Plasmid pAHC9, aspergillus niger unary editing system and construction method thereof
By constructing the monolithic editing system of Aspergillus niger with plasmid pAHC9 and gRNAcassette, the problem of low homologous recombination efficiency in Aspergillus niger was solved, and efficient single-gene and multi-gene editing was achieved, especially dual-gene synchronous editing.
Patent Information
- Application Number
- CN202510428153.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-07
- Publication Date
- 2025-08-05
AI Technical Summary
Homologous recombination in Aspergillus niger is inefficient and it is difficult to achieve synchronous editing of multiple genes. Especially under conditions that do not rely on homologous recombination, it is difficult for the prior art to efficiently perform gene knock-in or knock-out.
The plasmid pAHC9, which contains the Cas9 gene and gRNAcassette, was constructed, and targeted gene editing was achieved in Aspergillus niger by using PEG-mediated protoplast transformation method, and efficient editing was performed through non-homologous end ligation.
Under the condition of not relying on homologous recombination, efficient editing of single genes was achieved, with editing efficiency exceeding 90%, and synchronous editing efficiency of dual genes reached 24.7%.
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Figure CN120424969A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of genetic engineering and microbial technology. Technical Background
[0002] Aspergillus niger, belonging to the subphylum Deuteromycotina, class Hyphomycetes, orders Hyphomycetales, family Combretaceae, and genus Aspergillus, is a widely distributed asexual, saprophytic filamentous fungus. Due to its ability to produce a large number of exoproteins and metabolites, A. niger has become an important industrial producer of enzymes and various organic acids, and is widely used in the food industry. Improving its production performance through targeted genetic modification is a common technique used in A. niger. This approach traditionally relies on homologous recombination to knock in or out target genes. However, in filamentous fungi such as A. niger, nonhomologous end-joining primarily occurs, resulting in low homologous recombination efficiency. Even with homologous arms larger than 1,000 bp, high recombination efficiency is limited (Kupfer et al., 1997). Homologous recombination efficiency in A. niger has been improved by knocking out the ku70 and ku80 genes (Takahashi et al., 2006), but this technique still relies on plasmids with appropriate selection markers, making simultaneous editing of multiple genes difficult. Summary of the Invention
[0003] In view of this, the present invention provides a plasmid pAHC9, which is obtained by connecting a DNA fragment containing the Cas9 gene to the SmaⅠ site of the plasmid pUC19-AMA1-Hyg; the plasmid pUC19-AMA1-Hyg is obtained by assembling the DNA fragments of the trpC promoter and the hyg gene into the plasmid pUC19-AMA1; and the plasmid pUC19-AMA1 is obtained by inserting the DNA fragment containing AMA1 into the HindIII site of the plasmid pUC19.
[0004] Furthermore, the present invention provides an Aspergillus niger unified editing system, comprising the plasmid pAHC9 and a gRNA cassette; the gRNA cassette comprises an arginine tRNA promoter, a gene-targeting sgRNA, and a 6-T base sequence connected in series.
[0005] The unary editing system was used to carry out editing of Aspergillus niger target genes, wherein the target genes were: albA gene, pkaC gene, amyA gene or csA gene.
[0006] The gene-targeting sgRNA is a tandem combination of one or two gene-targeting sgRNAs.
[0007] In addition, the present invention also provides a strategy for constructing a gene-blocked mutant of Aspergillus niger, which is obtained by introducing the plasmid pAHC9-ΔX into Aspergillus niger CGMCC 3.17612 through a PEG-mediated protoplast transformation method.
[0008] Using the Aspergillus niger unified editing system provided by the present invention, under conditions independent of homologous recombination, except for the blocking efficiency of pkaC, which was 48.3%, the blocking efficiency of other target genes exceeded 90%, and the simultaneous blocking efficiency of two genes (albA and pkaC) reached 24.7%. BRIEF DESCRIPTION OF THE DRAWINGS
[0009] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for describing the embodiments or the prior art will be described in detail below.
[0010] Figure 1 sgRNA design for targeting amyA and csA.
[0011] Figure 2 Schematic diagram of plasmid pAHC9-ΔX.
[0012] The pUC19 vector is based on the pUC19 backbone and contains the Cas9 encoding gene driven by the gpd promoter, the hygromycin B encoding gene hyg, the autonomous replication sequence AMA1, the gRNA for targeting the target gene, and the arginine tRNA promoter that drives the transcription of the gRNA. X represents the target gene.
[0013] Figure 3 Phenotypes of albA and pkaC gene-disrupted mutants of Aspergillus niger.
[0014] The albA gene-blocked mutant of Aspergillus niger produced white or light yellow-brown conidia; the colony diameter of the pkaC gene-blocked mutant was reduced by 2-3 times.
[0015] Figure 4 PCR and enzyme digestion verification of amyA and csA gene disruption mutants of Aspergillus niger.
[0016] The amyA gene was edited at the target site, but due to the loss of the BamHI restriction site, it was not cut and remained as a 750 bp band. The csA gene was edited at the target site, but due to the loss of the BstXI restriction site, it was not cut and remained as an 829 bp band.
[0017] Figure 5 Schematic diagram of gRNA used for simultaneous editing of albA and pkaC genes.
[0018] Figure 6 Phenotype of the double-gene knockout mutant of albA and pkaC in Aspergillus niger.
[0019] When the albA and pkaC genes of Aspergillus niger are blocked at the same time, small white colonies appear. DETAILED DESCRIPTION
[0020] The present invention provides a unified editing system for Aspergillus niger gene interruption. The method comprises constructing a replicable plasmid pAHC9 containing the elements required for the editing system, inserting a gRNA targeting a target gene, and then introducing the plasmid into Aspergillus niger CGMCC No. 3.17612 to achieve Cas9-mediated targeted editing of the target gene and simultaneous editing of two genes.
[0021] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are based on conventional experimental conditions or the conditions recommended by the manufacturer's instructions.
[0022] The main experimental materials involved in the following examples are:
[0023] Table 1 Plasmids used in the present invention
[0024]
[0025] Table 2 Strains used in the present invention
[0026]
[0027] Table 3 Primer sequences used in the present invention
[0028]
[0029] Table 4 gRNA sequences targeting each gene used in the present invention
[0030]
[0031] The present invention will be further described below with reference to the embodiments.
[0032] Implementation Case 1
[0033] 1. Construction of Plasmid pAHC9
[0034] The shuttle vector Prg3-AMA1-NotI (Liu et al., 2004; Aleksenko & Clutterbuck, 1997) was digested with HindIII to recover the DNA fragment containing the autonomously replicating sequence AMA1.
[0035] Using plasmid pUC19 (purchased from Kangwei Century Company) as the basic skeleton, the DNA fragment containing AMA1 was inserted into the HindIII site of plasmid pUC19 to obtain plasmid pUC19-AMA1.
[0036] pUC19-AMA1 was digested with BamHI to obtain a linearized plasmid.
[0037] Primers Hyg-F and Hyg-R were designed based on the gene sequence of the hygromycin B phosphotransferase gene hyg. A 2,436 bp DNA fragment containing the trpC promoter and the complete hyg gene was amplified by PCR using plasmid pAg1-H3 (Khang et al., 2006) as a template. This fragment was then assembled into plasmid pUC19-AMA1 by Gibson assembly to obtain plasmid pUC19-AMA1-Hyg.
[0038] Using plasmid pLC1 as a template, primers Cas9-F and Cas9-R were designed, and a 4,266 bp DNA fragment containing the complete Cas9 gene (Chen et al., 2020) was obtained by PCR amplification (the Cas9 gene contains the NLS encoding the nuclear localization signal and the 3xFlag tag sequence).
[0039] The DNA fragment containing the Cas9 gene was ligated into the SmaⅠ site of the plasmid pUC19-AMA1-Hyg using the Gibson assembly method to obtain the plasmid pAHC9 containing a unified editing system for Aspergillus niger gene interruption.
[0040] Refer to the Gibson assembly reaction system (refer to NEB Gibson assembly kit) to assemble the enzyme-digested vector and exogenous fragments. The assembly system is as follows:
[0041] Table 5. Gibson assembly reaction system
[0042]
[0043] 2. Target gene selection and sgRNA design
[0044] The Aspergillus niger albA gene (NRRL3_00462) encodes a polyketide synthase (PKS) that is involved in the biosynthesis of dihydroxynaphthalene and naphthyl-γ-pyrone, which is required for the formation of melanin in A. niger spores (Jørgensen et al., 2011). Knockout of the albA gene in A. niger results in the disruption of melanin biosynthesis, resulting in the production of white or light yellow-brown conidia.
[0045] The Aspergillus niger pkaC gene (Gene ID: 4978914) encodes the catalytic subunit of cAMP-dependent protein kinase. Disruption of this gene results in a 2- to 3-fold decrease in the colony diameter of Aspergillus niger (Saudohar et al., 2002).
[0046] These phenotypic changes caused by gene deletion or blocking can be intuitively reflected in the transformants, thereby intuitively detecting the gene knockout efficiency.
[0047] The Aspergillus niger amyA gene (Gene ID: 4983845) encodes alpha-amylase A, and the csA gene (Gene ID: 4983406) encodes citrate synthase. Knockout of these genes does not result in phenotypic changes in Aspergillus niger. To facilitate the identification of gene knockout strains without phenotypic changes, we designed the sgRNA for the amyA gene with a BamHI cleavage site before the PAM site; when designing the sgRNA for the csA gene, we deliberately selected an sgRNA with a BstXI site before the PAM site. Figure 1 When the Cas9 protein mediates the creation of a double-strand break (DSB) in a gene, repair occurs through non-homologous end joining without the addition of a repair template. This leads to base loss, thus destroying the aforementioned BamHI and BstXI restriction sites. When a DNA band containing the target site is amplified and digested with the selected BamHI and BstXI restriction enzymes, if the DNA band cannot be cut, it indicates that the target gene has mutated. If the DNA band can be cut, it indicates that the target gene has not mutated. For each of the aforementioned genes, we designed corresponding gRNAs using the website http: / / crispor.tefor.net / crispor.py and evaluated their potential off-target cleavage sites. We then selected the arginine tRNA promoter from Aspergillus niger (GenBank: AM270178.1) to drive expression of the target gRNA in Aspergillus niger.
[0048] Using Aspergillus niger genomic DNA as a template, primer PtRNA arg21 -F and PtRNA arg21 -R amplification obtained the arginine tRNA promoter (172 bp).
[0049] The target gRNA containing the arginine tRNA promoter was amplified by fusion PCR using primers ΔalbA-gRNA-F, ΔpkaC-gRNA-F, ΔamyA-gRNA-F, ΔcsA-gRNA-F and gRNA-R, respectively.
[0050] The plasmid pAHC9 was linearized with NotI, and then the plasmid pAHC9 / NotI, the arginine tRNA promoter, the designed sgRNA (crRNA and tracrRNA) and the 6 T base sequence fragments were connected by Gibson assembly to form a plasmid pAHC9-ΔX ( Figure 2 ).
[0051] Example 2
[0052] 1. Single-gene knockout in Aspergillus niger based on a unified editing system
[0053] (1) Blocking of the albA gene
[0054] The plasmid pAHC9-∆albA, used for albA gene disruption, was introduced into Aspergillus niger CGMCC 3.17612 via PEG-mediated protoplast transformation (Campbell et al., 1989). Transformants were screened by plating on regeneration plates containing 100 µg / mL hygromycin B. After 3-5 days of incubation on the transformation plates, the A. niger transformants developed white colonies. Under a microscope, the color of the A. niger spores changed from dark brown to white ( Figure 3 Further sequencing confirmed that the albA gene in the white colonies had mutated. After three transformations under the same conditions, the number of Aspergillus niger transformants on the transformation plates was counted, and the ratio of the total number of transformants with mutations to the total number of transformants obtained was used as the editing efficiency. A total of 213 transformants containing pAHC9-ΔalbA were obtained, of which 211 were albA mutants (designated ΔalbA), with an editing efficiency of 99%.
[0055] (2) Blocking of the pkaC gene
[0056] As described above, the plasmid pAHC9-∆pkaC was introduced into Aspergillus niger CGMCC 3.17612 via PEG-mediated protoplast transformation, and the corresponding transformants were screened. The colony formation of the strain with the pkaC gene blocked was significantly reduced ( Figure 3 The pkaC gene-blocking mutant was identified based on colony size and ultimately verified by sequencing. After three co-transformations under identical conditions, 203 transformants containing pAHC9-ΔpkaC were obtained, 95 of which were pkaC-blocking mutants (designated ΔpkaC), with an editing efficiency of 46.8%.
[0057] (3) Blocking of the amyA gene
[0058] As described above, the plasmid pAHC9-∆amyA was introduced into Aspergillus niger CGMCC 3.17612 by PEG-mediated protoplast transformation, and the corresponding transformants were screened and obtained. Since the amyA gene-blocking mutant did not show obvious phenotypic changes, we designed primers amyA-F and amy-R and amplified the 750 bp amyA gene by PCR. If the amyA gene is not edited, the PCR product should be digested with BamHI to obtain two bands of 217 bp and 533 bp respectively; if the amyA gene is edited at the target site, due to the loss of the BamHI restriction site, it will still be a 750 bp band. The transformant verification results are shown in Figure 2. Figure 4 As shown, 11 of the 12 transformants produced gene editing, and sequencing verification further confirmed the result, with an editing efficiency of 91.7%.
[0059] (4) Blocking of the csA gene
[0060] As mentioned above, the plasmid pAHC9-∆csA was introduced into Aspergillus niger CGMCC 3.17612 by PEG-mediated protoplast transformation, and the corresponding transformants were screened and obtained. The csA gene blocking mutant also showed no obvious phenotypic changes. We designed primers csA-F and csA-R and amplified the 829 bp csA partial gene by PCR. If the csA gene has not been edited, two DNA bands of 375 bp and 454 bp should be obtained by digesting the PCR product with BstXI; if the csA gene has been edited at the target site, only an 829 bp band will be obtained due to the absence of the BstXI enzyme cleavage site. The verification results of the transformants are shown in Figure 2. Figure 4 As shown, 11 of the 12 transformants produced gene mutations, and sequencing verification further confirmed the result, with an editing efficiency of 91.7%.
[0061] 2. Simultaneous knockout of two genes in Aspergillus niger based on a unified editing system
[0062] To further verify the feasibility and editing efficiency of this single-gene editing system in the simultaneous knockout of two genes in Aspergillus niger, we continued to select genes albA and pkaC as target genes. Since knockout of both genes will lead to significant changes in the phenotype of the Aspergillus niger colony, the editing efficiency can be calculated based on the phenotypic changes. We sequentially connected the gRNAs for editing the albA and pkaC genes behind the arginine tRNA promoter ( Figure 5 ).
[0063] The DNA fragment containing the arginine tRNA promoter (172 bp), the sgRNA targeting the albA gene (100 bp), and the sgRNA targeting the pkaC gene (containing the gRNA and a terminator with 6 T base sequences, 106 bp) obtained by PCR amplification were ligated into the pAHC9 plasmid linearized with NotI by Gibson assembly to obtain the plasmid pAHC9-ΔalbAΔpkaC for simultaneous editing of the albA and pkaC genes, and the correct construction of the plasmid was verified by sequencing.
[0064] pAHC9-ΔalbAΔpkaC was introduced into Aspergillus niger CGMCC3.17612 via PEG-mediated protoplast transformation. Transformants were screened and the gene knockout was statistically analyzed. When both albA and pkaC genes were blocked simultaneously, the mutant Aspergillus niger colonies were small white colonies ( Figure 6 Sequencing further confirmed that the small white colonies were all double-gene knockout mutants of albA and pkaC. A total of 310 transformants were obtained from three transformation batches, of which 77 had simultaneous knockout of both genes, resulting in a 24.7% efficiency for simultaneous editing of both genes.
[0065] In summary, the present invention provides a unified system for Aspergillus niger gene editing, which can achieve efficient editing of single genes without relying on homologous recombination, and can also perform synchronous editing of multiple genes.
[0066] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the main contents of the present invention, are intended to be within the scope of protection claimed herein.
[0067] References
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Claims
1. Plasmid pAHC9, characterized in that The DNA fragment containing the Cas9 gene was ligated into the SmaⅠ site of the plasmid pUC19-AMA1-Hyg; The plasmid pUC19-AMA1-Hyg is obtained by assembling the DNA fragments of the trpC promoter and the hyg gene into the plasmid pUC19-AMA1; The plasmid pUC19-AMA1 is obtained by inserting a DNA fragment containing AMA1 into the HindIII site of the plasmid pUC19.
2. The Aspergillus niger mono-editing system is characterized by: comprising the plasmid pAHC9 and gRNA cassette of claim 1; The gRNA cassette includes an arginine tRNA promoter, a gene-targeting sgRNA, and 6 T base sequences connected in series.
3. The Aspergillus niger one-component editing system according to claim 2, characterized in that: The targeted genes are: albA gene, pkaC gene, amyA gene or csA gene.
4. The Aspergillus niger one-component editing system according to claim 2, characterized in that: The gene-targeting sgRNA is a tandem combination of one or two gene-targeting sgRNAs.
5. A gene-blocked mutant of Aspergillus niger, characterized in that: The Aspergillus niger unified editing system according to any of claims 2 to 4 is introduced into Aspergillus niger CGMCC 3.17612 through a PEG-mediated protoplast transformation method.
6. The method for constructing the plasmid pAHC9 according to claim 1, characterized in that: The steps include: The DNA fragment containing the Cas9 gene was ligated into the SmaⅠ site of the plasmid pUC19-AMA1-Hyg using the Gibson assembly method; The plasmid pUC19-AMA1-Hyg is constructed as follows: a DNA fragment containing the trpC promoter and the complete hyg gene is assembled into the plasmid pUC19-AMA1 using the Gibson assembly method to obtain the plasmid pUC19-AMA1-Hyg; The construction method of the plasmid pUC19-AMA1 is as follows: using the plasmid pUC19 as a basic skeleton, inserting a DNA fragment containing AMA1 into the HindIII site of the plasmid pUC19 to obtain the plasmid pUC19-AMA1.