Pilot editing system, recombinant strain containing pilot editing system, application of recombinant strain and gene editing method

Abstract

The invention relates to the field of gene engineering, and discloses a pilot editing system, a recombinant strain containing the pilot editing system, application of the recombinant strain and a gene editing method. The system comprises a coding gene of nicking enzyme nCas9, a coding gene of reverse transcriptase RT and pegRNA. Wherein the coding gene of the nicking enzyme nCas9 and the coding gene of the reverse transcriptase RT are connected through a Linker sequence, and the nucleotide sequence of the Linker sequence is as shown in SEQ ID NO. 1; the pegRNA comprises a gibberella zeylanica endogenous promoter 5SrRNA, sgRNA, a spacer sequence spacer, a primer binding site PBS (Phosphate Buffer Solution) and a reverse transcription template RT template; the reverse transcription template RT template carries an insertion sequence, and the insertion sequence is used for destroying the expression of a target gene. The method realizes efficient, accurate and safe gene editing of gibberella zeylanica, and has important scientific research and application values.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of genetic engineering, in particular to a prime editing system, a recombinant strain containing the prime editing system and application, and a gene editing method. BACKGROUND

[0002] Fusarium fujikuroi is an important filamentous fungus, and gibberellins produced by its metabolism as a key plant growth hormone are widely used in the agricultural field to promote plant growth, development and maturation, and have significant economic value. However, the genetic modification and functional gene research of Fusarium fujikuroi have been limited by the inefficiency of traditional genetic tools for a long time, making it difficult to achieve precise and efficient genome editing, which restricts the further exploration of its industrial application potential.

[0003] In recent years, the development of gene editing technology has provided new means for microbial genetic modification, but the existing technology has obvious limitations in Fusarium fujikuroi: gene editing technology represented by CRISPR-Cas9 relies on inducing DNA double-strand break (DSB) to start repair, which easily causes chromosomal abnormalities, large fragment deletion and other risks, leading to genome instability; RNA interference technology can only achieve temporary silencing of target genes and cannot achieve permanent editing, and the effect is unstable, which makes it difficult to meet the demand for precise modification.

[0004] Prime editing (PE) technology, as a new generation of gene editing tool, was first reported by David Liu's team in 2019. Its core design can reduce the dependence on DNA double-strand break, significantly reduce off-target risk and genome damage, and has higher editing safety and precision. The system mainly contains two parts: one is the fusion protein of Cas9 H840A nickase and Moloney murine leukemia virus (M-MLV) reverse transcriptase, and the other is prime editing guide RNA (pegRNA) containing target spacer sequence, sgRNA scaffold and primer binding site. Its mechanism of action is through single-strand cleavage, reverse transcription template-guided synthesis and DNA repair pathway to achieve precise editing of base substitution, insertion or deletion, which can avoid the potential risk of double-strand break in principle.

[0005] However, the currently reported lead editing system (PE system) is mainly applied to mammalian cells and bacteria, and there is no PE system suitable for Gibberella fujikuroi and other filamentous fungi. Due to the significant differences in genome structure, gene expression regulation mechanism and other aspects between filamentous fungi and other organisms, the core elements (such as fusion protein, pegRNA and regulatory elements) of the existing PE system cannot be directly adapted to Gibberella fujikuroi, which makes it difficult to function in the strain. Therefore, it is of great significance to develop an adaptive PE system for Gibberella fujikuroi to achieve efficient and precise genome editing, optimize its metabolic pathway, improve the fermentation level of target products and promote the improvement of industrial strains. SUMMARY

[0006] The purpose of the present application is to overcome the problems of low efficiency of traditional genetic tools for editing Gibberella fujikuroi and the inability of the reported lead editing system to be directly applied to Gibberella fujikuroi due to element adaptability, and to provide a lead editing system (PE system), a recombinant strain containing the PE system and its application, and a gene editing method. The PE system can achieve precise editing in the genome of Gibberella fujikuroi, is independent of DNA double-strand break, has high safety, can reduce the potential risk in the process of gene editing, and significantly improves the editing efficiency, providing an efficient and low-off-target precise editing means for the genetic modification of Gibberella fujikuroi, which helps to optimize its metabolic pathway, improve the fermentation level of target products, and promote the practical production and application of industrial strains.

[0007] In order to achieve the above-mentioned purpose, the present application provides a lead editing system in one aspect, which comprises a coding gene of a nicking enzyme nCas9, a coding gene of a reverse transcriptase RT and a pegRNA. The coding gene of the nicking enzyme nCas9 and the coding gene of the reverse transcriptase RT are connected by a Linker sequence, and the nucleotide sequence of the Linker sequence is shown in SEQ ID NO. 1; the pegRNA comprises an endogenous promoter 5SrRNA of Gibberella fujikuroi, an sgRNA, a spacer sequence spacer, a primer binding site PBS and a reverse transcription template RT template; the reverse transcription template RT template carries an insertion sequence, and the insertion sequence is used to destroy the expression of the target gene.

[0008] The second aspect of the present application provides a recombinant strain, which contains the lead editing system as described above.

[0009] The third aspect of the present application provides a method for gene editing, which inoculates the recombinant strain as described above into a culture medium for culture.

[0010] By the technical scheme, the application realizes efficient, precise and safe gene editing for Gibberella fujikuroi. The rigid Linker sequence shown in SEQ ID NO. 1 is designed to stably maintain the spatial conformation of the nicking enzyme nCas9 and the reverse transcriptase RT, ensuring that the two cooperate to perform single-strand cutting and reverse transcription functions, thereby improving the continuity and efficiency of the editing reaction; the introduction of the Gibberella fujikuroi endogenous 5SrRNA promoter in the pegRNA can adapt to the host's gene expression regulation mechanism and enhance the adaptability of the system in Gibberella fujikuroi; the PBS in the 3' extension region and the target gene reverse transcription template are precisely anchored to the editing site through base pairing, which can realize the targeted modification (such as insertion, deletion or base substitution) of the target gene, and does not need to rely on DNA double-strand breaks, thereby significantly reducing the risk of chromosomal abnormalities, off-target effects and the like. The gene editing method by culturing the recombinant strain can stably realize the precise modification of the Gibberella fujikuroi genome, and the editing efficiency is significantly improved, which has important scientific research and application value. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 is a phenotype verification diagram of the PE system editing the FCC1 gene in Example 3. DETAILED DESCRIPTION

[0012] The endpoints of the ranges and any values disclosed herein are not limited to the precise values recited as the exact dimensions are not critical to the characteristics of the application. The endpoints of the ranges and any numerical values are approximations only. Unless otherwise indicated, the endpoints of the ranges are not to be understood as being limited to the precise values recited as the exact dimensions are not critical to the characteristics of the application. Any numerical value, however, can include fractions of the value, ranges up to and including the value, and ranges that are larger than the value. For values that are less than one and for fractions of one, more precise numerical values can be actualized using a combination of fractions and decimals.

[0013] The first aspect of the application provides a pilot editing system, which comprises a coding gene of a nicking enzyme nCas9, a coding gene of a reverse transcriptase RT and a pegRNA; The coding gene of the nicking enzyme nCas9 and the coding gene of the reverse transcriptase RT are connected through a Linker sequence, the nucleotide sequence of the Linker sequence is shown in SEQ ID NO. 1; the pegRNA comprises a Gibberella fujikuroi endogenous promoter 5SrRNA, an sgRNA, a spacer, a primer binding site PBS and a reverse transcription template RT template; the reverse transcription template RT template carries an insertion sequence, and the insertion sequence is used to destroy the expression of a target gene.

[0014] The core element is optimized in the application to ensure that the pilot editing system performs optimally in Gibberella fujikuroi and improves the expression efficiency in the host. Through a large number of experiments, it is verified that the selected Linker sequence in the application is a rigid Linker sequence, which can effectively connect the two coding genes, ensure reasonable spatial conformation, maintain effective interaction, and improve the stability and efficiency of the system function. In the pegRNA, the endogenous 5SrRNA promoter in Gibberella fujikuroi can specifically start the expression of sgRNA, spacer, PBS and RT template, and adapt to the host regulation mechanism.

[0015] Through the above optimization, the pilot editing system (PE system) of the application can realize efficient, accurate and safe gene editing in Gibberella fujikuroi, and provide a powerful tool for genetic modification and industrial application.

[0016] The nucleotide sequence provided by the application can be obtained by polymerase chain reaction (PCR) amplification method, recombination method or artificial synthesis method. Once the nucleotide sequence is obtained, the relevant amino acid sequence can be obtained in large quantities by recombination method. In addition, the relevant nucleotide sequence can also be synthesized by a known artificial chemical synthesis method.

[0017] According to the application, preferably, the nucleotide sequence of the coding gene of the nCas9 is shown as SEQ ID NO. 2. The nCas9 coding gene with the nucleotide sequence shown in SEQ ID NO. 2 adopted by the application is the result of optimization for the codon preference of Gibberella fujikuroi, which can significantly improve the translation efficiency of nCas9 protein in host cells and ensure sufficient expression of the nicking enzyme. The sequence adapts to the gene expression system of Gibberella fujikuroi, so that nCas9 can be more efficiently folded into an active functional conformation. At the same time, the optimized coding sequence can reduce the interference of post-transcriptional modification on protein function, provide a reliable basis for the formation of a functional fusion protein by nCas9 and RT through a rigid Linker, and further improve the editing efficiency of the entire pilot editing system.

[0018] According to the present application, preferably, the nucleotide sequence of the coding gene of the reverse transcriptase RT is shown as SEQ ID NO. 3. The reverse transcriptase RT coding gene with the nucleotide sequence shown in SEQ ID NO. 3 is the result of modification according to the expression environment and functional requirements of Gibberella fujikuroi, can significantly enhance the reverse transcription activity of RT in host cells, and ensure efficient catalysis of DNA synthesis reaction with pegRNA as template. At the same time, its sequence compatibility with the nCas9 coding gene is optimized, and efficient fusion expression can be achieved through rigid Linker, ensuring the synergy of the two in spatial conformation, providing precise enzymatic support for reverse transcription after single-strand cleavage, and further improving the targeting modification efficiency of the whole editing system.

[0019] According to the present application, preferably, the target gene is FCC1 gene; further preferably, the nucleotide sequence of the sgRNA corresponding to the FCC1 gene is shown as SEQ ID NO. 4. The present application preferably takes FCC1 gene as target gene and adopts its corresponding sgRNA (nucleotide sequence shown as SEQ ID NO. 4), which can take advantage of the characteristic of Gibberella fujikuroi accumulating purple pigment after the gene knockout, and intuitively judge the editing effect through the color change of the colony, significantly improving the convenience of effective system verification; at the same time, specific sgRNA can enhance the precise targeting of target gene, which helps to improve the editing efficiency and reduce off-target risk.

[0020] According to the present application, preferably, the nucleotide sequence of the endogenous promoter 5SrRNA of Gibberella fujikuroi is shown as SEQ ID NO. 5. The present application adopts the Gibberella fujikuroi endogenous 5SrRNA promoter sequence shown in SEQ ID NO. 5, which has natural adaptability with the transcriptional regulation system of the host, can efficiently drive the specific transcription of pegRNA in Gibberella fujikuroi, and significantly improve the expression level of sgRNA. The promoter sequence is screened by long-term evolution of the host, which can respond to the physiological metabolic signals of Gibberella fujikuroi, ensure the stable expression of pegRNA in the key stage of editing, and avoid the expression disorder caused by the difference of regulation mechanism of exogenous promoter. At the same time, its sequence specificity can reduce the interference with the transcription of endogenous genes of the host, reduce the non-target effect caused by the cross action of the promoter, and further provide reliable transcription initiation guarantee for the precise operation of the whole guide editing system.

[0021] According to the present application, preferably, the spacer is a targeting sequence containing 15-25 nucleotides, which has sequence complementarity with a specific region of double-stranded DNA of the target gene. The inventors found that using a spacer with a length of 15-25 nucleotides as the targeting sequence of sgRNA can not only achieve precise recognition through high complementarity with a specific region of the target gene, but also avoid the off-target risk caused by too long sequence or the insufficient binding stability caused by too short sequence.

[0022] According to the present application, preferably, the length of the primer binding site PBS is 15-25 nucleotides, which has sequence complementarity with a specific region of the single-stranded DNA produced after the target gene is cut. The inventor found that the PBS in this length range can enhance the binding stability with the single-stranded DNA of the target gene, while avoiding non-specific pairing caused by excessive length, thereby providing an efficient and precise starting binding site for reverse transcriptase, ensuring the orderly initiation of reverse transcription reaction to introduce the insertion sequence.

[0023] According to the present application, preferably, the length of the insertion sequence is 1-100 bp. The inventor found that this length range can flexibly adapt to different gene editing needs, which can not only induce frameshift mutations through short sequences of 1-5 bp to interfere with the expression of target genes, but also can destroy the key functional domain of the target gene through longer sequences of 10-100 bp, while taking into account the extension efficiency of reverse transcriptase and the stability of the editing system, to ensure that the insertion sequence can be effectively introduced and function.

[0024] The second aspect of the present application provides a recombinant strain containing the lead editing system as described above.

[0025] According to the present application, preferably, the starting strain of the recombinant strain is Gibberella fujikuroi. The lead editing system of the present application has high adaptability to its natural genetic background, ensuring that the core elements of the system (such as nCas9, RT and pegRNA) can efficiently function, facilitating direct use in subsequent gene function research and metabolic pathway optimization, improving the genetic stability of the edited strain, and providing a reliable strain basis for industrial application.

[0026] The third aspect of the present application provides the use of the lead editing system as described above and the recombinant strain as described above for gene editing in the genome of Gibberella fujikuroi.

[0027] This application can use the lead editing system and recombinant strain adapted to Gibberella fujikuroi to achieve efficient, precise and safe gene editing in its genome, providing strong technical support for the functional gene research and industrial strain improvement of the fungus, and promoting its application and development in the fields of agriculture and industry.

[0028] The fourth aspect of the present application provides a method for gene editing, which inoculates the recombinant strain as described above into a culture medium for culture.

[0029] The gene editing method can naturally start the editing process in the metabolism environment of Gibberella fujikuroi by culturing the recombinant strain containing the lead editing system provided by the application, without the need for complex exogenous delivery steps, simplifying the operation process and reducing errors caused by human intervention. During the culture process, the physiological activities of the recombinant strain and the functions of the editing system are coordinated, which can realize efficient gene modification while maintaining the normal growth of the host, further improving the stability and repeatability of the editing operation.

[0030] According to the application, preferably, the culture conditions include: the temperature is 25-28℃, 90-110 μg / mL of hygromycin is added after 10-14 h of culture, and / or the soft agar regeneration layer is continuously cultured for 5-7 days.

[0031] The culture temperature can be specifically 25℃, 26℃, 28℃, 30℃, or any value between the above two values. This temperature range is suitable for the optimal growth requirements of Gibberella fujikuroi, and can ensure normal metabolism of the bacteria and efficient expression of the PE system components.

[0032] The addition of 90-110 μg / mL of hygromycin can quickly enrich positive strains successfully introduced with the editing system through resistance screening, reducing the interference of non-target strains.

[0033] The soft agar regeneration layer (soft agar components and contents: glucose 18-22 g / L, peptone 2-4 g / L, yeast powder 2-4 g / L, potassium phosphate dibasic 0.5-2 g / L, magnesium sulfate heptahydrate 0.2-1.2 g / L, potassium chloride 0.4-0.8 g / L, ferrous sulfate heptahydrate 0.005-0.015 g / L, sucrose 150-200 g / L, and agar 18-22 g / L) provides a suitable microenvironment for protoplast regeneration, helping the strain to restore its growth ability.

[0034] The duration of 5-7 days of continuous culture can ensure that the gene editing reaction is fully carried out, and can also ensure that the positive transformants form visible colonies, achieving the dual goals of efficient screening and obtaining edited strains, and overall improving the operability and success rate of gene editing.

[0035] The application will be described in detail through the following examples. In the following examples, the ClonExpress Ultra One Step Cloning Kit V2 cloning enzyme used for Gibson assembly was purchased from Novozyme Biotech Co., Ltd.; Gibberella fujikuroi was from the team of Professor Huang and Nanjing Normal University, disclosed in patent CN118813664A, with the number CCTCC NO. M20221848; the DNA extraction kit was purchased from Beijing Solabio Technology Co., Ltd. Other reagents and raw materials were all conventional market products.

[0036] Soft agar components and contents: glucose 20 g / L, peptone 3 g / L, yeast powder 3 g / L, potassium phosphate dibasic 1 g / L, magnesium sulfate heptahydrate 0.5 g / L, potassium chloride 0.5 g / L, ferrous sulfate heptahydrate 0.01 g / L, sucrose 171.4 g / L, and agar 20 g / L.

[0037] Example 1 The PE system containing four elements of nCas9, reverse transcriptase FfRT, pegRNA and hygromycin selection marker was constructed, and the specific steps were as follows: S1, construct plasmid pCMV-FfnCas9-FfRT: Take plasmid pUC-fFuCas9-HTB NLS -hph as template (reference literature: Shi, T. Q.; Gao, J.; Wang, W. J.; Wang, K. F.; Xu, G. Q.; Huang, H.; Ji, X. J. CRISPR / Cas9-based genome-editing in the filamentous fungus Fusarium fujikuroi and its application in strain engineering for gibberellic acid production. ACS Synthetic Biology, 2019, 8, 445 454), use primers Ala840-F and Ala840-R (primer sequences are shown in Table 1) to replace H840A of FfCas9, obtain plasmid PUC-FfnCas9; take plasmid PUC-FfnCas9 as template, use primers nCas9-F and nCas9-R (primer sequences are shown in Table 1) to amplify, obtain fragment FfnCas9; Retrieval of literature to dig out the gene sequences of reverse transcriptase M-MLV-RT and small RNA binding protein La (the codon-optimized gene sequence of reverse transcriptase M-MLV-RT is shown in SEQ ID NO: 3, and the codon-optimized gene sequence of small RNA binding protein La is shown in SEQ ID NO: 22, which can be referred to in the literature: Jun Yan, Paul Oyler-Castrillo, Purnima Ravisankar, Improving prime editing with an endogenous small RNA-binding protein. Nature, 2024, 628, 639-647), synthesize plasmid PUC-FfRT, and use it as a template, use primers RT-F and RT-R (primer sequences are shown in Table 1) to amplify, obtain fragment FfRT; The plasmid pCMV-PE1 was used as a vector (purchased from addgene website, number 132774), the vector was linearized by Not I and Pme I enzymes, the fragments FfnCas9 and FfRT obtained as described above were fused to obtain the fragment FfnCas9-FfRT, and the primer nCas9-R and RT-F were designed, and the coding region of the rigid Linker sequence (nucleotide sequence as shown in SEQ ID NO. 1) was introduced at the 3' end of the primer RT-F, so that the 5' end was complementary to the 3' end of the FfnCas9 fragment, and the 3' end was complementary to the 5' end of the FfRT fragment through the Linker sequence, so as to realize the sequence connection of FfnCas9 and FfRT. The above-mentioned primers were used to fuse the fragments FfnCas9 and FfRT obtained as described above to obtain the fusion fragment FfnCas9-Linker-FfRT containing the rigid Linker. Gibson assembly (one-step cloning kit purchased from Novozyme Biotech Co., Ltd., ClonExpress Ultra One Step Cloning Kit V2) was used to connect the vector and the fusion fragment to obtain the recombinant plasmid pCMV-FfnCas9-FfRT; S2, constructing plasmid pCMV-FfpegRNA-FfnCas9-FfRT: The plasmid pCMV-FfnCas9-FfRT obtained in step S1 was used as a vector, and the vector was linearized by Apo I enzyme. According to the genomic characteristics of G. fujikuroi and the editing requirements of the target gene, the functional elements of FfpegRNA were designed: first, the endogenous 5SrRNA sequence (nucleotide sequence as shown in SEQ ID NO. 5) of G. fujikuroi was selected as the starting element to adapt to the transcriptional regulation mechanism of the host and ensure the stable expression of pegRNA; the spacer sequence with a length of 15-25 nucleotides was designed to form strict sequence complementarity with the specific editing region of the target gene, so as to ensure the accuracy of targeted recognition; the scaffold sequence of sgRNA was optimized to enable efficient binding with FfnCas9 protein to realize precise guidance. After the nucleotide sequences of the above elements were designed and determined, they were synthesized by chemical synthesis, and then were assembled in the order of 5SrRNA, spacer and sgRNA to form the complete DNA fragment of FfpegRNA. This fragment contains sequence information for precise targeting of the target gene, and also has the structural characteristics suitable for the expression system of G. fujikuroi, laying a foundation for the subsequent construction of recombinant plasmids and the realization of the function of the lead editing system. The fragment FfpegRNA was connected with the vector plasmid pCMV-FfnCas9-FfRT by Gibson assembly to obtain the recombinant plasmid pCMV-FfpegRNA-FfnCas9-FfRT.

[0038] S3, resistance screening marker HPH insertion The plasmid pCMV-FfpegRNA-FfnCas9-FfRT obtained in step S2 is used as a vector, the vector is linearized by EcoR I enzyme, the plasmid pUC-fFuCas9-HTBNLS-hph is used as a template (reference literature: Shi, T. Q.; Gao, J.; Wang, W. J.; Wang, K. F.; Xu, G. Q.; Huang, H.; Ji, X. J. CRISPR / Cas9-based genome-editing in the filamentous fungus Fusarium fujikuroi and its application in strain engineering for gibberellic acid production. ACS Synthetic Biology, 2019, 8, 445454), the primers HPH-F and HPH-R (the primer sequences are shown in Table 1) are used for amplification to obtain a fragment HPH, Gibson assembly is adopted, the vector and the fragment are connected, and the plasmid pCMV-FfpegRNA-FfnCas9-FfRT-HPH is obtained, and this plasmid is named as PFf-PE. The obtained PFf-PE is a lead editing system of the application, and provides a core tool vector for subsequent gene editing research and application in Fusarium fujikuroi.

[0039]

[0040] Example 2 Construction of the purple PE system verification plasmid PFf-PE-FCC1, including the following steps: S1, the plasmid PFf-PE obtained in Example 1 is used as a vector, and the vector is linearized by Spe I enzyme. The primers FCC1N20-F and FCC1N20-R (the primer sequences are shown in Table 2) are designed, and the primers are used for PCR amplification to obtain a fragment FCC1-N20 containing a FCC1 gene specific spacer (20 nucleotides in length, designed through the website https: / / crispor.gi.ucsc.edu / crispor.py). Then, the plasmid PFf-PE linearized by Spe I enzyme is connected with the fragment FCC1-N20 obtained by amplification (T4 DNA ligase connection), and finally the plasmid PFf-PE-FCC1N20 is obtained.

[0041] S2, using the plasmid PFf-PE-FCC1N20 obtained in step S1 as a vector, linearizing the vector with Age I enzyme. According to the editing requirements of the target gene FCC1, a 3' extension region sequence containing a reverse transcription template (RT template) and a primer binding site (PBS) was designed, wherein the RT template carries a 13 bp insertion sequence "TCTGCCATCAAAG" for destroying the expression of the FCC1 gene; at the same time, specific primers RTT-PBS-F and RTT-PBS-R (primer sequences are shown in Table 2) were designed, and a 3' extension region fragment containing the RT template and the PBS was obtained by PCR amplification using the 3' extension region sequence as a template. Gibson assembly technology was used to connect the Age I linearized vector and the above-mentioned 3' extension region fragment, and a recombinant plasmid PFf-PE-FCC1 was obtained.

[0042] Through the operation of this embodiment, a specific purple PE system verification plasmid PFf-PE-FCC1 for the FCC1 gene of Gibberella fujikuroi was successfully constructed. The plasmid is inserted with a FCC1 gene specific spacer (FCC1-N20 fragment) by Spe I enzyme cutting, and a 3' extension region containing a 13 bp insertion sequence (containing an RT template and a PBS) is introduced by Age I enzyme cutting, achieving precise targeted design of the FCC1 gene. The expression of the gene can be destroyed by the insertion sequence. The construction of this plasmid provides a key tool for subsequent verification of the editing efficiency and specificity of the lead editing system on the FCC1 gene in Gibberella fujikuroi, and lays an experimental foundation for the application of the system in the functional study of target genes.

[0043]

[0044] Example 3 Transformation of Gibberella fujikuroi for verification of PE system editing efficiency and accuracy (1)Gibert transformation: Take 100 μL of Gibert Ff3 protoplast, add 10 μg of recombinant plasmid PFf-PE-FCC1 obtained in Example 2, mix evenly, then ice bath for 20 min, add 50 μL of PEG6000 solution (containing 1M sorbitol, 10mM Tris-HCl, 50mM CaCl2, pH=7.5), mix evenly, then ice bath for 20 min, add 1 mL of PEG6000 solution, mix evenly, then room temperature for 5 min, then add 2 mL of STC solution (sorbitol 18.217 g, anhydrous calcium chloride 0.555 g, Tris-base 0.121 g, adjust to pH 7.5 with HCl, STC solution is a commonly used buffer solution in microbial experiments, its core function is to maintain the stability of protoplast and assist the transformation process, widely used in gene editing or cell operation experiments of fungi, bacteria, etc.), mix evenly, then coat MYG solid regeneration plate, culture for 12 h, then cover 10 mL of hygromycin soft agar regeneration plate with a concentration of 100 μg / ml, wait for 5-7 days for the appearance of transformants.

[0045] (2) Editing efficiency calculation: Knockout of the cyclin C1 (FCC1) of Fusarium cell cycle can make Gibert Ff3 accumulate more purple pigment, which facilitates the calculation of the editing efficiency of the PE system, that is, the ratio of the number of purple fungi to the total number of fungi. As shown in Figure 1 , the successfully edited Gibert Ff3 transformants showed obvious purple phenotype due to the disruption of FCC1 gene expression, which directly verified that the PE system effectively disrupted the function of FCC1 gene by inserting a 13bp sequence.

[0046] (3) Genomic verification: Extract the genome of purple Gibert Ff3 by fungal genomic DNA extraction kit (purchased from Beijing Solabio Technology Co., Ltd.); use primers FCC1-YZ-F and FCC1-YZ-R (primer sequences are shown in Table 3) to perform genomic fragment amplification reaction in a 20 μL amplification system: 10 μL 2x Phanta Flash Master Mix (purchased from Nanjing Novozyme Bio-technology Co., Ltd.), to verify the accuracy of the PE system editing.

[0047] The amplification reaction conditions are: 95°C pre-denaturation for 3 min; 95°C denaturation for 15 s, 58°C annealing for 15 s, 72°C extension for 30 s, a total of 35 cycles; and 72°C final extension for 5 min. The amplification product is detected by 1% agarose gel electrophoresis, and a band of the expected size appears, indicating that the PE system edits the FCC1 gene accurately. Through the above examples, the effectiveness and reliability of the lead editing system suitable for G. fimbriatum constructed by the present application are fully demonstrated. The system not only can efficiently edit specific genes of G. fimbriatum, but also ensures the accuracy and operability of editing through the insertion and verification of the resistance screening marker, providing a powerful tool and method for the gene function research and related biotechnological applications of G. fimbriatum. Subsequently, based on this system, further editing research on other genes of G. fimbriatum can be carried out to explore its application potential in the fields of biosynthesis and metabolic regulation.

[0048] Comparative Example 1 The purple PE system verification plasmid PFf-PE-FCC1 was constructed according to the methods of Example 1 and Example 2, except that the endogenous promoter 5SrRNA of G. fimbriatum was adjusted to the non-endogenous U6 promoter from A. fumigatus (the nucleotide sequence is shown as SEQ ID NO. 20), and the rest remained unchanged. The editing efficiency was verified according to the method provided in Example 3, and the editing efficiency of the obtained PE system is shown in Table 4.

[0049] Comparative Example 2 The purple PE system verification plasmid PFf-PE-FCC1 was constructed according to the methods of Example 1 and Example 2, except that the rigid Linker sequence was adjusted to a flexible Linker sequence (the nucleotide sequence is shown as SEQ ID NO. 21), and the rest remained unchanged. The editing efficiency was verified according to the method provided in Example 3, and the editing efficiency of the obtained PE system is shown in Table 4.

[0050]

[0051] As can be seen from the comparison of the editing efficiency data in Table 4, the PE system provided by the present application has better reliability and effectiveness.

[0052] The above describes the preferred embodiments of the present application, but the present application is not limited thereto. Within the technical concept of the present application, various simple modifications can be made to the technical solutions of the present application, including the combination of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as disclosed by the present application and fall within the protection scope of the present application.

Claims

1. A pilot editing system, characterized in that, The system contains the coding gene for the nicking enzyme nCas9, the coding gene for the reverse transcriptase RT, and pegRNA. The gene encoding the nickase nCas9 and the gene encoding the reverse transcriptase RT are linked by a linker sequence, the nucleotide sequence of which is shown in SEQ ID NO.

1. The pegRNA contains the endogenous promoter 5S rRNA of Fusarium oxysporum, sgRNA, a spacer sequence, a primer binding site PBS, and a reverse transcription template RT template. The reverse transcription template RT template carries an insert sequence that is used to disrupt the expression of the target gene.

2. The pilot editing system according to claim 1, characterized in that, The nucleotide sequence of the gene encoding nCas9 is shown in SEQ ID NO.

2.

3. The pilot editing system according to claim 1 or 2, characterized in that, The nucleotide sequence of the gene encoding the reverse transcriptase RT is shown in SEQ ID NO.

3.

4. The pilot editing system according to claim 1 or 2, characterized in that, The target gene is the FCC1 gene; Preferably, the nucleotide sequence of the sgRNA corresponding to the FCC1 gene is shown in SEQ ID NO.

4.

5. The pilot editing system according to claim 1 or 2, characterized in that, The nucleotide sequence of the endogenous promoter 5S rRNA of *Fujikura fusarium* is shown in SEQ ID NO.

5.

6. The pilot editing system according to claim 1 or 2, characterized in that, The spacer sequence is a target sequence containing 15-25 nucleotides, which is sequence complementary to a specific region of the double-stranded DNA of the target gene. Preferably, the primer binding site PBS is 15-25 nucleotides in length and has sequence complementarity with a specific region of the single-stranded DNA generated after the target gene is cleaved; Preferably, the length of the inserted sequence is 1-100 bp.

7. A recombinant bacterial strain, characterized in that, The recombinant strain contains the lead editing system as described in any one of claims 1-6.

8. The recombinant strain according to claim 7, characterized in that, The recombinant strain originated from *Fujikura gibberellinii*.

9. The application of the lead editing system according to any one of claims 1-6 and the recombinant strain according to claim 7 in gene editing of the Fusarium oxysporum genome.

10. A method for gene editing, characterized in that, The method involves inoculating the recombinant strain of claim 7 into a culture medium for cultivation. Preferably, the culture conditions include: a temperature of 25-28℃, and after culturing for 10-14 h, adding 90-110 μg / mL of hygromycin and / or soft agar regeneration layer and continuing culture for 5-7 days.

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