阅读说明：本技术 一种基于crispr技术定向遗传改造米曲霉基因的方法 (Method for directionally genetically modifying aspergillus oryzae gene based on CRISPR technology ) 是由 张哲� 曾斌 龙传南 范俊侠 于 2019-09-25 设计创作，主要内容包括：本发明提供了利用一种可以在Aspergillus属真菌内进行自主复制的穿梭载体,并通过组成型启动子PgpdA启动表达cas9,定向遗传改造米曲霉基因的方法,该方法消除了载体插入位点和培养基组分对cas9表达的影响,提高了基因编辑效率,并且能够产生无标记(Marker-Free)的米曲霉工程菌,免除对转基因的担忧,提高转基因改造米曲霉的实用性。(The invention provides a method for directionally and genetically modifying an Aspergillus oryzae gene by utilizing a shuttle vector which can autonomously replicate in Aspergillus fungi and starting expression of cas9 through a constitutive promoter PgpdA, which eliminates the influence of a vector insertion site and culture medium components on cas9 expression, improves gene editing efficiency, can generate Marker-Free (Marker-Free) Aspergillus oryzae engineering bacteria, avoids the worry of transgenosis and improves the practicability of transgenic modified Aspergillus oryzae.)

1. A method for directionally genetically modifying an Aspergillus oryzae gene based on a CRISPR technology is characterized by comprising the following steps:

1) artificially synthesizing a cas9 gene by taking a cas9 sequence as reference, wherein the nucleotide sequence of the cas9 gene is a DNA molecule shown as a sequence 1; the artificially synthesized cas9 gene is used as a template, and a DNA fragment with a sequence of SEQ ID NO. 1 and a sequence of SEQ ID NO. 2 is used as a primer, a cas9 DNA molecule obtained by PCR amplification is recombined and connected to a vector pEX1, wherein the recombinant vector is a recombinant vector pEX1-cas9 obtained by replacing DNA between XhoI and BamHI recognition sequences of pEX1 with DNA molecule shown in a sequence 1, and the pEX1-cas9 recombinant vector contains a cas9 protein expression cassette shown in a sequence 2 and composed of three parts, namely PgpdA-cas9-TtrpC, of a gene promoter PgpdA of aspergillus nidulans, a cas9 gene shown in the sequence 1 and a trpC gene terminator;

2) the cas9 protein expression cassette obtained by PCR amplification is recombined and connected to the vector pPTR II by taking the cas9 protein expression cassette PgpdA-cas9-TtrpC as a template and taking DNA fragments with sequences SEQ ID NO 3 and SEQ ID NO 4 as primers. The recombinant vector is obtained by inserting a PgpdA-cas9-TtrpC expression cassette into a HindIII multiple cloning site of a vector pPTR II to obtain a recombinant vector pPTR II-cas9, wherein the vector pPTR II is a shuttle vector capable of autonomously replicating in Aspergillus fungi;

3) carrying out PCR amplification by taking Aspergillus oryzae RIB40 genome DNA as a template and taking DNA fragments with sequences of SEQ ID NO. 5 and SEQ ID NO. 6 as primers to obtain a guide sequence of a U6promoter U6promoter fusion target gene shown in sequence 3, wherein the DNA fragment of SEQ ID NO. 6 contains the guide sequence of the target gene, so that the guide sequence of the target gene is fused at the 3' end of the fusion DNA molecule to form the fusion DNA molecule of U6promoter + guide sequence; artificially synthesizing a sgRNA fusion U6terminator by taking guide RNA and a U6terminator sequence as reference, wherein the nucleotide sequence of the sgRNA fusion U6terminator is a DNA molecule shown in a sequence 4, and taking the sequence as a template, and taking DNA fragments of SEQ ID NO 7 and SEQ ID NO 8 as primers, carrying out PCR amplification to obtain a sgRNA fusion U6terminator shown in the sequence 4, wherein the DNA fragment of SEQ ID NO 7 contains a guide sequence of a target gene, so that the 5' end of the fusion DNA molecule is fused with the guide sequence of the target gene to form a guide sequence + sgRNA + U6terminator fusion DNA molecule; performing overlapping PCR amplification by using the amplified U6promoter + guide sequence, guide sequence + sgRNA + U6terminator fusion DNA molecule as a template and DNA fragments of SEQ ID NO. 5 and SEQ ID NO. 8 as primers to obtain a U6promoter + guide sequence + sgRNA + U6terminator expression cassette, recombining and connecting the U6promoter + guide sequence + sgRNA + U6terminator expression cassette into a vector pPTR II, and inserting the U6promoter + guide sequence + sgRNA + U6terminator expression cassette into a SmaI multi-cloning site of the vector pPTR II to obtain a recombinant vector pPTR II-cas9-guide sequence;

4) inoculating Aspergillus oryzae RIB40 or 3.042 spores into liquid DPY culture medium, culturing for 16-20h, collecting mycelia, washing with sterile water for 2 times, splitting the mycelia with Yatalase enzyme method to obtain protoplast, mixing the extracted recombinant vector with the protoplast, adding 60% PEG 4000 for protoplast transformation, standing at room temperature for 20min, centrifuging to collect the protoplast, mixing with MM culture medium containing 0.1. mu.g/ml pyridylthioamine and 0.8% agar, spreading on MM culture medium plate containing 0.1. mu.g/ml pyridylthioamine and 1.5% agar, and culturing at 30 deg.C for 3-4 days;

5) collecting mycelia grown from the MM medium, transferring to CD medium containing 0.1 μ g/ml pyrithione, and performing secondary screening;

6) selecting the CD plate with hyphae, picking hyphae, performing PCR amplification with DNA fragments of SEQ ID NO. 9 and SEQ ID NO. 10 as primers, sequencing the amplified product, and comparing the sequencing result to see whether mutation occurs on the target gene guide sequence；

7) To obtain a Marker-Free positive strain, the two screened positive strains were cultured on a CD medium containing NO pyrithione continuously for four more rounds, mycelia were picked, PCR amplification was performed using DNA fragments of SEQ ID NO:11 and SEQ ID NO:12 as primers, it was examined whether Aspergillus oryzae further contained pPTR II-cas9 vector, and the target gene guide sequence was sequenced again.

2. The method for directionally genetically modifying an aspergillus oryzae gene based on CRISPR technology according to claim 1, wherein:

in step 1), cas9 was expressed from the constitutive promoter PgpdA.

3. The method for directionally genetically modifying an aspergillus oryzae gene based on CRISPR technology according to claim 1, wherein:

step 2) autonomous replication in Aspergillus fungi was achieved using the AMA1 gene in the pPTR II vector.

4. The method for directionally genetically modifying an aspergillus oryzae gene based on CRISPR technology according to claim 1, wherein:

the sequence SEQ ID NO. 6 and SEQ ID NO. 7 in the step 3) contain complementary 20bp overlapping sequences, and the sequences are just target gene guide sequences.

5. The method for directionally genetically modifying an aspergillus oryzae gene based on CRISPR technology according to claim 1, wherein:

the specific program of the overlapping PCR in the step 3) is as follows: taking 2 mul of PCR products, adding Buffer, dNTPs and enzyme, annealing at 50 ℃, amplifying for 5 times, adding primers and supplementing 0.5 mul of enzyme, changing the annealing temperature to 60 ℃, and continuing to amplify for 30 cycles.

6. The method for directionally genetically modifying an aspergillus oryzae gene based on CRISPR technology according to claim 1, wherein:

the MM medium in the step 4) comprises the following components: 0.2% NH₄Cl，0.1％(NH₄)₂SO₄，0.05％KCl，0.05％NaCl，0.1％KH₂PO₄，0.05％MgSO₄·7H₂O，0.002％FeSO₄·7H₂O，2％glucose，0.15％methionine，l.2M sorbitol，pH 5.5。

7. The method for directionally genetically modifying an aspergillus oryzae gene based on CRISPR technology according to claim 1, wherein:

culturing spores by liquid DPY in the step 4) for no more than 20 hours.

8. The method for directionally genetically modifying an aspergillus oryzae gene based on CRISPR technology according to claim 1, wherein:

the plasmid and the recombinant vector extracted in the step 4) need to be extracted and purified by a method of removing endotoxin.

9. The method for directionally genetically modifying an aspergillus oryzae gene based on CRISPR technology according to claim 1, wherein:

and 7) obtaining the unmarked aspergillus oryzae engineering bacteria through multi-round screening.

Technical Field

The invention relates to the field of genetic engineering of biotechnology, in particular to a method for directionally genetically modifying an aspergillus oryzae gene by using a CRISPR (clustered regularly interspaced short palindromic repeats) technology.

Background

Aspergillus oryzae has been used for a long time in fermentation of food such as soy sauce and rice wine, and is therefore considered as an internationally recognized fungus of food safety class. In 2005, the completion of Aspergillus oryzae genome sequencing work established a foundation for accelerating genetic modification of Aspergillus oryzae to become an ideal strain for efficient production of enzyme preparations and fungal metabolites. However, aspergillus oryzae has multiple cores and variable phenotypes, and is not sensitive to common screening marker drugs, so that the progress of genetic modification of aspergillus oryzae is slow, and the industrial production and application are limited.

The success of current Aspergillus oryzae genetic manipulations depends on two factors: the first is selection of a screening marker; the second is the efficiency problem of homologous homologies. Because Aspergillus oryzae is not sensitive to various resistance drugs, the genetic transformation screening of Aspergillus oryzae is mainly dependent on the construction of auxotrophic strains, which are time-consuming and labor-intensive, and the efficiency of the genetic transformation screening depends on the efficiency of homologous recombination. The current research shows that the gene participating in the non-homologous recombination pathway is knocked out to promote the homologous recombination efficiency, and the genes mainly comprise ku70, ku80 and LigD. Therefore, to improve the genetic manipulation of Aspergillus oryzae, it is often necessary to construct ku70, ku80, LigD and auxotrophic strains. Moreover, for industrial applications, it is often necessary to transform multiple genes, which makes it necessary to construct strains containing multiple auxotrophs, which undoubtedly increases the difficulty of genetically engineering Aspergillus oryzae. In addition, the construction of multiple auxotrophic selection markers tends to result in alterations in the phenotype and metabolism of the host strain, which have an impact on the subsequent use of the genetically engineered strain.

The CRISPR-Cas system is utilized to carry out gene editing in fungi, so that the problems can be well solved. Firstly, the gene knockout efficiency of the CRISPR-Cas system does not depend on homologous homologies, so that the complicacy of knocking ku70, ku80 and LigD is avoided; secondly, the CRISPR-Cas system can complete genetic modification of multiple genes by using one auxotrophic screening marker. These solve exactly the above-mentioned difficulties of the conventional homologous recombination in the transformation of Aspergillus oryzae. However, CRISPR-Cas systems also have some drawbacks. For example, if the CRISPR-Cas expression cassette is inserted into heterochromatin, the expression level of Cas9 is low, resulting in low gene editing efficiency; the expression of Cas9 is started and promoted by an alpha amylase inducible promoter amyB in the aspergillus oryzae, the promoter is induced and expressed by a carbon source type in a culture medium, and the carbon source type has great influence on the growth of the aspergillus oryzae, so that the subsequent phenotype identification of the transgene is influenced; failure to form Marker-Free engineered strains raises concerns about transgenesis. In order to solve the problems of the CRISPR-Cas system, the invention utilizes a shuttle vector which can carry out autonomous replication in Aspergillus fungi and starts to express Cas9 through a constitutive promoter PgpdA, so that the expression of Cas9 is not influenced by an insertion site and culture medium components, the gene editing efficiency is improved, and a Marker-Free engineering strain can be formed.

Disclosure of Invention

The invention aims to solve the technical problem of how to efficiently genetically modify an aspergillus oryzae gene.

In order to solve the technical problems, the invention firstly provides a method for directionally mutating an aspergillus oryzae gene based on a CRISPR technology.

The method for directionally mutating the aspergillus oryzae gene based on the CRISPR technology comprises the following steps of:

1) artificially synthesizing a cas9 gene with a nucleotide sequence of a DNA molecule shown as a sequence 1 by taking a cas9 sequence (Katayama et al (2016) Biotechnol Lett 38: 637-642) as a reference; the cas9 DNA molecule obtained by PCR amplification using the artificially synthesized cas9 gene as a template and DNA fragments with the sequences SEQ ID NO:1 and SEQ ID NO:2 as primers was recombinantly ligated to the vector pEX1 (the vector was from Nguyen et al (2016) World J Microbiol Biotechnol, 32: 204). The recombinant vector is a recombinant vector pEX1-cas9 obtained by replacing DNA between XhoI and BamHI recognition sequences of pEX1 with DNA molecules shown in a sequence 1. The pEX1-cas9 recombinant vector contains a cas9 protein expression cassette shown in a sequence 2, which consists of three parts, namely a aspergillus nidulans gene gpdA gene promoter PgpdA, a cas9 gene shown in a sequence 1 and a trpC gene terminator (TtrpC), namely PgpdA-cas 9-TtrpC.

2) The cas9 protein expression cassette obtained by PCR amplification was recombinantly ligated to the vector pPTR II (TaKaRa, Japan, Code No.3622) using the above-mentioned cas9 protein expression cassette PgpdA-cas9-TtrpC as a template and DNA fragments having the sequences SEQ ID NO:3 and SEQ ID NO:4 as primers. The recombinant vector is obtained by inserting a PgpdA-cas9-TtrpC expression cassette into a HindIII multiple cloning site of a vector pPTR II to obtain the recombinant vector pPTR II-cas9, wherein the vector pPTR II is a shuttle vector capable of autonomously replicating in Aspergillus fungi.

3) Carrying out PCR amplification by taking Aspergillus oryzae RIB40 genome DNA as a template and taking DNA fragments with sequences of SEQ ID NO:5 and SEQ ID NO:6 as primers to obtain a U6promoter (U6 promoter) fusion target gene guide sequence (guide sequence) shown in sequence 3, wherein the DNA fragment with the sequence of SEQ ID NO:6 contains a guide sequence (20 bases) of a target gene, so that the guide sequence of the target gene is fused at the 3' end of the fusion DNA molecule to form a fusion DNA molecule of U6promoter + guide sequence; using artificially synthesized guide RNA (single-guide RNA, sgRNA) and a U6terminator sequence (Katayama et al (2016) Biotechnol Lett 38: 637-642) as reference, artificially synthesizing a fusion DNA molecule of sgRNA fusion U6terminator (U6terminator), the nucleotide sequence of which is a DNA molecule shown in sequence 4, and using the nucleotide sequence as a template, and using DNA fragments of SEQ ID NO:7 and SEQ ID NO:8 as primers, performing PCR amplification to obtain a sgRNA fusion U6terminator shown in sequence 4, wherein the DNA fragment of SEQ ID NO:7 contains a guide sequence (20 bases) of a target gene, so that the guide sequence of the target gene is fused at the 5' end of the fusion DNA molecule to form a guide sequence + sgRNA + U6terminator fusion DNA molecule; the U6promoter + guide sequence + sgRNA + U6terminator expression cassette obtained by performing overlapping PCR amplification is recombined and connected to the vector pPTR II by using the U6promoter + guide sequence, guide sequence + sgRNA + U6terminator fusion DNA molecule amplified as a template and DNA fragments of SEQ ID NO:5 and SEQ ID NO:8 as primers. The recombinant vector is obtained by inserting an expression cassette of U6promoter + guidessequence + sgRNA + U6terminator into SmaI multiple cloning site of a vector pPTR II to obtain a recombinant vector pPTR II-cas9-guide sequence.

4) Aspergillus oryzae (RIB40 or 3.042) spores were inoculated into liquid DPY medium, cultured for 16-20h, and mycelia were collected and washed 2 times with sterile water. And (4) cracking the hyphae by using a Yatalase enzyme method to obtain a protoplast. Mixing the extracted recombinant vector and the protoplast, adding 60% PEG 4000 for protoplast transformation, standing at room temperature for 20min, centrifuging to collect the protoplast, mixing with MM culture medium containing 0.1. mu.g/ml pyridylthioamine and 0.8% agar, spreading on MM culture medium plate containing 0.1. mu.g/ml pyridylthioamine and 1.5% agar, and culturing at 30 deg.C for 3-4 days.

5) Mycelia grown from the MM medium were picked up and transferred to a CD medium containing 0.1. mu.g/ml of pyrithione, followed by secondary screening.

6) Selecting the CD plate with the hyphae, picking the hyphae, performing PCR amplification by using DNA fragments of SEQ ID NO. 9 and SEQ ID NO. 10 as primers, sequencing the amplification product, and comparing the sequencing result to see whether mutation occurs on the guide sequence of the target gene.

7) To obtain a Marker-Free positive strain, the two selected positive strains were cultured on a CD medium containing NO pyrithione continuously for four more rounds, mycelia were picked, PCR amplification was performed using DNA fragments of SEQ ID NO:11 and SEQ ID NO:12 as primers, it was examined whether Aspergillus oryzae further contained pPTR II-cas9 vector, and the guide sequence of the target gene was sequenced again.

Preferably, in step 1) cas9 is expressed from the constitutive promoter PgpdA.

Preferably, step 2) uses the AMA1 gene in the pPTR II vector to effect autonomous replication in a fungus of the genus Aspergillus.

Preferably, the sequence SEQ ID NO 6 and SEQ ID NO 7 of step 3) contain a complementary 20bp overlap sequence, which is exactly the target gene guide sequence.

Preferably, the specific procedure of the overlapping PCR in step 3) is: taking 2 mul of PCR products, adding Buffer, dNTPs and enzyme, annealing at 50 ℃, amplifying for 5 times, adding primers and supplementing 0.5 mul of enzyme, changing the annealing temperature to 60 ℃, and continuing to amplify for 30 cycles.

Preferably, the MM medium in step 4) has the following composition: 0.2% NH₄Cl，0.1％(NH₄)₂SO₄，0.05％KCl，0.05％NaCl，0.1％KH₂PO₄，0.05％MgSO₄·7H₂O，0.002％FeSO₄·7H₂O，2％glucose，0.15％methionine，l.2M sorbitol，pH 5.5

Preferably, the liquid DPY in step 4) is used for culturing spores, preferably not more than 20h, otherwise the subsequent protoplast transformation efficiency is influenced.

Preferably, the plasmid (recombinant vector) extracted in step 4) must be purified by endotoxin removal, which is advantageous for improving the protoplast transformation efficiency.

Preferably, step 7) obtains the Marker-Free (Marker-Free) Aspergillus oryzae engineering bacteria through multiple rounds of screening.

The invention has the advantages of

The invention provides a method for directionally and genetically modifying an Aspergillus oryzae gene by utilizing a shuttle vector which can autonomously replicate in Aspergillus fungi and starting expression of cas9 through a constitutive promoter PgpdA, which eliminates the influence of a vector insertion site and culture medium components on cas9 expression, improves gene editing efficiency, can generate Marker-Free (Marker-Free) Aspergillus oryzae engineering bacteria, avoids the worry of transgenosis and improves the practicability of transgenic modified Aspergillus oryzae.

Drawings

FIG. 1 shows the construction of PgpdA-cas9-TtrpC expression cassette.

FIG. 2 shows the construction of pPTR II-cas9 vector.

FIG. 3 shows the construction of pPTR II-cas9-AozfpB vector.

FIG. 4 selection of transformants grown on medium.

FIG. 5 shows the identification of transformants.

FIG. 6 is detection of Marker-Free (Marker-Free) CRISPR-AozfpB engineering bacteria.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention.

The experimental procedures in the following examples are conventional unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.