WO2021032180A1 - 一种多靶点编辑重组曲霉菌株的可视化筛选方法 - Google Patents

一种多靶点编辑重组曲霉菌株的可视化筛选方法 Download PDF

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WO2021032180A1
WO2021032180A1 PCT/CN2020/110404 CN2020110404W WO2021032180A1 WO 2021032180 A1 WO2021032180 A1 WO 2021032180A1 CN 2020110404 W CN2020110404 W CN 2020110404W WO 2021032180 A1 WO2021032180 A1 WO 2021032180A1
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gene
aspergillus
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sgrna
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刘松
李岑
堵国成
陈坚
周景文
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江南大学
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Definitions

  • the invention relates to a visual screening method for multi-target editing of recombinant Aspergillus strains, and belongs to the technical field of genetic engineering.
  • Filamentous fungi have a long history of development in the traditional fermentation industry. Among them, Aspergillus is used in food fermentation to produce enzyme preparations and organic acids and other products. Commercially valuable Aspergillus species include Aspergillus niger and Aspergillus oryzae. However, the lack of efficient gene editing methods and rapid screening of recombinant strains are important factors that hinder the process of molecular transformation of industrial Aspergillus species.
  • the cells of Aspergillus are wrapped by hyphae, and the cell wall structure is more complicated than prokaryotic cells such as Escherichia coli and Bacillus subtilis. It is difficult to visually screen single colonies of Aspergillus.
  • a common strain visualization screening method can fusion expression of the target gene N-terminus with green fluorescent protein (GFP), and screen by fluorescence.
  • GFP green fluorescent protein
  • single colonies can be selected by bacterial green fluorescence deposition to screen recombinant bacteria, or single cells can be screened by fluorescence sorting by flow cytometry.
  • spore color can not only be used as a "visualization” feature to distinguish Aspergillus species, but also can be used to "visualize” recombinant Aspergillus strains by editing spore pigmentation related genes to change the host spore color. Filter markers. The color of mature spores of wild-type Aspergillus niger is mostly dark brown. Using CRISPR-Cas9 to simultaneously mutate spore color change related genes and target genes in Aspergillus may make the target gene editing visible.
  • the current gene knockout or blockade of filamentous fungi has disadvantages such as low homologous recombination efficiency and limited selection markers that can be used.
  • the first object of the present invention is to provide a method for visually screening gene editing strains in Aspergillus, using CRISPR-Cas9 gene editing technology to knock out genes (a) and (b) in Aspergillus at the same time; wherein, (a) is Genes that affect the color change of spores; (b) are genes whose Aspergillus phenotype remains unchanged before and after knockout.
  • the genes that affect the color change of spores include fwnA, pptA, and brnA.
  • the genes whose Aspergillus phenotype is unchanged before and after knockout include but are not limited to amyA, ammA, pepA, and kusA.
  • nucleotide sequence of fwnA is shown in SEQ ID NO. 24.
  • the Gene ID of pptA is 4985743
  • the Gene ID of brnA is 4987395.
  • the Aspergillus includes Aspergillus flavus, Aspergillus niger, Aspergillus fumigatus, Aspergillus versicolor, Aspergillus nidulans.
  • the host is Aspergillus niger.
  • the host is Aspergillus niger CCTCC M 2018881, which has been disclosed in the patent application document with the publication number CN110438018A.
  • the method is to construct a Cas9 expression plasmid and an sgRNA expression cassette, and transfer the Cas9 expression plasmid and sgRNA expression cassette into Aspergillus niger for co-expression.
  • the codon-optimized Cas9 gene sequence of Aspergillus niger is shown in SEQ ID NO.4.
  • the method is to construct an sgRNA expression cassette by using Pu3 or Pu6 promoter and the corresponding terminator Tu3 or Tu6.
  • the sgRNA is released using the endogenous tRNA of Aspergillus.
  • the endogenous tRNA includes tRNA Ala , tRNA Arg , tRNA Cys , tRNA Ile , tRNA Leu , tRNA Lys , tRNA Met , tRNA Phe , tRNA Ser , tRNA Thr , tRNA Val , tRNA Glu , tRNA Pro , tRNA Glu , tRNA Gln , tRNA Gly .
  • the first tRNA after the promoter is preferably tRNA Ala .
  • the first tRNA after the promoter is preferably tRNA Ala
  • the second tRNA is preferably tRNA Arg .
  • the first tRNA after the promoter is preferably tRNA Ala
  • the tRNA in the last sgRNA is preferably tRNA Arg .
  • the second object of the present invention is to provide a system for the visualization of gene knockout in Aspergillus, the system includes a gene encoding Cas9 protein, an sgRNA expression box and a selection marker; the sgRNA expression box contains changes that affect the color of spores The target sequence of the gene, and the target sequence of the gene that does not affect the phenotype of Aspergillus.
  • the sgRNA is released using the endogenous tRNA of Aspergillus.
  • the endogenous tRNA includes tRNA Ala , tRNA Arg , tRNA Cys , tRNA Ile , tRNA Leu , tRNA Lys , tRNA Met , tRNA Phe , tRNA Ser , tRNA Thr , tRNA Val , tRNA Glu , tRNA Pro , tRNA Glu , tRNA Gln , tRNA Gly .
  • the third objective of the present invention is to provide a method for improving the screening efficiency of Aspergillus gene editing, which is to use CRISPR-Cas9 gene editing technology to knock out genes (a) and (b) in Aspergillus at the same time; where (a) is The genes that affect the color change of spores; (b) are genes that do not change the phenotype of Aspergillus before and after knockout; the genes that affect the color change of spores include fwnA, pptA, and brnA; the genes that do not change the phenotype of Aspergillus before and after knockout include But not limited to amyA, ammA, pepA, kusA.
  • the present invention also protects the application of the method for visually screening gene editing strains in Aspergillus in Aspergillus gene editing.
  • the invention also protects the application of the system for visualizing gene knockout in Aspergillus in Aspergillus gene editing.
  • the invention also protects the application of the method for improving the screening efficiency of Aspergillus gene editing in Aspergillus gene editing.
  • the invention utilizes CRISPR-Cas9 to simultaneously shear spore color change related genes and target genes in Aspergillus, visualize target gene editing, and quickly and efficiently screen out Aspergillus niger multi-gene editing strains through spore phenotype.
  • CRISPR-Cas9 CRISPR-Cas9 to simultaneously shear spore color change related genes and target genes in Aspergillus, visualize target gene editing, and quickly and efficiently screen out Aspergillus niger multi-gene editing strains through spore phenotype.
  • the method of the present invention can also be commonly used in other Aspergillus, by combining the visualized gene of color change with the target gene to be knocked out, the target gene can be knocked out quickly and accurately.
  • Figure 1 is a map of the Aspergillus niger Cas9 expression plasmid pUC19-Cas9.
  • Figure 2 is a map of the Aspergillus niger sgRNA co-expression plasmid pUC19-sgRNA.
  • Figure 3 is a map of the co-expression plasmid pUC19-sgRNA-1/2/3 for the non-phenotypic gene sgRNA of Aspergillus niger and the visualized phenotypic gene sgRNA.
  • Figure 4 is a comparison diagram of the color of sporozoites transformed by visual gene editing of Aspergillus niger.
  • PDA medium 200g potatoes, 20g glucose, 15-20g agar, add water to dilute to 1L.
  • LB medium 10g peptone, 5g yeast powder, 10g NaCl, add water to make the volume to 1L.
  • STC buffer 1.2M sorbitol, 50mM CaCl 2, 10mM Tris, pH 7.5-8.
  • PEG buffer 25% PEG 6000, 50 mM CaCl 2 , 10 mM Tris, pH 7.5-8.
  • Primer name sequence pptA-F TAACCCAACCCCTCACTTCACCT
  • Example 1 Using CRISPR-Cas9 to construct a visualized single-gene editing recombinant strain of Aspergillus niger
  • the CRISPR-Cas9 system includes a gene encoding Cas9 protein (Cas9 gene), sgRNA and selection markers.
  • PglaA nucleotide sequence shown in SEQ ID NO. 7
  • Ptef1 nucleotide sequence shown in SEQ ID NO.6
  • Cas9 protein nucleotide sequence shown in SEQ ID NO. 1.
  • Plasmid pUC19-Cas9 Plasmid pUC19-Cas9 (see Figure 1 for the plasmid map). Among them, the N-terminal or C-terminal of the gene encoding Cas9 protein (shown in SEQ ID NO. 1) is added with a nuclear localization signal NLS sequence (CCCAAGAAGAAGCGCAAGGTC).
  • Pu3 promoter Use Pu3 promoter, target gene protospacers sequence (see Table 3), gRNA backbone sequence (nucleotide sequence shown in SEQ ID NO. 8), and terminator Tu3 to construct an sgRNA expression cassette.
  • Target gene Target gene sequence fwnA AGTGGGATCTCAAGAACTAC pptA GGCGGGTGTCGATGTACCAC brnA ACCATGCCAATGGATTCCGG kusA CGAGCACTGGTAGATGATGA
  • Selective markers include hygromycin B (hygB), orotidine-5'-phosphate dehydroxylase, acetamidase, which are commonly used in Aspergillus, and filamentous fungal markers with similar functions.
  • the hygromycin resistance gene in the recombinant plasmid is obtained from the plasmid PAN7-1.
  • the expression box primer Hyg-F/R is shown in Table 4. If you want to choose other resistance, you can replace hyg in the expression box for construction.
  • Hyg-F GAATTCCCTTGTATCTCTACACACAG Hyg-R TGAAGAACGAATACCGCGACATCCAACCCATC
  • Cultivate at 30°C for 5-7 days extract the genome of the transformant, and verify the editing of the targeted site of the target gene by PCR. Pick a positive single colony to transfer the plate, and transfer each single colony to the plate (that is, pick a single colony to culture in a new medium) three times.
  • Example 2 Screening of Aspergillus niger visualized double-targeted gene editing recombinant strains
  • step (1) of Example 1 For specific implementation, see step (1) of Example 1.
  • the nucleotide sequence is BsaI with the Pu3 promoter sequence shown in SEQ ID NO.2.
  • GAGACC site mutation is ACCCAC
  • target gene protospacers sequence pptA, fwnA, brnA see Table 1
  • amyA sequence is shown in Table 5
  • use tRNA Gly to release sgRNA expression box gRNA backbone sequence (nucleotide sequence such as SEQ ID No. 8), terminator Tu3 (nucleotide sequence shown in SEQ ID NO. 4) to construct an sgRNA expression cassette.
  • Target gene Target gene sequence
  • Selective markers include hygromycin B (hygB), orotidine-5'-phosphate dehydroxylase, acetamidase, which are commonly used in Aspergillus, and filamentous fungal markers with similar functions.
  • the hygromycin resistance gene in the recombinant plasmid is obtained from the plasmid PAN7-1.
  • the expression box primer Hyg-F/R is shown in Table 3. If you want to select other resistances, you can replace hygB in the expression box for construction.
  • the sgRNA expression box includes a visualized gene sgRNA and a non-phenotypic gene sgRNA.
  • the visualized gene sgRNA is pptA-sgRNA, fwnA-sgRNA or brnA-sgRNA, and the other non-phenotypic gene sgRNA is amyA-sgRNA (plasmid map See Figure 3).
  • Example 2 Using the same method as in Example 2, a single-copy gene-editing recombinant strain of the amyA gene was constructed. Using the same method, the recombinant strain was cultivated to grow a single colony. The single colony was picked and its gene editing efficiency was calculated. The results showed that The gene editing efficiency is 5%.
  • Example 3 Aspergillus niger visualized multi-targeted gene editing recombinant strain screening
  • Example 2 Specific implementation methods refer to Example 2, the difference is that the tRNA Gly after the promoter in Example 2 is replaced with tRNA Ala , and the second tRNA is replaced with tRNA Phe ;
  • tRNA Ala and tRNA Phe to connect to different sgRNAs.
  • One visualized gene sgRNA expression box is fwnA-sgRNA expression box, and the other non-phenotypic gene sgRNA expression box is amyA-sgRNA expression box (see Figure 3 for the plasmid map).
  • the fwnA and amyA double-gene editing recombinant strains were constructed using tRNA Gly .
  • the recombinant strains were cultivated to grow a single colony, and the single colony was picked and its gene editing efficiency was calculated. The results showed , 6% are white mutants, and their amyA gene editing efficiency is 36%.
  • Example 4 Aspergillus niger visualized multi-phenotype screening of gene-edited recombinant strains
  • Example 2 Specific implementation methods refer to Example 2. The difference is that the amyA gene in Example 2 is replaced with pepA or ammA (see Table 6 for the protospacers sequence list), and the tRNA Gly after the promoter in Example 2 is replaced with tRNA Ala , Change the second tRNA to tRNA Arg ;
  • Target gene Target gene sequence pepA CGGTGTCAAAGTCCAGATGG ammA CTGCCCCAGGATACTGCTGA
  • tRNA Ala and tRNA Arg to release different sgRNAs.
  • One of the visualized gene sgRNA expression box is fwnA-sgRNA, and the other non-phenotypic gene sgRNA expression box is pepA-sgRNA or ammA-sgRNA expression box (see Figure 3B for the plasmid map).
  • the Cas9 expression plasmid and sgRNA expression cassette were transferred into the host and obtained after sequencing.
  • Example 5 Visualized multi-phenotype screening of gene-edited recombinant strains of Aspergillus niger
  • Example 2 Specific implementation methods refer to Example 2, the difference is that the amyA gene in Example 2 is replaced with ammA, and the third amyA-sgRNA (see Table 7 for the protospacers sequence list) is connected with tRNA Arg , and the amyA gene in Example 2 The tRNA Gly after the promoter is replaced with tRNA Ala , and the second is replaced with tRNA Phe .
  • Target gene Target gene sequence ammA CTGCCCCAGGATACTGCTGA amyA TCTCTTCGGCCCTTCATGAG
  • tRNA Ala , tRNA Arg and tRNA Phe Use tRNA Ala , tRNA Arg and tRNA Phe to release different sgRNAs.
  • One of the visualized genes, sgRNA is fwnA-sgRNA, and the other two non-phenotypic genes, sgRNA, are pepA-sgRNA and ammA-sgRNA.
  • the Cas9 expression plasmid and sgRNA expression cassette were transferred to the host, and obtained after sequencing
  • Example 6 Visualized multi-phenotype screening of gene-edited recombinant strains of Aspergillus niger
  • Example 2 Specific implementation methods refer to Example 2, the difference is that the amyA gene in Example 2 is replaced with ammA, the tRNA Gly after the promoter in Example 2 is replaced with tRNA Ala , and the second tRNA Gly is replaced with tRNA Phe ; At the same time, an sgRNA expression box is added and released using tRNA Ile , and the Pu6 promoter (nucleotide sequence shown in SEQ ID NO. 3) and Tu6 terminator (nucleotide sequence shown in SEQ ID NO. 5) are used to express tRNA.
  • the Pu6 promoter nucleotide sequence shown in SEQ ID NO. 3
  • Tu6 terminator nucleotide sequence shown in SEQ ID NO. 5
  • tRNA Ala , tRNA Arg and tRNA Phe to release different sgRNAs.
  • One of the visualized gene sgRNA expression boxes is fwnA-sgRNA, and the other two non-phenotypic genes sgRNA are ammA-sgRNA and amyA-sgRNA.
  • the Cas9 expression plasmid and sgRNA expression cassette were transferred into the host and verified by sequencing.

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Abstract

提供了一种多靶点编辑重组曲霉菌株的可视化筛选方法，所述方法利用CRISPR-Cas9在曲霉中同时剪切孢子颜色变化相关基因与目的基因，使目的基因编辑可视化，可通过孢子表型快速高效筛选出黑曲霉多基因编辑菌株。通过可视化基因与无表型变化基因的不同组合，以实现多基因编辑菌株快速筛选和多个可视化基因同时筛选，可减少工业菌株中抗性基因的使用。

Description

一种多靶点编辑重组曲霉菌株的可视化筛选方法 技术领域
本发明涉及一种多靶点编辑重组曲霉菌株的可视化筛选方法，属于基因工程技术领域。
背景技术
丝状真菌在传统发酵行业中有悠久的发展历史，其中曲霉在工业中应用于食品发酵，生产酶制剂及其有机酸等产品，具有商业价值的曲霉菌种包括黑曲霉和米曲霉等。但是缺乏高效的基因编辑方法和快速筛选重组菌株的方法是阻碍着工业曲霉菌种分子改造进程的重要因素。
曲霉的细胞由菌丝包裹，细胞壁结构与大肠杆菌、枯草芽孢杆菌等原核细胞相比更复杂，曲霉可视化筛选单菌落难以实现。常见的菌株可视化筛选方法可将目的基因N端与绿色荧光蛋白(GFP)融合表达，通过荧光进行筛选。在大肠杆菌、枯草芽孢杆菌等原核细胞中可通过菌体绿色荧光沉积挑选单菌落进行筛选重组菌，或通过流式细胞仪的荧光分选筛分单细胞。但是曲霉中表达GFP需利用荧光显微镜进行观察菌体的荧光情况，难以达到分选的目的。而通过培养基的显色反应筛选曲霉的重组菌株不仅有使用的局限性，而且操作繁琐、效果不精确。所以需要在曲霉中应用更合理的进行“可视化”操作用于筛选重组菌株。
曲霉种类的不同，其孢子颜色有区别，孢子颜色的不同不仅可作为区分曲霉种类的“可视化”特征，也可以通过编辑孢子色素沉淀相关基因，改变宿主孢子颜色，用于曲霉重组菌株“可视化”的筛选标记。野生型黑曲霉成熟孢子颜色多为黑褐色，利用CRISPR-Cas9在曲霉中同时突变孢子颜色变化相关基因与目的基因，可能会使目的基因编辑可视化。但是，目前对丝状真菌进行基因敲除或阻断存在着同源重组效率低、可使用的筛选标记有限等缺点。
因此，提供一种无抗性可视化的曲霉基因重组菌株的筛选方法，有利于工业中对曲霉基因编辑菌株的筛选和利用。
发明内容
本发明的第一个目的是提供一种可视化筛选曲霉中基因编辑菌株的方法，利用CRISPR-Cas9基因编辑技术，同时敲除曲霉中的基因(a)和(b)；其中，(a)为影响孢子颜色变化的基因；(b)为敲除前后曲霉表型不变的基因。
在本发明的一种实施方式中，所述影响孢子颜色变化的基因包括fwnA、pptA、brnA。
在本发明的一种实施方式中，所述敲除前后曲霉表型不变的基因包括但不限于amyA、ammA、pepA、kusA。
在本发明的一种实施方式中，所述fwnA的核苷酸序列如SEQ ID NO.24所示。
在本发明的一种实施方式中，所述pptA的Gene ID:4985743，所述brnA的Gene ID:4987395。
在本发明的一种实施方式中，所述amyA的Gene ID:4980947，所述ammA的Gene ID:4984565，所述pepA的Gene ID:4987328，所述kusA的Gene ID:4987871。
在本发明的一种实施方式中，所述曲霉包括黄曲霉、黑曲霉、烟曲霉、杂色曲霉、构巢曲霉。
在本发明的一种实施方式中，宿主为黑曲霉(Aspergillus niger)。
在本发明的一种实施方式中，宿主为Aspergillus niger CCTCC M 2018881，已公开于公开号为CN110438018A的专利申请文件中。
在本发明的一种实施方式中，所述方法是通过构建Cas9表达质粒与sgRNA表达框，将Cas9表达质粒与sgRNA表达框转入黑曲霉中共表达。
在本发明的一种实施方式中，黑曲霉密码子优化的Cas9基因序列如SEQ ID NO.4所示。
在本发明的一种实施方式中，所述方法是利用Pu3或Pu6启动子以及相对应的终止子Tu3或Tu6构建sgRNA表达框。
在本发明的一种实施方式中，sgRNA利用曲霉内源的tRNA释放。
在本发明的一种实施方式中，所述内源tRNA包括tRNA Ala，tRNA Arg，tRNA Cys，tRNA Ile，tRNA Leu，tRNA Lys，tRNA Met，tRNA Phe，tRNA Ser，tRNA Thr，tRNA Val，tRNA Glu，tRNA Pro，tRNA Glu，tRNA Gln，tRNA Gly。
在本发明的一种实施方式中，当对单个基因编辑时，启动子后的第一个tRNA优选为tRNA Ala。
在本发明的一种实施方式中，当对两个基因编辑时，启动子后的第一个tRNA优选为tRNA Ala，第二个tRNA优选为tRNA Arg。
在本发明的一种实施方式中，当多个基因编辑时，启动子后的第一个tRNA优选为tRNA Ala，最后一个sgRNA中的tRNA优选为tRNA Arg。
本发明的第二个目的是提供一种用于曲霉中基因敲除可视化的系统，所述系统包括编码Cas9蛋白的基因、sgRNA表达框以及筛选标记；所述sgRNA表达框上含有影响孢子颜色变化的基因的靶序列，和不影响曲霉表型的基因的靶序列。
在本发明的一种实施方式中，sgRNA利用曲霉内源的tRNA释放。
在本发明的一种实施方式中，所述内源tRNA包括tRNA Ala，tRNA Arg，tRNA Cys，tRNA Ile， tRNA Leu，tRNA Lys，tRNA Met，tRNA Phe，tRNA Ser，tRNA Thr，tRNA Val，tRNA Glu，tRNA Pro，tRNA Glu，tRNA Gln，tRNA Gly。
本发明的第三个目的是提供一种提高曲霉基因编辑筛选效率的方法，是利用CRISPR-Cas9基因编辑技术，同时敲除曲霉中的基因(a)和(b)；其中，(a)为影响孢子颜色变化的基因；(b)为敲除前后曲霉表型不变的基因；所述影响孢子颜色变化的基因包括fwnA、pptA、brnA；所述敲除前后曲霉表型不变的基因包括但不限于amyA、ammA、pepA、kusA。
本发明还保护所述可视化筛选曲霉中基因编辑菌株的方法在曲霉基因编辑中的应用。
本发明还保护所述用于曲霉中基因敲除可视化的系统在曲霉基因编辑中的应用。
本发明还保护提高曲霉基因编辑筛选效率的方法在曲霉基因编辑中的应用。
本发明的有益效果：
本发明利用CRISPR-Cas9在曲霉中同时剪切孢子颜色变化相关基因与目的基因，使目的基因编辑可视化，可通过孢子表型快速高效筛选出黑曲霉多基因编辑菌株。通过可视化基因与无表型变化基因的不同组合，以实现多基因编辑菌株快速筛选和多个可视化基因同时筛选，减少工业菌株中抗性基因的使用。本发明的这种方法也可通用于其他的曲霉中，通过颜色变化的可视化基因与需要敲除的目的基因结合，可实现目的基因快速、准确的敲除。
附图说明
图1为黑曲霉Cas9表达质粒pUC19-Cas9图谱。
图2为黑曲霉sgRNA共表达质粒pUC19-sgRNA图谱。
图3为黑曲霉无表型基因sgRNA与可视化表型基因sgRNA共表达质粒pUC19-sgRNA-1/2/3的图谱。
图4为黑曲霉可视化基因编辑转化子孢子颜色对比图。
具体实施方式
(一)培养基
PDA培养基：土豆200g，葡萄糖20g，琼脂15-20g，加水定容至1L。
LB培养基：10g蛋白胨，5g酵母粉，10g NaCl，加水定容至1L。
(二)试剂配方
STC缓冲液：1.2M山梨糖醇，50mM CaCl 2，10mM Tris，pH 7.5-8。
PEG缓冲液：25％PEG 6000，50mM CaCl 2，10mM Tris，pH 7.5-8。
表1 靶向位点验证PCR引物
引物名 序列
pptA-F TAACCCAACCCCTCACTTCACCT
pptA-R TGGAGACGTATTCCAGGAAGGCT
brnA-F TGTTTGGATCTGATGCCGAGGC
brnA-R GGCTTGACGCTGATCTTGGT
fwnA-F GACCAATGACAAGACTCTGTGGGT
fwnA-R TCTTCTTCCCCTCCGCAGTGAC
kusA-F TCAAATGCGCCTATCACTTCATGC
kusA-R CCGCCGGTTAATACGATGTCATAT
amyA-F GCAGGGCATCATCGACAAGGT
amyA-R GGTGGTATCGAGATCAGGCAAGG
pepA-F TCCATCATGACGGCTGCCA
pepA-R CGAACTCGGAGCTGATCTTGC
ammA-F GACGCTGTTCTGTCGCTTTGT
ammA-R GATCAGGCAGTATGGGTGGAAGT
表2 tRNA序列
Figure PCTCN2020110404-appb-000001
Figure PCTCN2020110404-appb-000002
实施例1：利用CRISPR-Cas9构建黑曲霉可视化单基因编辑重组菌株
CRISPR-Cas9系统包括编码Cas9蛋白的基因(Cas9基因)、sgRNA以及筛选标记。
(1)Cas9表达载体的构建
利用曲霉启动子PglaA(核苷酸序列如SEQ ID NO.7所示)或Ptef1(核苷酸序列如SEQ ID NO.6所示)等曲霉强启动子表达Cas9蛋白(核苷酸序列如SEQ ID NO.1所示)。
利用Vazyme的
Figure PCTCN2020110404-appb-000003
II One Step Cloning Kit，以pUC19为载体骨架，将曲霉启动子序列、编码Cas9蛋白的基因序列、抗性基因与AMA1(GenBank:X78051.1)序列分两次进行同源重组合成，得到Cas9表达质粒pUC19-Cas9(质粒图谱见图1)。其中，编码Cas9蛋白的基因(如SEQ ID NO.1所示)的N端或C端添加核定位信号NLS序列(CCCAAGAAGAAGCGCAAGGTC)。
(2)sgRNA表达框的构建
利用Pu3启动子、目标基因protospacers序列(见表3)、gRNA backbone序列(核苷酸序列如SEQ ID NO.8所示)、终止子Tu3构建sgRNA表达框。
表3 目标基因protospacers序列表
目标基因 目标基因序列
fwnA AGTGGGATCTCAAGAACTAC
pptA GGCGGGTGTCGATGTACCAC
brnA ACCATGCCAATGGATTCCGG
kusA CGAGCACTGGTAGATGATGA
选择标记包括曲霉中常用的潮霉素B(hygB)、乳清酸核苷-5’-磷酸脱羟酶、乙酰胺酶等具有类似功效的丝状真菌标记。重组质粒中的潮霉素抗性基因由质粒PAN7-1中获得，表达框引物Hyg-F/R见表4，若要选择其他抗性可替换表达框中的hyg进行构建。
表4 引物表
引物名称 引物序列
Hyg-F GAATTCCCTTGTATCTCTACACACAG
Hyg-R TGAAGAACGAATACCGCGACATCCAACCCATC
利用Vazyme的
Figure PCTCN2020110404-appb-000004
II One Step Cloning Kit，以pUC19为载体骨架，与sgRNA表达框重组，构建sgRNA表达质粒(质粒图谱见图2)。
(3)Cas9表达质粒和sgRNA表达框的转化
采用原生质体转化方法将Cas9表达质粒和sgRNA表达框转入宿主：
在PDA培养基中过夜培养黑曲霉菌丝，收集菌丝体，用生理盐水清洗菌丝体三遍；用Lysozyme酶解3h，用四层擦镜纸过滤后制备原生质体；4℃，1000rpm离心收集原生质体，用预冷的STC洗涤原生质体2-3次；取制备好的原生质体100μL加入10μL Cas9表达质粒和10μL sgRNA表达框混匀，加入2mL PEG 6000，在培养基中加入相应抗性进行筛选。30℃培养5-7天，提取转化子的基因组，PCR验证目标基因靶向位点的编辑情况。挑取阳性单菌落转板，每个单菌落转板(即挑取单菌落至新的培养基培养)三次。
以Aspergillus niger CCTCC M 2018881出发菌株(菌株已公开于公开号为CN110438018A的专利申请文件中)的孢子颜色作为对照，观察转化后黑曲霉孢子颜色表型的变化，破坏fwnA或pptA后，孢子颜色由褐色变成白色，破坏brnA后，孢子颜色由褐色变成橄榄色，见图4。破坏无表型变化基因kusA时，菌体表型无变化。孢子颜色变化转化株的基因编辑效率显著提高，其中，颜色突变菌株占14％，颜色突变株中brnA的基因编辑效率为30％，fwnA或pptA的编辑效率达100％；而kusA的编辑效率为4.16％。
实施例2：黑曲霉可视化双靶向基因编辑重组菌株筛选
(1)Cas9表达载体的构建
具体实施方式参见实施例1步骤(1)。
(2)sgRNA表达框的构建
利用Pu3突变启动子(为方便多个sgRNA组装，将与之相关的BsaI位点进行突变，方便后续组装，具体为将核苷酸序列如SEQ ID NO.2所示的Pu3启动子序列的BsaI(GAGACC)位点突变为ACCCAC)、目标基因protospacers序列pptA、fwnA、brnA(见表1)、amyA序列见表5，利用tRNA Gly释放sgRNA表达框、gRNA backbone序列(核苷酸序列如SEQ ID NO.8所示)、终止子Tu3(核苷酸序列如SEQ ID NO.4所示)构建sgRNA表达框。
表5 目标基因protospacers序列表
目标基因 目标基因序列
amyA TCTCTTCGGCCCTTCATGAG
选择标记包括曲霉中常用的潮霉素B(hygB)、乳清酸核苷-5’-磷酸脱羟酶、乙酰胺酶等具有类似功效的丝状真菌标记。重组质粒中的潮霉素抗性基因由质粒PAN7-1中获得，表达框引物Hyg-F/R见表3，若要选择其他抗性可替换表达框中的hygB进行构建。
利用Vazyme的
Figure PCTCN2020110404-appb-000005
II One Step Cloning Kit，以pUC19为载体骨架，将曲霉启动子序列、编码Cas9蛋白的基因序列、抗性基因与AMA1序列分两次同源重组至pUC19，得到Cas9表达质粒pUC19-Cas9(质粒图谱见图1)。其中，编码Cas9蛋白的基因(如SEQ ID NO.1所示)的N端或C端添加核定位信号NLS序列(CCCAAGAAGAAGCGCAAGGTC)。
利用Vazyme的
Figure PCTCN2020110404-appb-000006
II One Step Cloning Kit，以pUC19为载体骨架，将sgRNA表达框重组至pUC19，构建包含两个protospacers序列的sgRNA表达框的双sgRNA表达质粒PUC19-sgRNA-1质粒，利用tRNA Gly进行不同sgRNA的释放。其中sgRNA表达框中包括一个可视化基因的sgRNA和一个无表型基因的sgRNA，可视化基因sgRNA为pptA-sgRNA、fwnA-sgRNA或brnA-sgRNA，另一个无表型基因sgRNA为amyA-sgRNA(质粒图谱见图3)。
(3)Cas9表达质粒和sgRNA表达框的转化
采用原生质体转化方法将Cas9表达质粒、双sgRNA表达框(包括pptA-sgRNA和amyA-sgRNA、或fwnA-sgRNA和amyA-sgRNA、或brnA-sgRNA和amyA-sgRNA中的任意一种)转入宿主，sgRNA直接与tRNA连接，构建得到含双sgRNA表达框的菌株。
在PDA培养基中过夜培养黑曲霉菌丝，收集菌丝体，生理盐水清洗三遍；用Lysozyme酶解3h，四层擦镜纸过滤后制备原生质体；4℃，1000rpm离心收集原生质体，用预冷的STC洗涤原生质体2-3次；取制备好的原生质体100μL加入10μL Cas9表达质粒和10μL sgRNA表达框混匀，加入2mL PEG 6000，在培养基中加入相应抗性进行筛选。30℃培养5-7天，挑白色孢子单菌落转化子进行测序验证。
以Aspergillus niger CCTCC M 2018881出发菌株的孢子颜色作为对照，观察转化后黑曲霉孢子颜色表型的变化，敲除fwnA后，孢子颜色由黑色变成白色，平板上共有4％的单菌落显示出白色表型，直接挑选这些白色单菌落，结果显示，在白色单菌落中，amyA基因双拷贝破坏的纯合转化子占25％，提高了多基因编辑转化子的阳性挑选率。对amyA基因双拷贝进行编辑时不需要使用新的抗性标记，有利于工业生产。
对比例1
利用与实施例2相同的方法，构建amyA基因的单拷贝基因编辑重组菌株，利用相同的方法，培养重组菌菌株至长出单菌落，挑取单菌落并计算其基因编辑效率，结果显示，其基因编辑效率为5％。
实施例3：黑曲霉可视化多靶向基因编辑重组菌株筛选
具体实施方式参见实施例2，区别在于，将实施例2中的启动子后的tRNA Gly替换为tRNA Ala，第二个tRNA换为tRNA Phe；
利用tRNA Ala和tRNA Phe与不同sgRNA相连。一个可视化基因sgRNA表达框为fwnA-sgRNA表达框，另一个无表型基因sgRNA表达框为amyA-sgRNA表达框(质粒图谱见图3)。
以Aspergillus niger CCTCC M 2018881出发菌株的孢子颜色作为对照，观察转化后黑曲霉孢子颜色表型的变化，敲除fwnA后，孢子颜色由黑色变成白色，36％为白色表型转化子，直接挑选这些白色单菌落提高了多基因编辑转化子的阳性挑选率并节约了菌株纯化的时间，其中amyA基因编辑效率为90％。对amyA基因进行编辑时不需要使用新的抗性标记，有利于工业生产。
对比例2
利用与实施例3相同的方法，利用tRNA Gly构建fwnA和amyA双基因编辑重组菌株，利用相同的方法，培养重组菌菌株至长出单菌落，挑取单菌落并计算其基因编辑效率，结果显示，6％为白色突变体，其个amyA基因编辑效率为36％。
对比例3
利用与实施例3相同的方法，利用tRNA Ala构建fwnA和amyA基因编辑重组菌株，利用相同的方法，培养重组菌菌株至长出单菌落，挑取单菌落并计算其基因编辑效率，结果显示，37％为白色突变体，其个amyA基因编辑效率为70％。
对比例4
利用与实施例3相同的方法，利用tRNA phe构建fwnA和amyA基因的单拷贝基因编辑重组菌株，利用相同的方法，培养重组菌菌株至长出单菌落，挑取单菌落并计算其基因编辑效率，结果显示，15％为白色突变体，其个amyA基因编辑效率为80％。
实施例4：黑曲霉可视化多表型筛选基因编辑重组菌株
具体实施方式参见实施例2，区别在于，将实施例2中的amyA基因替换为pepA或ammA(protospacers序列表见表6)，将实施例2中的启动子后的tRNA Gly替换为tRNA Ala，第二个tRNA换为tRNA Arg；
表6 目标基因protospacers序列表
目标基因 目标基因序列
pepA CGGTGTCAAAGTCCAGATGG
ammA CTGCCCCAGGATACTGCTGA
利用tRNA Ala和tRNA Arg进行不同sgRNA的释放。其中一个可视化基因sgRNA表达框为fwnA-sgRNA，另一个无表型基因sgRNA表达框为pepA-sgRNA或ammA-sgRNA表达框(质粒图谱见图3B)。
将Cas9表达质粒和sgRNA表达框转入宿主，测序后得到。
以Aspergillus niger CCTCC M 2018881出发菌株的孢子颜色作为对照，观察转化后黑曲霉孢子颜色表型的变化，敲除fwnA后孢子颜色由黑色变成白色，挑取白色菌落，计算基因编辑效率，pepA或ammA基因的编辑效率分别为75％和80％。有利于进行目标基因活性protospacers序列的黑曲霉体内筛选，节约分子操作时间和成本。
实施例5：黑曲霉可视化多表型筛选基因编辑重组菌株
具体实施方式参见实施例2，区别在于，将实施例2中的amyA基因替换为ammA，同时增加第三个amyA-sgRNA(protospacers序列表见表7)用tRNA Arg连接，将实施例2中的启动子后的tRNA Gly替换为tRNA Ala，第二个替换为tRNA Phe。
表7 目标基因protospacers序列表
目标基因 目标基因序列
ammA CTGCCCCAGGATACTGCTGA
amyA TCTCTTCGGCCCTTCATGAG
利用tRNA Ala，tRNA Arg和tRNA Phe进行不同sgRNA的释放。其中一个可视化基因sgRNA为fwnA-sgRNA，另两个无表型基因sgRNA分别为pepA-sgRNA和ammA-sgRNA。
将Cas9表达质粒和sgRNA表达框转入宿主，测序后得到
以Aspergillus niger CCTCC M 2018881出发菌株的孢子颜色作为对照，观察转化后黑曲霉孢子颜色表型的变化，敲除fwnA后孢子颜色由黑色变成白色。优化后的三基因敲除白色转化子中，ammA基因的编辑效率为50％，amyA基因的编辑效率为100％，双基因共编辑的效率为50％。有利于进行多目标基因活性protospacers序列的黑曲霉体内筛选，节约分子操作时间和成本。
实施例6：黑曲霉可视化多表型筛选基因编辑重组菌株
具体实施方式参见实施例2，区别在于，将实施例2中的amyA基因替换为ammA，将实 施例2中的启动子后的tRNA Gly替换为tRNA Ala，第二个tRNA Gly替换为tRNA Phe；同时增加一个sgRNA表达框利用tRNA Ile进行释放，利用Pu6启动子(核苷酸序列如SEQ ID NO.3所示)和Tu6终止子(核苷酸序列如SEQ ID NO.5所示)表达tRNA Ile和ammA-sgRNA
利用tRNA Ala，tRNA Arg和tRNA Phe进行不同sgRNA的释放。其中一个可视化基因sgRNA表达框为fwnA-sgRNA，另两个无表型基因sgRNA为ammA-sgRNA和amyA-sgRNA。
将Cas9表达质粒和sgRNA表达框转入宿主，并进行测序验证。
以Aspergillus niger CCTCC M 2018881出发菌株的孢子颜色作为对照，观察转化后黑曲霉孢子颜色表型的变化，敲除fwnA后孢子颜色由黑色变成白色。优化后的三基因敲除白色转化子中，ammA基因的编辑效率为69％，amyA基因的编辑效率为100％，两个基因共同编辑的效率为69％。有利于进行多目标基因活性protospacers序列的黑曲霉体内筛选，节约分子操作时间和成本。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (19)

  1. 一种可视化筛选曲霉中基因编辑菌株的方法，其特征在于，利用CRISPR-Cas9基因编辑技术，同时敲除曲霉中的基因(a)和(b)；其中，(a)为影响孢子颜色变化的基因；(b)为敲除前后曲霉表型不变的基因；所述影响孢子颜色变化的基因为fwnA，fwnA的核苷酸序列如SEQ ID NO.9所示；所述敲除前后曲霉表型不变的基因为amyA和/或ammA，所述amyA的Gene ID:4980947，所述ammA的Gene ID:4984565。
  2. 如权利要求1所述的方法，其特征在于，所述影响孢子颜色变化的基因包括fwnA、pptA、brnA。
  3. 所述敲除前后曲霉表型不变的基因包括但不限于amyA、ammA、pepA、kusA。
  4. 如权利要求2所述的方法，其特征在于，所述fwnA的核苷酸序列如SEQ ID NO.24所示。
  5. 如权利要求2所述的方法，其特征在于，所述pptA的Gene ID:4985743，所述brnA的Gene ID:4987395。
  6. 如权利要求3所述的方法，其特征在于，所述amyA的Gene ID:4980947，所述ammA的Gene ID:4984565。
  7. 如权利要求3所述的方法，其特征在于，所述pepA的Gene ID:4987328，所述kusA的Gene ID:4987871。
  8. 如权利要求1所述的方法，其特征在于，所述曲霉包括黄曲霉、黑曲霉、烟曲霉、杂色曲霉、构巢曲霉。
  9. 如权利要求1所述的方法，其特征在于，sgRNA利用曲霉内源的tRNA释放；所述内源tRNA包括tRNA Ala，tRNA Arg，tRNA Cys，tRNA Ile，tRNA Leu，tRNA Lys，tRNA Met，tRNA Phe，tRNA Ser，tRNA Thr，tRNA Val，tRNA Glu，tRNA Pro，tRNA Glu，tRNA Gln，tRNA Gly。
  10. 如权利要求9所述的方法，其特征在于，当对单个基因编辑时，启动子后的第一个tRNA为tRNA Ala。
  11. 如权利要求9所述的方法，其特征在于，当对两个基因编辑时，启动子后的第一个tRNA为tRNA Ala，第二个tRNA为tRNA Arg。
  12. 如权利要求9所述的方法，其特征在于，当多个基因编辑时，启动子后的第一个tRNA为tRNA Ala，最后一个sgRNA中的tRNA为tRNA Arg。
  13. 一种用于曲霉中基因敲除可视化的系统，其特征在于，所述系统包括编码Cas9蛋白的基因、sgRNA表达框以及筛选标记；所述sgRNA表达框上含有影响孢子颜色变化的基因的靶序列，和不影响曲霉表型的基因的靶序列。
  14. 如权利要求13所述的系统，其特征在于，sgRNA利用曲霉内源的tRNA释放；所述 内源tRNA包括tRNA Ala，tRNA Arg，tRNA Cys，tRNA Ile，tRNA Leu，tRNA Lys，tRNA Met，tRNA Phe，tRNA Ser，tRNA Thr，tRNA Val，tRNA Glu，tRNA Pro，tRNA Glu，tRNA Gln，tRNA Gly。
  15. 如权利要求14所述的系统，其特征在于，当对单个基因编辑时，启动子后的第一个tRNA为tRNA Ala。
  16. 如权利要求14所述的系统，其特征在于，当对两个基因编辑时，启动子后的第一个tRNA为tRNA Ala，第二个tRNA为tRNA Arg。
  17. 如权利要求14所述的系统，其特征在于，当多个基因编辑时，启动子后的第一个tRNA为tRNA Ala，最后一个sgRNA中的tRNA为tRNA Arg。
  18. 一种提高曲霉基因编辑筛选效率的方法，其特征在于，是利用CRISPR-Cas9基因编辑技术，同时敲除曲霉中的基因(a)和(b)；
    其中，(a)为影响孢子颜色变化的基因；(b)为敲除前后曲霉表型不变的基因，所述影响孢子颜色变化的基因包括fwnA、pptA、brnA，所述敲除前后曲霉表型不变的基因包括但不限于amyA、ammA、pepA、kusA。
  19. 权利要求1～12任一所述方法，或权利要求13～17任一所述系统，或权利要求18所述方法在曲霉基因编辑中的应用。
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