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing).

The invention provides an endogenous U6 promoter derived from Fusarium venetianus, which is characterized in that the expression of sgRNA can be induced in Fusarium venetianus, so that Cas9 cuts the target sequence at the correct position, and the endogenous U6 promoter The nucleotide sequence of the sub is shown in SEQ ID NO:1 or SEQ ID NO:2.

Further, the present invention provides an expression cassette containing the endogenous U6 promoter of Fusarium venetianus. Preferably, the expression cassette comprises the endogenous U6 promoter, and the sequences in the sgRNA operably linked thereto that are involved in recognizing the target of the gene of interest and the gRNA scaffold. More preferably, the nucleotide sequence of the gRNA scaffold is shown in SEQ ID NO:3.

The present invention also provides a recombinant vector containing the endogenous U6 promoter or the expression cassette derived from Fusarium venetianus.

The present invention further provides a method for gene editing of Fusarium venetianus, which comprises the following steps: co-mingling the sgRNA fragment containing the endogenous U6 promoter-derived editing target gene mediated by the Fusarium venetianus with a Cas9 expression vector Transformed Fusarium venetianus protoplasts, and further screened the successfully edited transformants.

In a preferred embodiment, the regulatory elements in the Cas9 expression vector include the PgpdA promoter shown in SEQ ID NO: 5; the Cas9 protein; SV40 NLS is the nuclear localization signal of the virus SV40; the promoter Ptrpc of Aspergillus nidulans trpc and terminator Ttrpc; hygromycin resistance gene Hyg; filamentous fungus autonomous replicon AMA1; wherein, the sgRNA fragment of the endogenous U6 promoter-mediated editing target gene includes the endogenous U6 promoter, sgRNA The sequence involved in identifying the target of the target gene and the gRNA scaffold, further preferably, the nucleotide sequence of the gRNA scaffold is shown in SEQ ID NO: 3.

In a specific embodiment, the Fusarium venetianus protoplast is prepared by the following method: collecting Fusarium venetianus spores, washing with sterile 0.7 M sodium chloride, enzymatic hydrolysis for 2 hours, and the enzymatic lysate used in the enzymatic hydrolysis To be: 20 mg of crash enzyme + 40 mg of helicase dissolved in 10 ml of 0.7 M sodium chloride and filter sterilized.

Further preferably, the Fusarium venetianus is Fusarium venetianus TB01.

In addition, preferably, the above-mentioned method further comprises verifying the successful transformants of the target gene editing by PCR or sequencing.

The beneficial effect of the invention lies in that the CRISPR/Cas9 gene editing system based on the endogenous U6 promoter is successfully established in Fusarium venetianus, and the targeted editing of the target gene is realized, thus realizing the low homologous recombination rate in Fusarium venetianus. Convenient, fast and efficient gene editing with high application value.

Other advantages, objects, and features of the present invention will appear in part from the description that follows, and in part will be appreciated by those skilled in the art from the study and practice of the invention.

Description of drawings

Fig. 1 Construction of fusion fragment of Fusarium venetianus endogenous U6 promoter to regulate sgRNA _GFP expression. A is the electrophoresis result of each endogenous U6 promoter fragment and sgRNA _GFP fragment amplified. B is the electrophoresis result of each endogenous U6 promoter fragment fused with sgRNA _GFP fragment. C is the sequencing comparison result of each endogenous U6 promoter fragment fused with the sgRNA _GFP fragment.

Figure 2 Construction of Cas9 expression vector. A is the schematic diagram of pCas9 expression vector. PgpdA is the endogenous promoter of F. venezia TB01; Cas9 is the codon-optimized Cas9 protein of Aspergillus niger; SV40 NLS is the nuclear localization signal of virus SV40; Ptrpc and Ttrpc are the promoter and terminator of trpc of Aspergillus nidulans; Hyg is Hygromycin resistance gene; AMA1 is an autonomous replicon of filamentous fungi. B is the PCR amplification electrophoresis result of pCas9 expression vector transformants. 1-10 are pCas9 transformants, and H ₂ 0 is a negative control. C is the structural diagram of the amplified fragment of the promoter and Cas9 protein indicated by the sequencing results of the transformant of the pCas9 expression vector.

Figure 3 Schematic diagram of the CRISPR/Cas9 gene editing system in Fusarium venezia.

Figure 4. Representative photographs of fluorescence of GFP gene-edited transformants of CRISPR/Cas9 edited GFP fluorescent strains.

Figure 5. Electrophoresis results after PCR amplification of GFP -edited transformants of fluorescent strains. M is DNA marker; WT is wild-type Fusarium venetianus (negative control); 1-4 are edited non-fluorescent transformants; 5 is unedited GFP fluorescent strain.

Detailed ways

The present invention will be further described below through specific examples, so as to better understand the present invention, but it does not constitute a limitation of the present invention.

Example 1: Construction of fusion fragments for the regulation of sgRNA _GFP expression by different endogenous U6 promoters of Fusarium venetianus TB01

Through sequence comparison and retrieval with other species, the present invention excavates and clones endogenous U6 promoters U6 ₉₄ and U6 ₃₇₄ (sequences as SEQ ID NO: 1 and SEQ ID NO: 2) in Fusarium venetianus TB01 (sequences are respectively SEQ ID NO: 1 and SEQ ID NO: 2), belonging to Class II promoters are therefore particularly suitable for inducing sgRNA expression in gene editing.

1. Primers

U6₉₈-1 CTCACGTGCCAGTGCCAAAT U6₉₈-2 TGATATAGACGTTGTGGCTGCATATTCGGTCAGTATCGTGATATCAGG U6₉₄-1 CCAGGCGGAATTAATCAGTCAG U6₉₄-2 TGATATAGACGTTGTGGCTGCGTAGTGATGGTCTTGTGTTAAC U6₈₆-1 GCTGGAGTGTGATAGAATAG U6₈₆-2 TGATATAGACGTTGTGGCTGCCCATAGACCGCTGGACCAAAC U6₃₇₄-1 ATGCGGCGGATGTTTTGCGAGTCT U6₃₇₄-2 TGATATAGACGTTTGTGGCTGCGAAGGGTTGGTCTTATTTTAAC sgRNA-1 GCAGCCACAACGTCTATATCAGTTTTAGAGCTAGAAATAGC sgRNA-2 AAAAAAAGCACCGACTCGGTGCC

2. Fragment Amplification Procedure

3. Experimental method

Using primer pairs U6 ₉₈ -1/2, U6 ₉₄ -1/2, U6 ₈₆ -1/2, U6 ₃₇₄ -1/2 to amplify the corresponding endogenous U6 promoter sequence U6 from the DNA genome of Fusarium venezia TB01, respectively ₈₆ , U6 ₉₄ , U6 ₉₈ , U6 ₃₇₄ , the sgRNA _GFP sequence (SEQ ID NO: 4) was amplified based on the synthetic gRNA scaffold fragment (SEQ ID NO: 3) with primer pair sgRNA-1/2. Subsequently, two rounds of amplification were performed by fusion PCR, the U6 fragment was fused with the sgRNA _GFP fragment, and the fusion fragment was confirmed by sequencing.

The experimental results are shown in Figure 1, where A is the electrophoresis result of each endogenous U6 promoter fragment and sgRNA _GFP fragment amplified. B is the electrophoresis result of each endogenous U6 promoter fragment fused with sgRNA _GFP fragment. C is the sequencing comparison result of each endogenous U6 promoter fragment fused with the sgRNA _GFP fragment. The results of electrophoresis and sequence sequencing showed that each endogenous U6 promoter was successfully fused to the sgRNA _GFP fragment, and then the fusion fragment was stored at -20°C.

Example 2: Construction of Cas9 protein expression vector

1. Primers

PgpdA1 TATGACCATGATTACgaattcGGTGTTGATCGTCAACCAAGTCC PgpdA2 tatacttcttgtccatAAGCTTTTTGTTAAGGAGGTTCTGTTTGAGG PCas9-T1 atggacaagaagtatagcatcgg PCas9-T2 catcttctgtcgacactagtgCCTCTAAACAAGTGTACCTG Pgc1 TGTGTGTTCGTGGGATGATG Pgc2 gtcagaccaagtgacaacgc

2. Fragment Amplification Procedure

Pgpda fragment 98℃ 10s, 55℃ 15s, 72℃ 50s; 35 cycles primestar, TaKaRa Cas9-T fragment 98℃ 10s, 55℃ 15s, 72℃ 3min; 35 cycles primestar, TaKaRa Transformer Validation 98℃ 10s, 55℃ 15s, 72℃ 50s; 35 cycles primestar, TaKaRa homologous recombination 50℃ 30min Minerva Super Fusion Cloning Kit, US Everbright

3. Experimental method

The sequence of the endogenous strong promoter PgpdA (SEQ ID NO: 5) was amplified from the DNA genome of Fusarium venetianus TB01 with primer pair PgpdA1/2, and the sequence of the endogenous strong promoter PgpdA (SEQ ID NO: 5) was amplified with primer pair pCas9-T1/2 from containing optimized codons from Aspergillus niger The Cas9-Ttrpc sequence was expanded in the latter Cas9 backbone vector. The pCas9 expression vector was then obtained by homologous recombinase ligation to the backbone vector containing the autoreplicon AMA1 and Hyg resistance in filamentous fungi. After transforming it into E. coli DH5α, a single colony was selected for PCR verification (with the fragment spanning gpdA and Cas9 as the detection object, and the primer pair was Pgc1/2) to obtain a positive transformant.

A in Figure 2 shows a schematic diagram of the pCas9 expression vector. The experimental results are shown in B and C in Figure 2. The electropherogram (B in Figure 2) and the structure indicated by the sequencing results (C in Figure 2) showed that the Pgpda fragment and the Cas9-T fragment had been successfully connected to the backbone vector, and then the positive transformant strains were stored at -80 °C, For subsequent extraction of Cas9 expression plasmid.

Example 3: Co-transformation of protoplasts of Fusarium venetianus GFP fluorescent strains with different endogenous U6 promoters regulating sgRNA _GFP fusion fragments and Cas9 expression vectors

This example describes the specific experimental process of CRISPR/Cas9 gene editing in Fusarium venezia. Figure 3 shows a schematic diagram of the Fusarium venezia CRISPR/Cas9 gene editing system of the present invention.

1. Culture medium

YEPD: Yeast powder 3g, peptone 10g, glucose 20g, make up to 1L

Buffer to dissolve enzyme: 0.7 M NaCl

STC: 0.8 M sorbitol; 50 mM CaCl ₂ , 50 mM Tris-HCl (Ph 8.0)

SPTC: STC with 40% PEG6000

Regeneration medium: yeast extract 1g, tryptone 1g, sucrose 274g, agarose 10g, constant volume 1L

Screening medium: glucose 30g, yeast powder 6g, agar powder 15g, make up to 1L.

2. Experimental method

1) The spore suspension of Fusarium venezia was prepared with Tween 80, spread on GY solid medium, and cultured at 28°C for 7-10 days to produce spores;

2) Prepare the spore suspension and inoculate it in YEPD liquid medium (add glass beads), cultivate at 28°C, 200rmp until the length of the germinated mycelium is 3-4 times that of the spore (16h);

3) Collect spores at 4°C, 13000rpm, 15min, and wash once with sterile 0.7 M sodium chloride;

4) Preparation of protoplasts from enzyme lysate (20mg crash enzyme + 40mg helicase dissolved in 10ml 0.7 M sodium chloride, and sterilized by filtration), lysed at 30°C and 100 rpm for 2h;

5) After filtering with three layers of lens paper, 4℃, 7000rpm, 10min;

6) Wash twice with STC, reselect protoplasts (concentration 10 ⁶ ) with STC after centrifugation as above, and put them on ice for later use;

7) Take 80 μl of the above protoplast suspension, add 20 μl SPTC, mix gently, then add 10 μg fusion U6::sgRNA fragment and Cas9 expression vector, mix gently and place on ice for 30 minutes;

8) Add 1ml SPTC and mix gently, leave at room temperature for 20min;

9) Add the mixture to the regeneration medium at about 40°C, shake well and pour the plate, and culture at 28°C overnight (about 12h);

10) Pour on the screening medium and grow the transformants at 28°C (3-4d) to further screen the GFP gene editing transformants.

Example 4: Fluorescence observation of GFP gene editing transformants

The GFP gene editing transformants obtained in Example 2 were irradiated with a hand-held fluorescence exciter LUYOR-3415GR, and photographed through a filter. The experimental results are shown in the following table and Figure 4.

Comparison of GFP gene editing efficiency by CRISPR/Cas9 editing systems based on different endogenous U6 promoters

promoter name number of transformants The number of non-fluorescent transformants Editing efficiency (%) U6₉₈ 11 0 0 U6₉₄ 9 2 twenty two U6₈₆ 9 0 0 U6₃₇₄ 6 4 67

The results showed that under the irradiation of the fluorescent exciter, only after the CRISPR/Cas9 gene editing system based on the U6 ₃₇₄ promoter and the U6 ₉₄ promoter transformed the fluorescent strains, non-fluorescent transformants (GFP was edited) appeared, and U6 ₃₇₄ The proportion of non-fluorescent transformants presented in the promoter (67%) was significantly higher than that of the U6 ₉₄ promoter (22%), indicating that the CRISPR/Cas9 gene editing based on the endogenous U6 ₃₇₄ promoter and U6 ₉₄ promoter of F. venezia was established The system was able to work in Fusarium venetianus, and the promoter U6 ₃₇₄ had the strongest induction activity.

Example 5: PCR amplification and sequencing of GFP -edited transformants of fluorescent strains

1. Reagents

Mycelial lysate: dissolve 1.2 g NaOH in deionized water and dilute to 100 mL.

Mycelial neutralization solution: 10 mL 1M Tris-HCl (pH8.0), 40 mL 0.3 M HCl, and make up to 800 mL with deionized water.

2. Primers

edit validation -1 ATGGTGAGCAAGGGCGAGGAGCTGTT edit validation-2 cgcctggacgactaaaccaaaataggc

3. Fragment Amplification Procedure

Edit verification snippet 98℃ 10s, 55℃ 15s, 72℃ 45s; 35 cycles primestar, TaKaRa

4. Experimental method

For the non-fluorescent transformants in Example 3, a simple template was prepared by using mycelium lysate and neutralization solution (a small amount of mycelium was taken in 8 μl of mycelium lysate, treated at 98°C for 2 min, and then added to 170 μl of mycelium and solution is a simple template), and the fragment containing the GFP gene was amplified by primer pair editing to verify-1/2, and then the fragments were sequenced and compared.

The experimental results are shown in Figure 5. The results of electrophoresis after PCR amplification of the GFP -edited transformants of fluorescent strains are shown. The results of PCR amplification and sequencing comparison of non-fluorescent transformants showed that after the introduction of Cas9 and sgRNA _GFP , the GFP gene had base deletion at the specific sgRNA binding position, resulting in frameshift mutation of the entire gene. This result further confirms that the endogenous U6 promoter and the corresponding CRISPR/Cas9 gene editing system can be effectively applied in Fusarium venezia.

<110> Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences

<120> Fusarium venetianus endogenous U6 promoter and its CRISPR/Cas9-based gene editing method

<130> 2022

<160> 5

<170> PatentIn version 3.5

<210> 1

<211> 543

<212> DNA

<213> Fusarium venenatum

<400> 1

ccaggcggaa ttaatcagtc agcgccccaa ttctttattt catcccgagt cttgacggag 60

agatgggata gtgaacatat ttagacctaa aaaaatacac aaaaaacatg ctaatatcgt 120

aaactgactc gataagcacg tccctcattc cctcagccgt caatccaaat gcgccacctc 180

gtctccaagt acgaaacggt tgtcttgatg ttgcggagtt taggcttggg gttttcgtac 240

aagtttaagc atgcttctta gcgcgtggga gctgacatct actgcgaggc gtatgcaaag 300

acctatataa atacccgcca acaggaggag ttttcgggac cgaactgaag cttgtctgtt 360

gcttctgcga gtataggttt gtcacaatca atcactccca ggcatatggc aaagaatgga 420

gtgagtttga caagtcatgc tgatagtgac aatgattcaa aaccttgata atatactttg 480

ctgctgctgc ctggaaaata ggcgttggga agggtttata agttaacaca agaccatcac 540

tac 543

<210> 2

<211> 303

<212> DNA

<213> Fusarium venenatum

<400> 2

atgcgggcgg atgttttgcg agtctggcac tgatgaccag ctgttgttgt tccgctatat 60

ttagaaccta gcaaatgctc actcttcaca acaacgtgag atgatttatt gatcagtaat 120

tcctttttaa tgagcaggac tctttttgtg aagatgtgaa gatgtgccgt gctgctgtgg 180

tgatgacaga aactcaaaaa cagactttgt tgctgcctag agcatgccta ggtacctaga 240

caatgcatgc ctggacaata catgctgggt aaggcttata agttaaaata agaccaaccc 300

ttc 303

<210> 3

<211> 83

<212> DNA

<213> Artificial sequences

<400> 3

gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 60

ggcaccgagt cggtgctttt ttt 83

<210> 4

<211> 104

<212> DNA

<213> Artificial sequences

<400> 4

gcagccacaa cgtctatatc agttttagag ctagaaatag caagttaaaa taaggctagt 60

ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt tttt 104

<210> 5

<211> 1450

<212> DNA

<213> Synthetic

<400> 5

ggtgttgatc gtcaaccaag tccgttcacc ggagcttccg gagaggaaga aaaaaaaaac 60

atctgatatg tttgtaacaa atttgctgaa tccattgaac tgaattgaaa gacatcacgg 120

agagttgcac ggtagaatac ggtgtcttat tcggcggcac ctacagaaaa aacagtatcc 180

atccatttcg tctctgtgag gaggaggatg gatggacaga aatggaatca actgaggtgg 240

catggcatgg catgaggggt agaaaaggta ctttttaata gagtgaggcg tgcataatgg 300

gacatgtatc tttttcaggg catcaatcat caccccacca taatgtattg tagctattgt 360

cagtagtggg gaccttagct caccgagctg tggaaagtga gcatgatatg ctctgtacct 420

aaggacaatt aacttgtaaa gctgtaaata tattagttag ttcttataaa acattgaaag 480

gttaagtatt accaagtcat ggcaacggga ttctatgtcc agctatcaag gtactagaca 540

gccgttgaga ggggaaggaa aaaaaaaaag tctcccaccc tctcacccga tattccatgt 600

tccattggcc cctgttatga tacgctacct acactacgat tacctaccta cctactatac 660

tgtacgcccc cttgtctggc ctggcctagc accctggcag tctgggtctg gtctggggtg 720

tgtgttcgtg ggatgatgaa tgatgtggtg ggtgggttac taaactaaac atgagcttga 780

tgagagagtc catggttttc gattattagt atttgtactc ccaccctttc tcctcgacct 840

ttttttctttt tcctctcttt tcttttcatt ctcatcatcc tcaacaacac aacaactact 900

tctttcttca ccaaaaggta tgtgtcttac ctgtctagct ttagctctct agctttgcct 960

gtctctgccc ctccattctc aggattgctt ggctggttct acccctccat tccgcgcgac 1020

tacatcacca tcataacatc acaacatgat ctcgttgctt ccatgctaca tcatgtcata 1080

tcatacctgc ttcttatcac cactctgtga tgatacgtca actattggca ttcatccatt 1140

gagcggataa caaggggagg ggagcaattg gaaggcaaaa aaaaaaaggg acaagaaaaa 1200

agacagagct tcagagctca tatcctgccc ctcctttgca ccaacgttgt cccctcccct 1260

ccactttctt ctactgtggc aacacccgtc ctcaccaaaa aaagctctct ttacatacca 1320

taccatacca taccatacca taccatacct gcctctgatc tgatctgtcc cactccgtca 1380

tacagacggc atataaaata ccccactccc tcgcctcaac ctttgctctt cctcctcatc 1440

ttcttctcta 1450

Claims (10)

1. An endogenous U6 promoter from fusarium venenatum, wherein expression of sgRNA can be induced in fusarium venenatum to enable Cas9 to cut a target sequence at a correct position, and a nucleotide sequence of the endogenous U6 promoter is shown as SEQ ID NO: 1 or SEQ ID NO: 2, respectively.

2. An expression cassette comprising the endogenous U6 promoter of Fusarium venenatum of claim 1.

3. The expression cassette of claim 2, comprising the endogenous U6 promoter, and operably linked thereto sequences in the sgRNA involved in recognition of a target gene and the gRNA scaffold.

4. The expression cassette of claim 3, wherein the nucleotide sequence of the gRNA scaffold is as set forth in SEQ ID NO: 3, respectively.

5. A recombinant vector comprising the endogenous U6 promoter derived from Fusarium venenatum of claim 1 or the expression cassette of any one of claims 2 to 4.

6. A method for gene editing of Fusarium venenatum comprising the steps of: co-transforming the sgRNA fragment containing the endogenous U6 promoter-mediated editing target gene derived from Fusarium venenatum of claim 1 and the Cas9 expression vector into Fusarium venenatum protoplasts, and further screening successfully edited transformants.

7. The method of claim 6, wherein the regulatory element in the Cas9 expression vector comprises a sequence as set forth in SEQ ID NO:5 under the condition of no promoter; a Cas9 protein; SV40 NLS is the nuclear localization signal of virus SV 40; the promoter Ptrpc and terminator Ttrpc of A.nidulans trpc; hygromycin resistance gene Hyg; the filamentous fungal autonomous replicon AMA 1;

the sgRNA fragment of the target gene edited by the endogenous U6 promoter includes the endogenous U6 promoter, a sequence in the sgRNA involved in recognizing the target of the target gene, and a gRNA scaffold.

8. The method of claim 7, wherein the Fusarium venenatum protoplast is prepared by: collecting fusarium venenatum spores, washing the fusarium venenatum spores by using sterile 0.7M sodium chloride, and performing enzymolysis for 2 hours by using an enzyme lysate: 20mg of crash enzyme +40mg of snailase are dissolved in 10ml of 0.7M sodium chloride and sterile filtered.

9. The method of claim 8, wherein the fusarium venenatum has a accession number of CGMCC No. 20740.

10. The method according to any one of claims 7 to 9, further comprising verifying transformants with successful editing of the gene of interest by PCR or sequencing.

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