CN114317580A - Specific gene knockout CRISPR/Cas9 editing plasmid containing double sgRNAs and application thereof - Google Patents

Abstract

The invention discloses a CRISPR/Cas9 editing plasmid with double sgRNAs and specific gene knockout, which comprises double sgRNAs and upstream and downstream homologous arms, wherein the sequences of the double sgRNAs are shown as SEQ ID No.1 and SEQ ID No. 2; the upstream and downstream homologous arms are obtained by respectively carrying out PCR amplification by using primers shown by SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10 by using A.keratiniphila HCCB10007 genome as a template. The invention also discloses application of the CRISPR/Cas9 editing plasmid in large fragment gene knockout in amycolatopsis keratinase hydrolysis. The invention realizes the accurate knockout of the gene of a large fragment of more than 80kb in the hydrolyzed keratin amycolatopsis.

Description

A CRISPR/Cas9 editing plasmid for specific gene knockout containing double sgRNA and its application

technical field

The invention belongs to the technical field of genetic engineering, and in particular relates to a CRISPR/Cas9 editing plasmid containing double sgRNA specific gene knockout and its application.

Background technique

Microbial secondary metabolites are an important source of various antibiotics, antiviral drugs, antifungal drugs, immunosuppressants and other drugs that are widely used in clinical practice. Due to the complexity of the microbial genome, in addition to the essential genes related to growth and reproduction, there are many secondary metabolic biosynthetic gene clusters in the genome. However, many secondary metabolic biosynthesis gene clusters are silent or even redundant. Their existence will compete with the biosynthesis of target products, and their expression will consume a large amount of energy and precursors produced by primary metabolism, and also Lead to the generation of impurities, reduce the yield of the target product and increase the difficulty of separation and purification. Therefore, the engineering of microbial cells to remove redundant large-segment genes to efficiently synthesize high value-added natural products has received more and more attention.

Traditional methods of genome simplification include homologous recombination, double-strand break repair recombination, site-specific recombination, and transposition recombination, but they all have more or less complex construction of gene editing tools, and residual fragments on the genome cannot achieve seamless knockout. There are uncertainties in deletion and gene deletion. The CRISPR/Cas9 system is a new technology that is widely used, efficient, simple, and capable of site-directed editing of genomes. This system only requires the participation of sgRNAs linked by Cas9 protein, crRNA and tracrRNA. The CRISPR/Cas9 system can efficiently edit genes, and use the method of homologous recombination repair to achieve seamless gene knockout.

Vancomycin is currently the drug of choice for the clinical treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections. It is also used to treat serious infections that are ineffective against all antibiotics, and is of great significance in clinical treatment. A.keratiniphila HCCB10007 is a high-yielding vancomycin strain selected by physical and chemical mutagenesis. The genome contains a circular chromosome and a circular endogenous plasmid. The full length of the chromosomal genome is 8,948,591 bp, and the GC content is 69%. Through bioinformatics analysis, it is predicted that there are at least 26 secondary metabolic biosynthesis gene clusters in the genome. Although the CRISPR/Cas9 system has been successfully applied to microorganisms such as Escherichia coli, Saccharomyces cerevisiae, Streptococcus pneumoniae, and Streptomyces, large-scale gene editing in Streptomyces is also highly efficient. Although A. keratiniphila HCCB10007 belongs to Actinomyces However, compared with Streptomyces, its genome structure is more complex, and the secondary metabolite network is larger. It is still extremely challenging to apply CRISPR/Cas9 technology to knock out large fragments of genes in this strain.

SUMMARY OF THE INVENTION

The first object of the present invention is to provide a specific gene knockout CRISPR/Cas9 editing plasmid containing double sgRNA. The second object of the present invention is to provide a method for constructing a CRISPR/Cas9 editing plasmid containing a specific gene knockout of double sgRNA. The third object of the present invention is to provide the application of a CRISPR/Cas9 editing plasmid containing double sgRNA specific gene knockout for gene knockout of large fragments in A. keratinolyticus. The fourth aspect of the present invention is to provide a double sgRNA for knocking out the large fragment gene of A. keratiniphila HCCB10007. The fifth aspect of the present invention is to provide an upstream and downstream homology arm for homologous recombination repair after knocking out a large segment gene in A. keratiniphila HCCB10007. The sixth aspect of the present invention is to provide a mutant strain of A. keratiniphila HCCB10007 with a large segment gene knocked out.

To achieve the above object, the present invention adopts the following technical solutions:

As a first aspect of the present invention, a CRISPR/Cas9 editing plasmid for specific gene knockout containing double sgRNA, including double sgRNA for knocking out the large fragment gene of A. keratiniphila HCCB10007 and knocking out A. keratiniphila HCCB10007 The upstream and downstream homology arms used for homologous recombination repair after the large fragment gene on the The downstream homology arm was based on the A. keratiniphila HCCB10007 genome as a template, and PCR amplification was performed with the primers shown in SEQ ID NO. Sequencing verification obtained.

According to the present invention, the CRISPR/Cas9 editing plasmid is constructed and obtained through the following steps:

Step 1. Use Hind III to digest the pLYNY04 plasmid to obtain a digested vector; connect the digested linearized vector and the upstream and downstream homology arms by overlapping recombination to obtain a backbone plasmid;

Step 2: Use SpeI to cut the backbone plasmid to obtain a linearized backbone vector; anneal and connect the two oligos shown in SEQ ID NO. It is annealed and connected with the two oligos shown in SEQ ID NO.6 to form double-stranded sgRNA-2, and then the linearized backbone vector is connected with double-stranded sgRNA-1 and double-stranded sgRNA-2 by overlapping recombination to obtain a mixture containing Editing plasmids for single sgRNA-1 and editing plasmids containing single sgRNA-2;

Step 3. Use XbaI to digest the editing plasmid containing a single sgRNA-1 to obtain a linearized sgRNA-1 vector; take the editing plasmid containing a single sgRNA-2 as a template, and use the editing plasmids as shown in SEQ ID NO.11 and SEQ ID NO.12. The primers shown were used to amplify the gene fragment containing the ermE ^* promoter and sgRNA-2 by PCR; then the gene fragment of the ermE ^* promoter and sgRNA-2 was combined with the linearized sgRNA-1 of the edited plasmid containing a single sgRNA-1 The vectors were ligated, and overlapping recombination was used to obtain the editing plasmid pHMYECO4-26 containing double sgRNAs, which was verified by sequencing.

As a second aspect of the present invention, a method for constructing a CRISPR/Cas9 editing plasmid containing a specific gene knockout of double sgRNA, comprising the following steps:

Step 1. Using the A. keratiniphila HCCB10007 genome as a template, PCR amplification was performed with the primers shown in SEQ ID NO. The upstream and downstream homology arms of recombination repair;

Step 2: Use Hind III to digest the pLYNY04 plasmid to obtain the digested vector; then connect the digested linearized vector and the amplified upstream and downstream homology arms by overlapping recombination to obtain the backbone plasmid;

Step 3. Use SpeI to cut the backbone plasmid to obtain a linearized backbone vector; anneal and connect the two oligos shown in SEQ ID NO. It is annealed and connected with the two oligos shown in SEQ ID NO.6 to form double-stranded sgRNA-2, and then the linearized backbone vector is connected with double-stranded sgRNA-1 and double-stranded sgRNA-2 by overlapping recombination to obtain a mixture containing Editing plasmids for single sgRNA-1 and editing plasmids containing single sgRNA-2;

Step 4. Use XbaI to digest the editing plasmid containing a single sgRNA-1 to obtain a linearized sgRNA-1 vector; take the editing plasmid containing a single sgRNA-2 as a template, and use the editing plasmids as shown in SEQ ID NO.11 and SEQ ID NO.12. The primers shown were used to amplify the gene fragment containing the ermE ^* promoter and sgRNA-2 by PCR; then the gene fragment of the ermE ^* promoter and sgRNA-2 was combined with the linearized sgRNA-1 of the edited plasmid containing a single sgRNA-1 The vectors were ligated, and overlapping recombination was used to obtain the editing plasmid pHMYECO4-26 containing double sgRNAs, which was verified by sequencing.

According to the present invention, step 1 further includes purifying the amplified product: subjecting the amplified product to agarose gel electrophoresis, obtaining products of corresponding size fragments, performing gel recovery, obtaining purified front and rear homology arms, and performing sequencing verification .

As a third aspect of the present invention, a CRISPR/Cas9 editing plasmid containing a specific gene knockout of double sgRNA is used for large fragment gene knockout in A. keratinolyticus.

According to the present invention, the keratin-hydrolyzing Amycolatopsis comprises A. keratiniphila HCCB10007.

According to the present invention, the described application is to transform the CRISPR/Cas9 editing plasmid containing the specific gene knockout of double sgRNAs into E.coli JM110 for demethylation treatment, and then electroporate into A.keratiniphilaHCCB10007 for sensing in state.

As the fourth aspect of the present invention, a double sgRNA for knocking out the large fragment gene of A. keratiniphila HCCB10007, the sequences of which are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

As the fifth aspect of the present invention, an upstream and downstream homology arm used for homologous recombination repair after knocking out a large fragment gene on A. keratiniphila HCCB10007 is based on the A. keratiniphila HCCB10007 genome as a template. The primers shown in ID NO.7, SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10 were amplified by PCR and verified by sequencing.

As the sixth aspect of the present invention, a large segment gene knockout A. keratiniphila HCCB10007 mutant strain is used to knock out the glycoside polyketone using the CRISPR/Cas9 editing plasmid containing the specific gene knockout of double sgRNA The mutant strain obtained after the 87.5kb large fragment gene on the secondary metabolite ECO-0501 biosynthetic gene cluster, the specific location of the large fragment gene is AORI_2930-AORI_2954 on the ECO-0501 biosynthetic gene cluster.

According to the present invention, the A. keratiniphila HCCB10007 mutant strain with large fragment gene knockout is used to knock out the secondary metabolism of glycoside polyketides using the CRISPR/Cas9 editing plasmid containing the specific gene knockout of double sgRNA The mutant strain obtained after the product ECO-0501 biosynthesizes large fragments of genes in the gene cluster.

As the seventh aspect of the present invention, a method for identifying a mutant strain after knockout of a CRISPR/Cas9 editing plasmid containing a specific gene knockout of double sgRNA, comprising the following steps:

Step 1. Inoculate a single colony into a medium containing apramycin for resistance screening;

Step 2, using bacterial liquid PCR amplification, design the inner primers shown in SEQ ID NO.13 and SEQ ID NO.14 and the outer primers shown in SEQ ID NO.15 and SEQ ID NO.16 for verification, and detected by gel electrophoresis;

Step 3: Use the genomic DNA of the mutant strain whose inner and outer primers are correct in PCR verification as a template, and use the PCR products amplified by SEQ ID NO. 15 and SEQ ID NO. 16 for sequencing verification.

The beneficial effects of the present invention are as follows: the present invention realizes for the first time the precise knockout of a large fragment gene of more than 80 kb in Amycobacterium keratinolyticum, and the constructed CRISPR/Cas9 editing of specific gene knockout containing double sgRNA The plasmid effectively improves the gene knockout efficiency, simplifies the genetic engineering operation, and lays a good foundation for the simplification of the genome of the subsequent strains.

Description of drawings

Figure 1 shows the positions of two sgRNA design sites on the ECO-0501 gene cluster of A. Keratiniphila HCCB10007 strain.

Figure 2(1) and Figure 2(2) are schematic diagrams of the structures of the editing plasmids synthesized in Example 3 and containing a single sgRNA-1 and sgRNA-2, respectively.

FIG. 3 is a schematic structural diagram of the editing plasmid pHMYECO4-26 containing double sgRNA synthesized in Example 3. FIG.

Figure 4 is used to verify whether the transformant has achieved gene knockout in Example 5, the upper picture is the design site of the primer Verify-inside-F/R; the lower picture is the use of the primer Verify-inside-F/R to the transformant The results of PCR verification; wherein, the numbers 1-7 are different transformants, Con is A. Keratiniphila HCCB10007 (negative control), and M is 1kb DNA Ladder marker (1kb DNA molecular weight gradient scale). If the target band (1491bp) could not be amplified, it proved that the gene had been knocked out.

Figure 5 shows the correct verification of the primers Verify-inside-F/R transformants No. 3 and No. 5 using the Verify-outside-F/R primer for secondary verification. The top picture is the schematic diagram of the primer design site; the bottom picture is the use of the primer Verify -outside-F/R results of PCR verification of transformants; among them, Nos. 3 and 5 are transformants to be verified, Con is A.KeratiniphilaHCCB10007 (negative control), M is 1kb DNA Ladder marker (1kb DNA molecular weight gradient scale) . If the target band (6777bp) can be amplified, the gene has been knocked out.

FIG. 6 shows the sequencing results of the mutant strains verified by PCR in Example 5. FIG.

Figure 7 is the HPLC detection result of the fermentation product of the positive mutant bacteria in Example 7, and the arrow indicates the peak position of the ECO-0501 product.

Detailed ways

The present invention will be further described below with reference to specific embodiments. It should be understood that the following examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods that do not specify specific conditions in the following examples are usually carried out in accordance with conventional conditions or conditions provided by manufacturers.

Sources of reagents and plasmids used in the following examples:

1. The plasmid pLYNY04 was constructed by the method of Chinese patent application document CN109609537A. Built for this lab.

2. Both E.coli DH5α and E.coli JM110 are commercially available;

3. A.Keratiniphila HCCB10007 was deposited in the General Microbiology Center of the China Microorganism Collection and Administration Commission, and the deposit number is CGMCC No.6023.

Example 1 sgRNA design for targeted knockout

Step 1. The selected knockout fragment is an 87.5kb fragment located on the ECO-0501 biosynthetic gene cluster of A. Keratiniphila HCCB10007 strain, and the specific location is AORI_2930-AORI_2954.

Step 2. Use the sgRNA online design tool CCTop-CRISPR/Cas9 target online predictor to design and screen sgRNA fragments based on the fragments to be knocked out. The two sgRNA sequences are:

sgRNA-1:ACTCGGGATCTCCTGACTTG(PAM:GGG), (SEQ ID NO. 1);

sgRNA-2: CAAAGGACAGAAAAGAAAGG (PAM:TGG), (SEQ ID NO. 2).

Step 3. Synthesize the corresponding oligonucleotide fragments (the lowercase part is the homology arm when connected to the vector)

sgRNA-1 oligo-F:atttctagctctaaaacCAAGTCAGGAGATCCCGAGTactagttcctaccaaccggcacg, (SEQ ID NO. 3);

sgRNA-1 oligo-R: cgtgccggttggtaggaactagtACTCGGGATCTCCTGACTTGgttttagagctagaaat, (SEQ ID NO. 4);

sgRNA-2 oligo-F:atttctagctctaaaacCCTTTCTTTTCTGTCCTTTGactagttcctaccaaccggcacg, (SEQ ID NO. 5);

sgRNA-2 oligo-R: cgtgccggttggtaggaactagtCAAAGGACAGAAAAGAAAGGgttttagagctagaaat, (SEQ ID NO. 6).

Step 4. The two oligos of sgRNA1 and sgRNA2 are respectively annealed and connected to form double strands, which are used for subsequent connection with the carrier. The reaction conditions are: 95°C water bath for 5 minutes, and then naturally cooled to room temperature; the reaction system is shown in Table 1:

Table 1 Reaction system

Oligo-F (10μM) 5μl Oligo-R (10μM) 10μl T4 ligase buffer (T4 ligase buffer) 10μl ddH₂O 25μl Total 50μl

Example 2 Synthesis of upstream and downstream homology arms for homologous recombination repair after gene knockout

Step 1. Design and synthesize amplification primers with the genome sequence of A. keratiniphila HCCB10007 as a template:

arm-aF: acgacggccagtgccaagcttCCGGATACACCAAGAGCACATCA, (SEQ ID NO. 7);

arm-aR: acgggcgatcTTGCCGAGGAGCCTAGAGGAC, (SEQ ID NO. 8);

arm-zF: tcctcggcaaGATCGCCCGTCCCACCGAGCGT, (SEQ ID NO. 9);

arm-zR: ggtgctttttttgagaagcttGGATCAAGGCAACCTGCTGTG, (SEQ ID NO. 10).

Step 2. Using the A.keratiniphila HCCB10007 genome as a template, PCR amplification was performed with the primers shown in SEQ ID NO.7, SEQID NO.8, SEQ ID NO.9, and SEQ ID NO.10 respectively. As shown in Table 2 (taking the amplification of arm-aF and arm-aR as an example, the amplification of arm-zF and arm-zR is the same as in Table 2, and arm-aF and arm-aR are replaced by arm-zF and arm- zR):

Table 2 Reaction system and PCR program

In step 3, the amplified products are subjected to agarose gel electrophoresis (1%, 120V, 40min), and the products with corresponding size fragments are recovered by gel using the kit to obtain purified fragments and upstream and downstream homology arms.

Example 3 Construction of a CRISPR/Cas9 editing plasmid for specific gene knockout containing double sgRNA

Step 1. Use Hind III to digest the pLYNY04 plasmid to obtain the digested vector. The reaction conditions are: 37°C, 4h; the reaction system is shown in Table 3:

Table 3 Reaction system

Hind III 2.5μl 10×M buffer 5μl pLYNY04 2.5μg ddH₂O Xμl Total 50μl

Step 2: Use a gel recovery kit to purify the enzyme-digested plasmid product, and operate according to the instructions.

Step 3. Connect the purified linearized vector with the amplified upstream and downstream homology arms to obtain a backbone plasmid. The overlap recombination method is used. Place on ice to cool; the reaction system is shown in Table 4:

Table 4 Reaction system

Remarks: The optimal amount of each fragment used = [0.02×fragment base pairs]ng (X, Y1, Y2 are all calculated from this).

Step 4. After the ligation reaction, add 10 μl of the recombinant product to 100 μl of DH5α competent cells, flick the tube wall to mix, let stand on ice for 25 min, heat shock in a 42°C water bath for 45 s, and quickly put it back on ice for 2 min. Add 700 μl of anti-LB medium to the centrifuge tube, mix well, recover at 37°C, 200 rpm for 60min, spread it on the LB solid plate with apramycin, and invert overnight at 37°C.

Step 5. The grown single colony is verified by PCR with primers of SEQ ID NO.7 and SEQ ID NO.10, and the single colony of the corresponding band can be amplified, and the homology arm part is verified by sequencing, and the verification is correct. Obtain the backbone plasmid.

Step 6. Use SpeI to digest the backbone plasmid to obtain a linearized backbone vector. The reaction conditions are: 37°C, 4h; the reaction system is shown in Table 5:

Table 5 Reaction system

Step 7. Connect the linearized backbone vector with the two oligo annealed and connected fragments of sgRNA-1 and sgRNA-2 of Example 1 respectively, adopt the method of overlap recombination, and the experimental method is the same as that described in step 3 of this embodiment, An edited plasmid containing a single sgRNA-1 and an edited plasmid containing a single sgRNA-2 were obtained.

Step 8. Use Xba I to digest the edited plasmid containing a single sgRNA-1 to obtain a linearized sgRNA-1 vector. The reaction conditions are: 37°C, 4h; the reaction system is shown in Table 6:

Table 6 Reaction system

Step 9, using the editing plasmid containing a single sgRNA-2 as a template, using the primers shown in SEQ ID NO.11 and SEQ ID NO.12 to amplify the gene fragment containing the ermE ^* promoter and sgRNA-2 by the method of PCR, The primer sequences are as follows:

sgRNA2-F: gcatgcatactagagaatctAAAAAAAGCACCGACTCG, (SEQ ID NO. 11);

sgRNA2-R: ggaaaactctcagcttctagGATTCTCTAGTATGCATG, (SEQ ID NO. 12).

The PCR reaction system and program are shown in Table 7:

Table 7 PCR reaction system and program

Step 10. Connect the purified ermE ^* promoter and the gene fragment of sgRNA-2 with the linearized sgRNA-1 vector, adopt the method of overlap recombination, and the experimental method is the same as that described in step 3 of this embodiment. The plasmid pHMYECO4-26 was edited for CRISPR/Cas9 containing dual sgRNAs. The schematic diagram is shown in Figure 3.

Example 4 CRISPR/Cas9 editing plasmid was electroporated into A. Keratiniphila HCCB10007

Step 1. Transfer the constructed editing plasmid pHMYECO4-26 into E.coli JM110 for demethylation: add 10 μl of the plasmid to 100 μl of JM110 competent cells, flick the tube wall to mix, and let stand on ice for 25 minutes , heat shock in a water bath at 42°C for 45s, quickly put back on ice for 2min, then add 700μl of anti-LB medium to the centrifuge tube, mix well, recover at 37°C, 200rpm for 60min, and then spread on the LB solid plate with apramycin Inverted and cultured overnight at 37°C, the single colonies grown were subjected to liquid amplification, and the plasmid was extracted using the kit to obtain the demethylated pHMYECO4-26 plasmid.

Step 2: Take 2-5 μg (volume <6 μl) of demethylated pHMYECO4-26 plasmid and add it to 60 μl of A.Keratiniphila HCCB10007 competent cells, gently pipette and mix, and immediately put it into an ice-cooled electroporation cup (BTX, Φ2mm) medium-electric conversion conditions: 600Ω, 25μF, 7.5kV/cm, and the electric shock duration is about 13ms.

Step 3: After electrotransformation, resuspend the bacterial cells with 1 ml of TSB medium, transfer to a 15 ml test tube, and shake and culture in a constant temperature incubator for 7 hours, 28° C., 220 rpm.

Step 4: Centrifuge the activated bacterial liquid at 4000 rpm for 2 min, discard 900 ul of supernatant, resuspend the remaining bacterial liquid and spread it on the bennet plate medium containing apramycin, and cultivate in a constant temperature incubator at 28°C for 5 days.

Example 5 Identification of mutant strains

Step 1. Based on the genome sequence of A.Keratiniphila HCCB10007, two pairs of primers were designed inside and outside the knockout gene to verify the mutant strain: the inside primers (SEQ ID NO. 13 and SEQ ID NO. 14) were not able to amplify the target. The band (1491bp) proves that the gene has been knocked out; if the outer primers (SEQ ID NO.15 and SEQ ID NO.16) can amplify the target band (6777bp), it proves that the gene has been knocked out. The primer sequences are as follows :

Verify-inside-F: ATCCTACTGTCGGGTTCATCGG, (SEQ ID NO. 13);

Verify-inside-R: GGTCCACCTTTCTTTTCTGTCCTTT, (SEQ ID NO. 14);

Verify-outside-F:AGTTTCCTTTGACGCCATGC, (SEQ ID NO. 15);

Verify-outside-R: TGGACTCTGTCTGGGGTGTG, (SEQ ID NO. 16).

Step 2: Select the transformants on the plate, inoculate them in 3 ml of TSB liquid medium containing apramycin, and culture with shaking in a constant temperature incubator for 2 days, 28° C., 220 rpm.

Step 3. The transformant bacterial liquid is used for PCR experiment with inner primers (SEQ ID NO.13 and SEQ ID NO.14) and outer primers (SEQ ID NO.15 and SEQ ID NO.16) respectively, and detected by gel electrophoresis (1%, 120V, 40min), the results are shown in Figure 4 and Figure 5.

Step 4. Take the genomic DNA of the mutant strain whose inner and outer primers PCR verification is correct as a template, and use the PCR products amplified by SEQ ID NO. 15 and SEQ ID NO. 16 for sequencing verification. The positive mutant strain of gene knockout, the results are shown in Figure 6.

Example 6 Elimination of CRISPR/Cas9 Editing Plasmids in Positive Mutants

Step 1. The bacterial liquid of the positive mutant strain was streaked and separated on Ben's medium without antibiotics. After overnight culture at 37°C, a single colony was picked and spread on Ben's medium without antibiotics and apramycin. It was cultured at 28°C for 3 days, and it grew normally on the plate without antibiotics, and the single colony that did not grow on the resistant plate was the positive strain that successfully eliminated the plasmid.

Step 2, confirm again by the mode of PCR amplification, use the primers SEQ ID NO.7 and SEQ ID NO.8 (or SEQ ID NO.79 and SEQ ID NO.10) of the amplification homology arm to carry out PCR verification, The inability to amplify a band confirms the plasmid elimination.

Example 7 Metabolite analysis of mutant strains

Step 1. Spread the bacterial liquid of the starting bacteria A.Keratiniphila HCCB10007 and the gene knockout mutant bacteria evenly on the Gao's No. 1 solid medium plate, and invert them in a constant temperature incubator at 28°C for 4 days until they are covered with white spore;

Step ² : Use a sterile flat shovel to excavate about 1 cm of the bacterial mass and transfer it to 25 ml of seed medium, and shake it in a shaker at 220 rpm and 28 ° C for 48 hours;

Step 3, transfer the seed bacteria liquid (10% of the total volume) after the cultivation into 25ml fermentation medium F1, and shake and cultivate for 5 days in a shaker at 220rpm and 28°C;

Step 4: Take 2 ml of fermentation broth, centrifuge at 12,000 rpm for 5 min, discard the supernatant, add 500 μl of absolute ethanol to the cells and ultrasonicate for 30 min, soak overnight, and perform HPLC detection on the extract. HPLC detection conditions are as follows:

Mobile phase preparation: use 10mM ammonium acetate solution and acetic acid to adjust pH to 4.0 as buffer. Mix buffer and acetonitrile in a 45:55 ratio and shake well.

Chromatographic column: Agilent ZORBAX SB-C18 column (5μm×250mm); column temperature: 35°C; detector: DAD (HP1100) detector; detection wavelength: 360nm; flow rate: 1ml/min; injection volume: 20μl; elution Method: Isocratic elution.

The results showed that the peak of ECO-0501 could be detected in the starting strain A.Keratiniphila HCCB10007, while the peak of ECO-0501 product could not be detected in the knockout mutant strain, which further proved that the CRISPR/Cas9 editing plasmid pHMYECO4- 26 successfully knocked out a fragment on the ECO-0501 gene cluster, preventing the synthesis of ECO-0501.

The above are only examples of the embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the technical principles of the present invention, several improvements and modifications can be made. These improvements and Variations should also be considered within the scope of protection of the present invention.

sequence listing

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Shanghai Laiyi Biological Drug Research and Development Center Co., Ltd.

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Claims (9)

1. A specific gene-knocked-out CRISPR/Cas9 editing plasmid containing double sgRNAs, which is characterized by comprising double sgRNAs for knocking out a large fragment gene of A.keratiniphila HCCB10007 and upstream and downstream homologous arms for homologous recombination repair after knocking out the large fragment gene on the A.keratiniphila HCCB10007, wherein the sequences of the double sgRNAs are shown as SEQ ID No.1 and SEQ ID No. 2; the upstream and downstream homology arms for homologous recombination and repair are obtained by respectively carrying out PCR amplification by using primers shown by SEQ ID NO.7 and SEQ ID NO.8 and primers shown by SEQ ID NO.9 and SEQ ID NO.10 by using A.keratiniphila HCCB10007 genome as a template.

2. The CRISPR/Cas9 editing plasmid of claim 1, wherein the CRISPR/Cas9 editing plasmid is constructed by the following steps:

step one, carrying out enzyme digestion on pLYNY04 plasmid by using Hind III to obtain an enzyme digested vector; connecting the linearized vector after enzyme digestion with the upstream and downstream homologous arms in an overlap recombination manner to obtain a skeleton plasmid;

step two, using SpeI to enzyme-cut the skeleton plasmid to obtain a linearized skeleton vector; annealing and connecting two oligos shown as SEQ ID No.3 and SEQ ID No.4 to form a double-chain sgRNA-1, annealing and connecting two oligos shown as SEQ ID No.5 and SEQ ID No.6 to form a double-chain sgRNA-2, and then connecting a linear framework vector with the double-chain sgRNA-1 and the double-chain sgRNA-2 respectively in an overlap recombination manner to obtain an editing plasmid containing a single sgRNA-1 and an editing plasmid containing a single sgRNA-2;

step three, using XbaI to enzyme-cut an editing plasmid containing single sgRNA-1 to obtain a linear sgRNA-1 vector; using an editing plasmid containing single sgRNA-2 as a template, and amplifying ermE-containing plasmid by using primers shown as SEQ ID NO.11 and SEQ ID NO.12 by using a PCR method^*A promoter and a gene fragment of sgRNA-2; then e is mixedrmE^*The promoter and the gene fragment of the sgRNA-2 are connected with a linearized sgRNA-1 vector of an editing plasmid containing single sgRNA-1 to obtain an editing plasmid pHMYECO4-26 containing double sgRNAs.

3. A construction method of a specific gene knockout CRISPR/Cas9 editing plasmid containing double sgRNAs is characterized by comprising the following steps:

step one, taking A.keratiniphila HCCB10007 genome as a template, and respectively carrying out PCR amplification by using primers shown in SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10 to obtain an upstream and downstream homologous arm for homologous recombination and repair;

step two, carrying out enzyme digestion on pLYNY04 plasmid by using Hind III to obtain an enzyme-digested vector; then connecting the linearized vector after enzyme digestion with the upstream and downstream homologous arms obtained by amplification in an overlap recombination manner to obtain a skeleton plasmid;

step three, using SpeI to enzyme-cut the skeleton plasmid to obtain a linearized skeleton vector; annealing and connecting two oligos shown as SEQ ID No.3 and SEQ ID No.4 to form a double-chain sgRNA-1, annealing and connecting two oligos shown as SEQ ID No.5 and SEQ ID No.6 to form a double-chain sgRNA-2, and then connecting a linear framework vector with the double-chain sgRNA-1 and the double-chain sgRNA-2 respectively in an overlap recombination manner to obtain an editing plasmid containing a single sgRNA-1 and an editing plasmid containing a single sgRNA-2;

step four, using XbaI to enzyme-cut the editing plasmid containing single sgRNA-1 to obtain a linear sgRNA-1 vector; using an editing plasmid containing single sgRNA-2 as a template, and amplifying ermE-containing plasmid by using primers shown as SEQ ID NO.11 and SEQ ID NO.12 by using a PCR method^*A promoter and a gene fragment of sgRNA-2; then the ermE^*The promoter and the gene fragment of the sgRNA-2 are connected with a linearized sgRNA-1 vector of an editing plasmid containing single sgRNA-1, and the editing plasmid pHMYECO4-26 containing double sgRNAs is obtained by adopting an overlap recombination mode.

4. Use of the double sgRNA-containing specific knockout CRISPR/Cas9 editing plasmid of claim 1 or 2 for large fragment gene knockout in amycolatopsis keratinolyticus.

5. The use of claim 4, wherein the hydrolyzed kerateiomycotic comprises A.keratiniphila HCCB 10007.

6. The use according to claim 4, wherein the CRISPR/Cas9 editing plasmid verifying correct specific knockout containing double sgrnas is transferred to e.coli JM110 demethylation treatment and then electrotransformed into a.keratiniphila HCCB10007 competence.

7. A double sgRNA for knocking out a 10007 large fragment gene of A.keratiniphila HCCB is characterized in that sequences of the double sgRNA are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2.

8. An upstream and downstream homology arm for homologous recombination and repair after a large fragment gene on A.keratiniphila HCCB10007 is knocked out is characterized in that the upstream and downstream homology arm is obtained by taking the A.keratiniphila HCCB10007 genome as a template and performing PCR amplification by using primers shown in SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10 respectively.

9. A large fragment gene knockout a.keratiniphila HCCB10007 mutant strain, which is obtained by knocking out a large fragment gene on a glycoside polyketone secondary metabolite ECO-0501 biosynthetic gene cluster using the double sgRNA-containing specific gene knockout CRISPR/Cas9 editing plasmid of claim 1 or 2, wherein the specific position of the large fragment gene is AORI _2930-AORI _2954 on the ECO-0501 biosynthetic gene cluster.

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