CN-122012571-A - System and method for traceless gene editing in Cephalosporium acremonium based on CRISPR-Cas9 system

CN122012571ACN 122012571 ACN122012571 ACN 122012571ACN-122012571-A

Abstract

The invention discloses a system and a method for traceless gene editing in cephalosporium acremonium based on a CRISPR-Cas9 system, and belongs to the technical field of genetic engineering. According to the invention, a CRISPR-Cas9 gene editing DNA fragment containing a Cas9 nuclease and a sgRNA expression cassette is firstly constructed, then homologous recombination donor plasmids of corresponding genes are designed, the editing fragment and the donor plasmids are co-transformed into the Cephalosporium acremonium protoplast through a PEG-mediated protoplast transformation method, ku70 and sorB gene knockout and cefR gene promoter in-situ replacement are sequentially realized, and meanwhile, the resistance screening mark and the Cas9 gene are removed through constructing a traceless knockout donor plasmid, so that traceless editing is realized. The traceless editing system constructed by the method can realize multiple genetic transformation, provides a new way for improving the production efficiency of cephalosporin C, and is suitable for the genetic engineering transformation of the industrial strain of the Cephalosporium acremonium.

Inventors

GAO SHUSHAN
Niu Weijing
DING ZHONGTAO
Ju Xiaozhi
LIANG YE
Peng Mengliu
HOU ZHIPING
WU JIAFENG

Assignees

中国科学院天津工业生物技术研究所

Dates

Publication Date: 20260512
Application Date: 20260414

Claims (10)

1. A method for performing traceless gene editing in cephalosporium acremonium based on a CRISPR-Cas9 system is characterized by comprising the following steps of constructing a CRISPR-Cas9 gene editing DNA fragment containing a Cas9 nuclease coding gene expression cassette and a targeting target sequence sgRNA expression cassette, designing a homologous recombination donor plasmid containing a target sequence upstream and downstream homology arm sequence, co-transforming the CRISPR-Cas9 gene editing DNA fragment and the donor plasmid into cephalosporium acremonium protoplast by a PEG-mediated protoplast transformation method to realize target gene editing, constructing and transforming the traceless knockout donor plasmid, and removing residual resistance screening marker genes in the edited strain.
2. The method of claim 1, wherein the Cas9 nuclease-encoding gene expression cassette consists of a Cas9 gene, a promoter Pmbf1 operably linked to the Cas9 gene, the nucleotide sequence of the promoter Pmbf is shown as SEQ ID No.1, the nucleotide sequence of the Cas9 gene is shown as SEQ ID No.2, the nucleotide sequence of the terminator TtrpC1 is shown as SEQ ID No.3, the sgRNA expression cassette consists of a P5S promoter from a cephem, a 5 sRNA-encoding fragment, and a sgRNA fragment derived from aspergillus nidulans, the nucleotide sequence of the P5S promoter is shown as SEQ ID No.4, the nucleotide sequence of the 5 sRNA-encoding fragment is shown as SEQ ID No.5, the nucleotide sequence of the sgRNA fragment is shown as SEQ ID No.6, and bases 1 to 20 of the sgRNA nucleotide sequence shown as SEQ ID No.6 can be substituted according to the specific target gene.
3. The method according to claim 1, wherein the Cephalosporium acremonium is an industrial high-yield strain of Cephalosporium acremonium, the target sequence is any functional gene coding region or gene regulatory region of Cephalosporium acremonium, and the gene regulatory region is a gene promoter region.
4. The method of claim 1, wherein the constructing of the homologous recombination donor plasmid comprises the steps of selecting 1000-1500bp homology arms, a resistance marker gene expression cassette and a sgRNA complementary target DNA sequence and/or a fluorescent protein expression cassette which are edited by a next round of genes according to requirements on the upstream and downstream of the target sequence, amplifying the plasmid pUC19 as a template to obtain a vector skeleton, and seamlessly cloning and connecting the upstream and downstream homology arms, the resistance screening marker gene expression cassette and the sequences which are introduced according to requirements with the pUC19 vector skeleton to obtain the homologous recombination donor plasmid.
5. The method according to claim 4, wherein the resistance selection marker gene expression cassette comprises a resistance gene, a promoter and a terminator, wherein the resistance gene is hygromycin phosphotransferase hph gene or bleomycin resistance gene ble, the sequence of the hygromycin phosphotransferase hph gene is shown as SEQ ID NO.7, the sequence of bleomycin resistance gene ble is shown as SEQ ID NO.8, the promoter is PtrpC promoter, the nucleotide sequence of the promoter is shown as SEQ ID NO.9, the terminator is TtrpC1 terminator or TtrpC2 terminator, the nucleotide sequences of the TtrpC1 terminator and the TtrpC2 terminator are shown as SEQ ID NO.3 and SEQ ID NO.10 respectively, the fluorescent protein expression cassette is an expression cassette consisting of red fluorescent protein mScarlet gene operably linked with the promoter PgpdA and Ttef as the gene terminator, the nucleotide sequence of the promoter PgpdA is shown as SEQ ID NO.11, the nucleotide sequence of the promoter is shown as SEQ ID NO. 34, and the nucleotide sequence of the TtrpC2 terminator is shown as SEQ ID NO. 12.
6. The method of claim 1, wherein the construction of the traceless knockout donor plasmid comprises selecting a 1000bp-1500bp homology arm on the upstream and downstream of a site where a resistance selection marker gene to be removed is located, amplifying by using a plasmid pUC19 as a template to obtain a vector skeleton, and performing seamless cloning connection on the upstream and downstream homology arm and the pUC19 vector skeleton to obtain the traceless knockout donor plasmid without the resistance selection marker gene, a fluorescent protein expression cassette and a next round of gene editing sgRNA complementary target DNA sequence.
7. The method according to claim 1, wherein the identification of the strain after gene editing adopts a PCR method, and the homozygous genetically edited cephalosporium acremonium engineering bacteria are obtained by designing verification primers which span the outer side of the homology arm of the target sequence and the gene region of the resistance gene and/or the fluorescent protein, verifying the editing effect according to the amplified fragment size, and performing PCR verification again after multiple passages of the positive transformant.
8. The method according to claim 1, wherein when multiple rounds of sequential gene editing are performed, the sgRNA complementary target DNA sequence of the next round of gene editing is introduced into the homologous recombination donor plasmid constructed in each round, and the traceless knockout donor plasmid of the previous round of resistance selection marker gene is synchronously added in each round of gene editing, so that the multiple rounds of gene editing and the traceless removal of the previous round of resistance selection marker are synchronously performed.
9. The recombinant cephalosporium acremonium engineering bacteria prepared by the method of any one of claims 1-8, wherein a target sequence is subjected to gene editing in a genome, the target sequence does not contain a resistance screening marker gene and a fluorescent protein coding gene, the target sequence is any functional gene coding region or gene regulatory region of cephalosporium acremonium, and the gene regulatory region is a gene promoter region.
10. Use of the method according to any one of claims 1-8 in genetic engineering of industrial strains of cephalosporanges, characterized in that the metabolic pathway of cephalosporanges is optimized by knocking out, knocking in or replacing any gene regulatory region of any functional genes of cephalosporanges, improving the fermentation production efficiency of cephalosporin C.

Description

System and method for traceless gene editing in Cephalosporium acremonium based on CRISPR-Cas9 system Technical Field The invention belongs to the technical field of genetic engineering, and particularly relates to a method for traceless gene editing in cephalosporium acremonium based on a CRISPR-Cas9 system. Background Cephalosporin C is an important precursor of cephalosporin antibiotics widely used clinically, and has the advantages of stronger stability, broad-spectrum resistance, lower toxicity and the like. The filamentous fungus Cephalosporium acremonium (Acremonium chrysogenum) is an important industrial microorganism for the production of cephalosporin antibiotics, from which cephalosporin C is currently produced by fermentation in the pharmaceutical industry. At present, the high-yield synthesis regulation mechanism of cephalosporin C in Cephalosporium acremonium is not completely resolved, and becomes the bottleneck for further genetic engineering modification of the strain. Unlike wild type cephalosporium acremonium and other common filamentous fungi, the industrial strain of cephalosporium acremonium has no perfect genetic operation system, and the modern genetic engineering modification of the industrial strain of cephalosporium acremonium needs further technical optimization by applying gene targeting methods such as gene fixed-point knock-in, gene replacement, gene knockout, promoter replacement and the like. In view of the importance of cephalosporin antibiotics in the pharmaceutical industry, the strain of the Cephalosporium acremonium industry is modified by metabolic engineering and other synthetic biological technologies, so that the fermentation production level of the cephalosporin C is further improved, the production cost of the cephalosporin C is reduced, and the method has important significance and great potential. The CRISPR-Cas9 system has been widely used for genome editing of a variety of microorganisms as a highly efficient gene editing tool. At present, accurate gene manipulation has been successfully realized in industrial filamentous fungi such as yellow-producing penicillium, aspergillus nidulans, aspergillus oryzae and the like, and research in the industrial strain of cephalosporium acremonium has been relatively slow at present. With the rapid development of genome editing technology, the gene targeting of the industrial strain of the Cephalosporium acremonium is currently performed by constructing a CRISPR-Cas9 system, but the defects that a resistance screening marker gene and a Cas9 nuclease coding gene remain in the genome of the engineering bacteria after gene editing exist, so that the fermentation production level of cephalosporin C of the engineering bacteria of the industrial strain of the Cephalosporium is hindered, and the industrial application of the engineering bacteria of the industrial strain of the Cephalosporium acremonium is hindered (CN 119709817A, publication day: 2025.03.28). Therefore, development of a traceless gene editing system is needed to carry out efficient strain genetic engineering transformation such as gene targeting of a cefuroxime axetil industrial strain. An efficient traceless gene editing system is established in the cephalosporanic industrial strain, a multiple genetic transformation strategy is realized, and new possibility is provided for further directionally transforming the cephalosporanic industrial strain and improving the production efficiency of cephalosporin C. Disclosure of Invention In order to solve the problems in the prior art, the invention provides a method for traceless gene editing in Cephalosporium acremonium based on a CRISPR-Cas9 system. On one hand, the invention provides a method for performing traceless gene editing in cephalosporium based on a CRISPR-Cas9 system, which comprises the following steps of constructing a CRISPR-Cas9 gene editing DNA fragment containing a Cas9 nuclease coding gene expression cassette and a targeting target sequence sgRNA expression cassette, designing a homologous recombination donor plasmid containing a target sequence upstream and downstream homology arm sequence, co-transforming the CRISPR-Cas9 gene editing DNA fragment and the donor plasmid into cephalosporium protoplast by a PEG-mediated protoplast transformation method, realizing target gene editing, constructing and transforming the traceless knockout donor plasmid, and removing residual resistance screening marker genes in the edited strain. In one embodiment, the Cas9 nuclease-encoding gene expression cassette consists of a Cas9 gene, a promoter Pmbf1 operably linked to the Cas9 gene, and a terminator TtrpC1, wherein the nucleotide sequence of the promoter Pmbf is shown as SEQ ID No.1, the nucleotide sequence of the Cas9 gene is shown as SEQ ID No.2, the nucleotide sequence of the terminator TtrpC1 is shown as SEQ ID No.3, the sgRNA expression cassette consists of a sequence nucleotide sequence shown as SEQ ID No.4 w