A helper plasmid for dual sgRNA expression, its preparation method and application

Abstract

This invention provides a helper plasmid for dual sgRNA expression, its preparation method, and its application, belonging to the field of gene editing technology. The helper plasmid of this invention includes plasmid pTargetF, which carries sgRNA-1 and sgRNA-2; sgRNA-1 targets the Cas9 gene, and sgRNA-2 targets the wild-type replicon pSC101; plasmid pTargetF does not carry any homologous arms and can be used in CRISPR-Cas9 system gene editing to remove plasmid pCasM, which carries the wild-type replicon pSC101. This invention achieves a 70% success rate in removing plasmid pCasM, shortens the processing time to 4 days, and maintains the strain yield at over 70% of the original yield after removing plasmid pCasM, thus preserving the overall production performance of high-yielding strains and making it suitable for gene editing of high-yielding strains.

Description

Technical Field

[0001] This invention relates to the field of gene editing technology, specifically to a helper plasmid for dual sgRNA expression, its preparation method, and its application. Background Technology

[0002] CRISPR / Cas9 gene editing technology is now widely used in various microorganisms, plants, and animals for targeted genome modification. Its application in *Escherichia coli* is the most mature, with the most typical example being the dual-plasmid pCas / pTargetF-X (X representing the genome editing site) gene editing system developed by Yang Sheng's team at the Chinese Academy of Sciences. (Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S. *Appl Environ Microbiol*. 2015) (Apr;81(7):2506-14) In the pCas / pTargetF-X system, plasmid pCas carries constitutively expressed cas9 gene, arabinose-induced λ-Red system, thermosensitive replicon protein RepA101ts, and IPTG-induced sgRNA targeting pMB1 replicon. Plasmid pTargetF-X carries sgRNA targeting the target gene, pMB1 replicon, and upstream and downstream homologous arms. The general gene editing process of this system is as follows: first, transform plasmid pCas, add arabinose-induced λ-Red system, then transform plasmid pTargetF-X. After gene editing is completed by the sgRNA carried by plasmid pTargetF-X targeting the target gene, add IPTG and the sgRNA carried by plasmid pCas will target and excise plasmid pTargetF-X. Finally, change the temperature to discard plasmid pCas using thermosensitive replicon. This method is very simple and efficient, and therefore widely used.

[0003] The biggest drawback of the above-mentioned dual-plasmid pCas / pTargetF-X gene editing system is that the strain can only be cultured at 30℃ after transforming the pCas plasmid. At this temperature, the growth rate is significantly lower than that of normal culture at 37℃. In particular, during metabolic engineering, the strain's synthesis rate increases significantly after multiple rounds of editing, while its growth rate decreases significantly.

[0004] Therefore, to improve the growth rate and editing efficiency of the strain after transformation with plasmid pTargetF-X, the thermosensitive replicon protein RepA101ts was replaced with the normal replicon protein RepA101 to construct plasmid pCasM. After the culture temperature was restored to 37℃, the growth rate and editing efficiency of the strain were significantly improved. However, after gene editing and excision of plasmid pTargetF-X, plasmid pCasM could only be removed by adding sodium dodecyl sulfate (SDS) and culturing at 42℃. However, adding SDS and culturing at 42℃ essentially damages the cell membrane, causing the cytosol, including plasmid pCasM, to "flow out" to achieve the purpose of removing pCasM. The damage to cells during the removal process is sometimes not negligible, especially for high-yielding strains, often resulting in a significant reduction in cell viability and yield after removal. Moreover, this method has a relatively low efficiency in removing pCasM, often requiring repeated culture and multiple rounds of screening to obtain strains with successful removal.

[0005] In summary, both temperature-sensitive plasmid pCas and normal plasmid pCasM have significant drawbacks in subsequent editing of high-yield strains, greatly limiting their application. Therefore, in order to retain the editing convenience brought by plasmid pCasM, it is urgent to develop a new and efficient method for removing plasmid pCasM. Summary of the Invention

[0006] After using plasmid pCasM (carrying the wild-type replicon pSC101) to improve the growth rate and editing efficiency of strains during gene editing, problems arise, such as the difficulty in efficiently discarding pCasM and a significant reduction in cell viability and yield after discarding it. To address this issue, this invention provides a helper plasmid for dual sgRNA expression, its preparation method, and its applications.

[0007] To achieve the above objectives, the present invention provides a helper plasmid for dual sgRNA expression, comprising plasmid pTargetF, wherein plasmid pTargetF carries sgRNA-1 and sgRNA-2; sgRNA-1 targets the cas9 gene, and sgRNA-2 targets the wild-type replicon pSC101.

[0008] The plasmid pTargetF does not carry any homologous arms; the use of dual sgRNAs, sgRNA-1 targeting the cas9 gene and sgRNA-2 targeting the wild-type replicon pSC101, is to improve the efficiency of plasmid pCasM excision, making the excision of plasmid pCasM faster and more thorough; if only one sgRNA is used, the efficiency of plasmid pCasM excision is lower.

[0009] Furthermore, the nucleotide sequence of sgRNA-1 is shown in SEQ ID No. 1, and the nucleotide sequence of sgRNA-2 is shown in SEQ ID No. 2.

[0010] Furthermore, the plasmid pTargetF also carries the sacB gene.

[0011] The sacB gene encodes a sucrose-glucanyltransferase, which is lethal in sucrose medium, facilitating the subsequent removal of the helper plasmid.

[0012] Furthermore, the plasmid pTargetF also exhibits resistance to zearalenone.

[0013] The method for constructing a helper plasmid for dual sgRNA expression includes the following steps: Add one copy of sgRNA to plasmid pTargetF to construct plasmid pTargetDouble; Design sgRNA-1 and sgRNA-2, sgRNA-1 targets the cas9 gene of plasmid pCasM, and sgRNA-2 targets the wild-type replicon pSC101 of plasmid pCasM to construct a helper plasmid for dual sgRNA expression (pTargetDouble-pCasM).

[0014] Furthermore, the sacB gene is introduced onto the helper plasmid.

[0015] A CRISPR-Cas9 system, including the aforementioned helper plasmid.

[0016] Furthermore, the CRISPR-Cas9 system also includes plasmid pCasM, which contains the cas9 gene, wild-type replicon pSC101, and arabinose-induced λ-Red system.

[0017] The plasmid pCasM contains the wild-type replicon pSC101, which allows the strain to be cultured at 37°C during the gene editing process, thereby increasing the growth rate of the strain after transformation with plasmid pCasM. Moreover, the wild-type replicon pSC101 can also cooperate with the aforementioned helper plasmid, making it easier to discard plasmid pCasM.

[0018] Furthermore, plasmid pCasM also exhibits kanamycin resistance.

[0019] The application of the aforementioned helper plasmid or the aforementioned CRISPR-Cas9 system in gene editing loss plasmid pCasM.

[0020] Furthermore, during the gene editing process, the strain transformed with plasmid pCasM was cultured at 37°C.

[0021] The method for losing plasmids in gene editing using the CRISPR-Cas9 system includes the following steps: Strains containing plasmid pCasM and with plasmid pTargetF-X excised were screened, induced with arabinose, transformed with the helper plasmid, plated on azithromycin plates, and pCasM was cut to verify whether pCasM was successfully excised.

[0022] Using this method to remove plasmid pCasM not only improves the success rate of plasmid removal but also shortens the time required for plasmid removal, while maintaining more than 70% of the original yield of the strain.

[0023] Further, single colonies of bacteria with successfully removed plasmid pCasM were cultured at 37°C without antibiotics and streaked onto sucrose-containing antibiotic-free plates. Then, the removal of the helper plasmid was verified to be successful, thus completing the removal of all plasmids. All plasmids here include plasmid pCasM and helper plasmids.

[0024] The above-described technical solution of the present invention has at least the following beneficial effects: Compared to the SDS-42℃ method, the pCasM plasmid removal method of this invention significantly improves the removal success rate, increasing the average removal success rate from approximately 8% to 70%, and reducing the average processing time from approximately 5 days to 4 days. If the SDS-42℃ method is used to remove the plasmid, the strain yield after removing the pCasM plasmid is almost zero. Using the pCasM plasmid removal method of this invention, the strain yield after removing the pCasM plasmid can be increased to more than 70% of the original yield (the yield without removing the pCasM plasmid), while preserving the overall production performance of high-yielding strains, making it suitable for gene editing of high-yielding strains. Attached Figure Description

[0025] Figure 1 The image of plasmid pTargetDouble-pCasM-sacB; Figure 2 The bar chart shows the NeuAc yield of the control group and the strains obtained in Example 3 and Comparative Example 2. Figure 3 The bar chart shows the NeuAc yield of the control group and the strains obtained in Example 4 and Comparative Example 3. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the described embodiments of the present invention are within the scope of protection of the present invention.

[0027] The exogenous genes neuB and neuC required for the experiment were derived from Campylobacter jejuni and synthesized through codon optimization; the exogenous gene AnsAGE was derived from Anabaena and synthesized through codon optimization; and the exogenous gene PpGNA1 was derived from Pichia pastoris and synthesized through codon optimization. All of the above gene synthesis was completed by Suzhou Genewiz Company.

[0028] The strains, vectors, and PCR reaction systems involved in the examples are as follows: The cloning host bacterium was Escherichia coli DH5α, and the expression host bacterium was Escherichia coli BL21(DE3). Both strains were purchased from Weidi Biotechnology.

[0029] Plasmids pCas, pEcCas, and pTargetF were purchased from Tolo Biotech. The purchased pCas plasmid carries the Cas9 gene, a mutant thermosensitive replicon pSC101 (encoding the thermosensitive replicon protein RepA101ts, cultured at 30°C), an arabinose-induced λ-Red system, and an IPTG-induced sgRNA targeting the pMB1 replicon, and also exhibits kanamycin resistance. The purchased pTargetF plasmid inherently possesses zizomycin resistance and, compared to pTargetF-X, lacks upstream and downstream homologous arms.

[0030] The PCR reaction system consisted of: Phanta Flash high-fidelity enzyme from Nanjing Novizan Biotechnology Co., Ltd., 25 μL of 2×Phanta Flash Master Mix, 20 μL of ddH2O, 2 μL of upstream primer (10 μM), 2 μL of downstream primer (10 μM), 1 μL of template, and a total volume of 50 μL. The reaction conditions were: 98 ℃ for 3 min, one cycle; 98 ℃ for 10 s, 58 ℃ for 10 s, 72 ℃ for 30 s, 32 cycles; 72 ℃ for 1 min, one cycle; and storage at 4 ℃.

[0031] After PCR, the products were purified using a column PCR product purification kit from Shanghai Sangon Biotech Co., Ltd., and the band sizes were determined by agarose gel electrophoresis.

[0032] Plasmids were constructed using the Seamless Cloning Kit from Shanghai Sangon Biotech Co., Ltd.

[0033] Example 1 A CRISPR-Cas9 system includes a helper plasmid expressing dual sgRNAs and plasmid pCasM. Plasmid pCasM contains the Cas9 gene, the wild-type replicon pSC101, an arabinose-induced λ-Red system, and an IPTG-induced sgRNA targeting the pMB1 replicon. The Cas9 gene, the arabinose-induced λ-Red system, and the IPTG-induced sgRNA targeting the pMB1 replicon are all included with the purchased plasmid pCasM, and it also contains kanamycin resistance (Kana). The method for constructing plasmid pCasM involves replacing the mutant thermosensitive replicon pSC101 carried on plasmid pCas back with the wild-type replicon pSC101, specifically as follows: Plasmid pCas carries a mutant thermosensitive replicon protein RepA101ts, the amino acid sequence of which is shown in SEQ ID No. 8. Compared to the wild-type replicon protein RepA101, the mutant thermosensitive replicon protein RepA101ts has a mutant Ala56Val, the amino acid sequence of which is shown in SEQ ID No. 9. Primers were designed to mutate Val (valine) at position 56 back to Ala (alanine). Specifically, using plasmid pCas as a template, a linear fragment was amplified using primers RepA101-F and RepA101-R, the primer sequences of which are shown in SEQ ID No. 10 and SEQ ID No. 11. The single fragment was seamlessly cloned and transformed into E. coli DH5α and plated on a kanamycin plate. After successful sequencing, the resulting plasmid was named pCasM. The strain transformed with plasmid pCasM could grow normally at 37℃.

[0034] Primer RepA101-F, SEQ ID No. 10: atcatctgaAcgtattggttataagtgaacgataccgt; Primer RepA101-R, SEQ ID No. 11: ttataaccaatacgTtcagatgatgaacatcagtagggaa.

[0035] The method for constructing a helper plasmid for dual sgRNA expression includes the following steps: To enhance CRISPR / Cas9 excision efficiency, a copy of sgRNA was added to plasmid pTargetF to construct plasmid pTargetDouble; sgRNA-1 and sgRNA-2 were designed. sgRNA-1 targets the cas9 gene of plasmid pCasM, and its N20 sequence is shown in SEQ ID No. 1, specifically gatggattgatggataagag. sgRNA-2 targets the wild-type replicon pSC101 of plasmid pCasM, and its N20 sequence is shown in SEQ ID No. 2, specifically ggtctagcggaatttacaga. The resulting plasmid was named pTargetDouble-pCasM and does not carry any homologous arms. The specific preparation method for this step is the same as that described in Chinese patent document CN202010950403.8 (patent application number).

[0036] The sacB gene was added to plasmid pTargetDouble-pCasM for expression. Specifically, using plasmid pEcCas as a template, the sacB fragment was amplified using primers sacB-F and sacB-R (primer sequences shown in SEQ ID No. 12 and SEQ ID No. 13). Using plasmid pTargetDouble-pCasM as a template, the vector fragment was amplified using primers pTD-F and pTD-R (primer sequences shown in SEQ ID No. 14 and SEQ ID No. 15). Seamless cloning and ligation were performed, and the resulting plasmid was transformed into *E. coli* DH5α and plated on a zithromycin plate. After successful sequencing, the plasmid was named pTargetDouble-pCasM-sacB. The amino acid sequence of the protein encoded by the sacB gene is shown in SEQ ID No. 7. A helper plasmid for double sgRNA expression (named pTargetDouble-pCasM-sacB) was constructed. The sacB gene encodes a sucrose-glucanyltransferase, which is lethal in sucrose medium. Plasmid pTargetF inherently carries zithromycin resistance.

[0037] Primer sacB-F, SEQ ID No. 12: Gaagcttagatctattgaattcgaatgaggtcgaaaagtaaatcgcgc; Primer sacB-R, SEQ ID No. 13: gtACCCTGCAGactagttctagatttttttgaggaatacggttagccat; Primer pTD-F, SEQ ID No. 14: TCTAGAACTAGTCTGCAGGGTAC; Primer pTD-R, SEQ ID No. 15: GAATTCAATAGATCTAAGCTTC.

[0038] Example 2 The CRISPR-Cas9 system also includes plasmid pTargetF-X. Plasmid pCasM and plasmid pTargetF-X constitute the dual plasmid pCasM / pTargetF-X gene editing system. First, the dual plasmid pCasM / pTargetF-X gene editing system is used to edit the strain's genes, and the specific gene editing method is the same as the existing technology. Then, the helper plasmid expressing dual sgRNA (pTargetDouble-pCasM-sacB) is used to remove plasmid pCasM.

[0039] The method for losing plasmids in gene editing using the CRISPR-Cas9 system described in Example 1 includes the following steps: After gene editing of the strain, IPTG was added. The sgRNA carried by plasmid pCasM (targeting the pMB1 replicon) was used to target and excise plasmid pTargetF-X. Strains containing plasmid pCasM were kanamycin resistant (Kana). Strains containing plasmid pCasM were screened by streaking kanamycin plates. Then, arabinose was induced, and plasmid pTargetDouble-pCasM-sacB was transformed. The plasmids were then plated with azithromycin (Sm). At this time, the Cas9 protein, guided by two sgRNAs, sgRNA-1 and sgRNA-2, targeted plasmid pCasM for complete cleavage. The cleavage fragments were degraded by the cells. Strains containing plasmid pTargetDouble-pCasM-sacB were screened by azithromycin. Finally, colony PCR was used to verify whether plasmid pCasM was successfully excised. (4) Remove plasmid pTargetDouble-pCasM-sacB Single colonies of bacteria that successfully lost plasmid pCasM were cultured overnight at 37°C without antibiotics. They were then streaked onto antibiotic-free plates containing 10 g / L sucrose, which killed the plasmid pTargetDouble-pCasM-sacB. Colony PCR was then performed to verify whether plasmid pTargetDouble-pCasM-sacB had been successfully lost, thus completing the loss of all plasmids.

[0040] Comparative Example 1 The CRISPR-Cas9 system used in Comparative Example 1 includes plasmid pCasM and plasmid pTargetF-X. Plasmid pCasM carries the cas9 gene, the arabinose-induced λ-Red system, the wild-type replicon pSC101, and the sgRNA induced by IPTG that targets the pMB1 replicon. Plasmid pTargetF-X carries the sgRNA that targets the target gene, the pMB1 replicon, and upstream and downstream homologous arms.

[0041] The difference between this CRISPR-Cas9 system and the existing dual-plasmid pCas / pTargetF-X gene editing system (see Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System. Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S. Appl Environ Microbiol. 2015Apr;81(7):2506-14) is that the mutant thermosensitive replicon pSC101 (which encodes the thermosensitive replicon protein RepA101ts, and the strain is cultured at 30℃) is replaced with the wild-type replicon pSC101 (which encodes the normal replicon protein RepA101, and the strain is cultured at 37℃), and everything else is the same.

[0042] The method for plasmid loss in gene editing using the CRISPR-Cas9 system in this comparative example includes the following steps: After gene editing was completed and plasmid pTargetF-X was successfully removed, strains containing plasmid pCasM were kanamycin resistant (Kana). Strawberries containing plasmid pCasM were screened by streaking kanamycin plates. Then, sodium dodecyl sulfate (SDS) was added and the culture was carried out at 42°C to remove plasmid pCasM.

[0043] Construction of recombinant strain Escherichia coli BL21(DE3)△nanATEK△nagEBA / pCasM nanATEK: Escherichia coli nan gene cluster, encoding NeuAc degradation-related genes; nagEBA: nag gene cluster, encoding N-acetylglucosamine degradation-related genes. The following gene editing techniques were employed to knock out NeuAc lysin nanA (NCBI Gene ID: 947742), NeuAc transporter nanT (NCBI Gene ID: 947740), N-acetylglucosamine-2-epimerase nanE (NCBI Gene ID: 947745), N-acetylglucosamine kinase nanK (NCBI Gene ID: 947757), N-acetylglucosamine-specific IIABC component nagE (NCBI Gene ID: 945292), glucosamine-6-phosphate deaminase nagB (NCBI Gene ID: 945290), and N-acetylglucosamine-6-phosphate deacetylase nagA (NCBI Gene ID: 945289) in *E. coli* BL21(DE3) using CRISPR / Cas9 gene editing technology (dual plasmid pCasM / pTargetF-X gene editing system). The specific methods are as follows: First, the pCasM plasmid containing the Cas9 gene was transformed into *E. coli* BL21(DE3) using electroporation. Next, knockout plasmids were constructed. The N20 sequence for knocking out nanATEK was designed as shown in SEQ ID No. 3 (gttgttgatcaaaaggagtc), and the N20 sequence for knocking out nagEBA was designed as shown in SEQ ID No. 4 (gctcagggccatgttgtggc). Using the *E. coli* BL21(DE3) genome as a template, approximately 1000 bp upstream and downstream fragments of the knockout site were amplified by PCR. Then, using a seamless cloning kit, the pTargetF-X vector fragment containing the sequence shown in SEQ ID No. 3 (for constructing the nanATEK knockout plasmid) and the pTargetF-X vector fragment containing the sequence shown in SEQ ID No. 3 were used. The pTargetF-X vector fragment (constructing the nagEBA knockout plasmid) shown in No. 4 was ligated, transformed into E. coli DH5α, and plated on antibiotic resistance plates (azithromycin plates). Single colonies were picked and sequenced. After successful sequencing, E. coli BL21(DE3) / pCasM electrocompetent cells were prepared. The knockout plasmid was electrotransformed into these cells, plated on antibiotic resistance plates (azithromycin plates), and single colonies were picked for colony PCR verification. After successful knockout verification, IPTG was added to culture and the knockout plasmid pTargetF-X was removed. The nanATEK and nagEBA gene clusters were knocked out sequentially using this method. After successful knockout of all genes, the host strain BL21(DE3)△nanATEK△nagEBA / pCasM was obtained.

[0044] Construction of NeuAc (N-acetylneuraminic acid) producing strains Using CRISPR / Cas9 gene editing technology (dual plasmid pCasM / pTargetF-X gene editing system), the following enzymes were integrated into *E. coli* BL21(DE3)ΔnanATEKΔnagEBA: *Campylobacter jejuni*-derived hydrolytic UDP-N-acetylglucosamine-2-epomerase neuC (NCBI Gene ID: 905433, neuC amino acid sequence as shown in SEQ ID No. 4), *Campylobacter jejuni*-derived NeuAc synthase neuB (NCBI Gene ID: 905432, neuB amino acid sequence as shown in SEQ ID No. 3), *AnsAGE*-derived N-acetylglucosamine-2-epomerase AnsAGE (NCBI GeneBank: ABG57043.1, AnsAGE amino acid sequence as shown in SEQ ID No. 5), *E. coli*-derived 1-phosphofructophosphatase yqaB (NCBI Gene ID: 945776), and *Pichia pastoris*-derived 6-phosphoglucosamine-N-acetyltransferase PpGNA1 (NCBI Gene ID: 945776). ID: 8201148, PpGNA1 amino acid sequence is shown in SEQ ID No. 6.

[0045] The N20 sequence for integration at the wcaK site is designed as shown in SEQ ID No. 5, specifically cgccacgattgccgcaagtg, and the N20 sequence for integration at the recA site is designed as shown in SEQ ID No. 6, specifically gtcttcacccaggcgcatga. Unlike knockout plasmid construction, when constructing the integration plasmid, the integrating gene fragment (Ptac-neuB-neuC fragment, where Ptac is the tac promoter or T7-neuB-AnsAGE-yqaB-PpGNA1 fragment, where T7 is the T7 promoter) needs to be ligated between the upstream and downstream homologous fragments. The rest is the same, integrating the Ptac-neuB-neuC fragment and the T7-neuB-AnsAGE-yqaB-PpGNA1 fragment, respectively. The B-PpGNA1 fragment yielded the NeuAc-producing strain: BL21(DE3)△nanATEK△nagEBA,△wcaK::Ptac-neuBC / pCasM, where wcaK is pyruvyltransferase synthesized from collamerate, and neuBC is neuB-neuC; BL21(DE3)△nanATEK△nagEBA,△recA::T7-neuB-AnsAGE-yqaB-PpGNA1 / pCasM, where recA is the recombinant protein RecA.

[0046] Example 3 The BL21(DE3)△nanATEK△nagEBA,△wcaK::Ptac-neuBC / pCasM strain was transformed with plasmid pTargetDouble-pCasM-sacB using the method described in Example 2. The specific method for discarding plasmid pCasM is as follows: Strains containing the plasmid pCasM were screened by streaking BL21(DE3)△nanATEK△nagEBA,△wcaK::Ptac-neuBC / pCasM onto kanamycin plates. Single colonies were inoculated into LB liquid medium and cultured overnight at 37°C. The next day, the colonies were transferred and arabinose was added for induction to create electrocompetent cells. The plasmid pTargetDouble-pCasM-sacB was electrotransformed once and plated onto azithromycin (Sm) plates. At this time, the Cas9 protein, guided by sgRNA-1 and sgRNA-2, targeted the plasmid pCasM for cleavage. The cleavage fragments were degraded by the cells. Strains containing the plasmid pTargetDouble-pCasM-sacB were screened by azithromycin. On the third day, colony PCR confirmed that the success rate of removing the plasmid pCasM was 70% (7 / 10). Remove plasmid pTargetDouble-pCasM-sacB The single colony with successfully removed plasmid pCasM was inoculated into antibiotic-free LB liquid medium and cultured overnight at 37°C. It was then streaked onto antibiotic-free plates containing 10 g / L sucrose. Plasmid pTargetDouble-pCasM-sacB was lethal under the action of sucrose. On the fourth day, colony PCR was performed to verify that the success rate of removing plasmid pTargetDouble-pCasM-sacB was 100% (10 / 10), that is, all plasmids were removed, resulting in BL21(DE3)△nanATEK△nagEBA,△wcaK::Ptac-neuBC strain-1. The above process took a total of 4 days.

[0047] Comparative Example 2 The BL21(DE3)△nanATEK△nagEBA,△wcaK::Ptac-neuBC / pCasM strain was subjected to the method described in Comparative Example 1, with the plasmid pCasM removed at SDS-42℃. The specific method for removing the pCasM plasmid using this method is as follows: Strains containing the pCasM plasmid were screened by streaking the BL21(DE3)△nanATEK△nagEBA,△wcaK::Ptac-neuBC / pCasM strain onto kanamycin plates. Single colonies were inoculated into LB broth containing 5% sodium dodecyl sulfate (SDS) and incubated overnight at 42°C. Substitution was performed every 12 hours, and after 7 subcultures, the colonies were streaked onto antibiotic-free plates. Colony PCR verification showed a rejection rate of 8% (2 / 25). The total incubation time was 5 days (4 days for subculture + 1 day for streaked single colony growth), yielding strain BL21(DE3)△nanATEK△nagEBA,△wcaK::Ptac-neuBC-2. The "7 subcultures" refers to 7 consecutive cultures of the same colony; testing showed that 7 consecutive cultures were necessary for the rejection of the pCasM plasmid.

[0048] The NeuAc yield shake flask test method is as follows: The BL21(DE3)△nanATEK△nagEBA,△wcaK::Ptac-neuBC / pCasM strain (control group) was inoculated into 5 ml LB liquid medium, and kanamycin was added as needed. The culture was incubated overnight at 37 ℃ and 220 rpm. The next day, the culture was transferred to 50 ml LBG liquid medium in shake flasks for testing. The inoculation amount was 2%, and the culture was carried out at 37 ℃ and 220 rpm for 5 h. The culture temperature was then lowered to 25 ℃, and the NeuAc yield was determined by high performance liquid chromatography-differential refractive index (HPLC-RIA). The results are as follows: Figure 2 As shown.

[0049] The BL21(DE3)△nanATEK△nagEBA,△wcaK::Ptac-neuBC strain-1 obtained in Example 3 and the BL21(DE3)△nanATEK△nagEBA,△wcaK::Ptac-neuBC strain-2 obtained in Comparative Example 2 were inoculated into 5 ml LB liquid medium and cultured overnight at 37 ℃ and 220 rpm. The next day, they were transferred to 50 ml LBG liquid medium in shake flasks for testing. The inoculation amount was 2%, and the culture was carried out at 37 ℃ and 220 rpm for 5 h. The culture temperature was then reduced to 25 ℃, and the NeuAc yield was determined by high performance liquid chromatography-differential refractive index. The results are as follows. Figure 2 As shown.

[0050] Depend on Figure 2It can be seen that in Comparative Example 2, after removing plasmid pCasM using the SDS-42℃ method, the yield of the engineered strain NeuAc almost dropped to 0, verifying that both the knockout and integrated genes were sequenced correctly. The suspected reason is that the strain was significantly damaged during the removal of plasmid pCasM, resulting in a substantial decrease in production performance to almost complete loss. In contrast, in Example 3, after removing plasmid pCasM using the transformation plasmid pTargetDouble-pCasM-sacB method, the decrease in NeuAc yield was less severe, and the overall production performance of the strain was preserved. The yield of the strain obtained in Example 3 was 78.57% of that of the control group strain, thus it was superior.

[0051] Example 4 Using the method described in Example 2, the plasmid pCasM in BL21(DE3)△nanATEK△nagEBA,△recA::T7-neuB-AnsAGE-yqaB-PpGNA1 / pCasM was removed. The specific method is as follows: Strawberries of strain BL21(DE3)△nanATEK△nagEBA,△recA::T7-neuB-AnsAGE-yqaB-PpGNA1 / pCasM were streaked onto kanamycin plates to screen for strains containing plasmid pCasM. Single colonies were inoculated onto LB broth and cultured overnight at 37°C. The next day, the colonies were transferred and arabinose was added for induction to create electrocompetent cells. The plasmid pTargetDouble-pCasM-sacB was then electrotransformed and plated onto azithromycin (Sm) plates. At this point, the Cas9 protein, guided by sgRNA-1 and sgRNA-2, targeted and cleaved the plasmid pCasM. The cleaved fragments were degraded by the cells. Strawberries containing plasmid pTargetDouble-pCasM-sacB were screened using azithromycin. On the third day, colony PCR confirmed a 70% (7 / 10) success rate in removing plasmid pCasM. Remove plasmid pTargetDouble-pCasM-sacB Single colonies with successfully removed plasmid pCasM were inoculated into antibiotic-free LB liquid medium and cultured overnight at 37°C. They were then streaked onto antibiotic-free plates containing 10 g / L sucrose, which caused the plasmid pTargetDouble-pCasM-sacB to die under sucrose. On the fourth day, colony PCR confirmed that the success rate of removing plasmid pTargetDouble-pCasM-sacB was 100% (10 / 10), meaning that all plasmids had been removed, resulting in strain BL21(DE3)△nanATEK△nagEBA,△recA::T7-neuB-AnsAGE-yqaB-PpGNA1-1. The entire process took 4 days.

[0052] Comparative Example 3 Using the method of Comparative Example 1, the plasmid pCasM in BL21(DE3)△nanATEK△nagEBA,△recA::T7-neuB-AnsAGE-yqaB-PpGNA1 / pCasM was removed. The specific method is as follows: Strawberry strain BL21(DE3)△nanATEK△nagEBA,△recA::T7-neuB-AnsAGE-yqaB-PpGNA1 / pCasM was streaked onto kanamycin plates to screen for strains containing plasmid pCasM. Single colonies were inoculated into LB broth containing 5% sodium dodecyl sulfate (SDS) and incubated overnight at 42°C. Substitution was performed every 12 hours, and after 7 subcultures, the culture was streaked onto antibiotic-free plates. Colony PCR verification showed a rejection rate of 8% (2 / 25). The entire process took 5 days, including 4 days of subculture and 1 day of streaked colony growth, yielding strain BL21(DE3)△nanATEK△nagEBA,△recA::T7-neuB-AnsAGE-yqaB-PpGNA1-2. Here, 7 subcultures refer to 7 consecutive cultures of the same colony; testing showed that 7 consecutive cultures were necessary for the rejection of plasmid pCasM.

[0053] The NeuAc yield of strain BL21(DE3)△nanATEK△nagEBA,△recA::T7-neuB-AnsAGE-yqaB-PpGNA1 / pCasM (control group) was tested. Inoculated into 5 ml LB liquid medium, kanamycin was added as needed, and the culture was incubated overnight at 37°C and 220 rpm. The next day, the culture was transferred to 50 ml LBG liquid medium in shake flasks for testing. The inoculation amount was 2%, and the culture was carried out at 37°C and 220 rpm for 5 h. 0.2 mM IPTG was added for induction, and the culture temperature was lowered to 25°C. After 48 h of culture, the NeuAc yield was determined by high-performance liquid chromatography-differential refractive index (HPLC-RIA). The results are as follows: Figure 3 As shown.

[0054] The BL21(DE3)△nanATEK△nagEBA,△recA::T7-neuB-AnsAGE-yqaB-PpGNA1 strain-1 obtained in Example 4 and the BL21(DE3)△nanATEK△nagEBA,△recA::T7-neuB-AnsAGE-yqaB-PpGNA1 strain-2 obtained in Comparative Example 3 were inoculated into 5 ml LB liquid medium and cultured overnight at 37 ℃ and 220 rpm. The next day, they were transferred to 50 ml LBG liquid medium in shake flasks for testing. The inoculation amount was 2%, and the culture was carried out at 37 ℃ and 220 rpm for 5 h. 0.2 mM IPTG was added for induction, and the culture temperature was lowered to 25 ℃. After 48 h of culture, the yield of NeuAc was determined by high performance liquid chromatography-differential refractive index. The results are as follows. Figure 3 As shown.

[0055] Depend on Figure 3 It can be seen that in Comparative Example 3, after removing plasmid pCasM using the SDS-42℃ method, the yield of the engineered strain NeuAc also dropped to almost zero. Furthermore, the sequencing of both the knockout and integrated genes was verified to be error-free. The suspected reason is that the strain was significantly damaged during the removal of plasmid pCasM, resulting in a substantial decrease in production performance to almost complete loss. In contrast, in Example 4, after removing plasmid pCasM using the pTargetDouble-pCasM-sacB transformation method, the decrease in NeuAc yield was less pronounced, and the overall production performance of the strain was preserved. The yield of the strain obtained in Example 4 was 72.92% of that of the control group strain, thus demonstrating superior performance.

[0056] The culture media used in the examples and comparative examples are as follows: LB liquid medium: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L.

[0057] LBG liquid medium: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, glucose 15 g / L.

[0058] LB solid medium: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar 20 g / L. This invention uses LB solid medium for both coating and streaking.

[0059] The method for determining NeuAc yield by high performance liquid chromatography-differential refractive index is as follows: The NeuAc content was determined using high-performance liquid chromatography (HPLC) (Agilent 1260 series, USA). Specifically, 1 mL of fermentation broth was centrifuged at 12,000 rpm for 5 min at room temperature, the supernatant was collected, and then filtered through an aqueous membrane with a pore size of 0.22 μm. The product concentration was then determined by HPLC. The HPLC instrument was equipped with a UV detector (195 nm) and a differential refractive index detector (RID-10A). The detection was performed using the differential refractive index detector. The chromatographic column was an Aminex HPX-87H (300 mm × 7.8 mm), the mobile phase was 25 mM dilute sulfuric acid, the detection temperature was 65 ℃, and the flow rate was 0.6 mL / min.

[0060] neuB amino acid sequence, SEQ ID No. 3: MKEIKIQNIIISEEKAPLVVPEIGINHNGSLELAKIMVDAAFSAGAKIIKHQTHIVEDEMSKAAKKVIPGNAKISIYEIMQKCALDYKDELALKEYTEKLGLVYLSTPFSRAGANRLEDMGVSAFKIGSGECNNYPLIKHIAAFKKPMIVSTGMNSIESIKPTVKILLDNEIP FVLMHTTNLYPTPHNLVRLNAMLELKKEFSCMVGLSDHTTDNLACLGAVVLGACVLERHFTDSMHRSGPDIVCSMDTKALKELIIQSEQMAIIRGNNESKKAAKQEQVTIDFAFASVVSIKDIKKGEVLSMDNIWVKRPGLGGISAAEFENILGKKALRDIENDAQLSYEDFA.

[0061] neuC amino acid sequence, SEQ ID No. 4: MKKILFITGSRADYSKIKSLMYRVQNSSEFELYIFATGMHLSKNFGYTVKELYKNGFKNIYEFINYDKYYQTDKALATTIDGFSRYANELKPDLIVVHGDRIEPLAAAIVGALNNILVAHIEGGEISGTIDDSLRHAISKLAHIHLVNDEFAKRRLMQLGEDEKSIFIIGSPDLELLNDNKISLSEAKKYYDINYENYALLMFHPVTTEITSIKNQADNLVKALIQSNKNYIVIYPNNDLGFELILQSYEEFKNNPRFKLFPSLRFEYFITLLKNADFIIGNSSCILKEALYLKTAGILVGSRQNGRLGNENTLKVNANSDEILKAINTIHKKQDLFSAKLEILDSSKLFFEYLQSGDFFKLSTQKVFKDIK。

[0062] AnsAGE amino acid sequence, SEQ ID No. 5: MGKNLQALAQLYKNALLNDVLPFWENHSLDSEGGYFTCLDRQGKVYDTDKFIWLQNRQVWTFSMLCNQLEKRENWLKIARNGAKFLAQHGRDDEGNWYFALTRGGEPLVQPYNIFSDCFAAMAFSQYALASGEEWAKDVAMQAYNNVLRRKDNPKGKYTKTYPGTRPMKALAVPMILANLTLEMEWLLPQETLENVLAATVQEVMGDFLDQEQGLMYENVAPDGSHIDCFEGRLINPGHGIEAMWFIMDIARRKNDSKTINQAVDVVLNILNFAWDNEYGGLYYFMDAAGHPPQQLEWDQKLWWVHLESLVALAMGYRLTGRDACWAWYQKMHDYSWQHFADPEYGEWFGYLNRRGEVLLNLKGGKWKGCFHVPRAMYLCWQQFEALS。

[0063] PpGNA1 amino acid sequence, SEQ ID No. 6: MQPVSVPALPQGYNLRRVGKEDFQDKNLFKTLSILTTVGDIPEPKFHALIEYWNDRKEIYNPMVITNAENVIIATGMLFVEHKLIHGGGKVGHIEDISVNPSEQGKKLGLIMIRNLIQIAQTEGCYKVILDCDEKNVRFYEKCGMKIEGVEMGYRF。

[0064] Amino acid sequence of sacB, SEQ ID No.7: MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISHITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYHIVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEWSGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDGKTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKAYYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDEIERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLKMDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSVVKDSILEQGQLTVNK。

[0065] Amino acid sequence of RepA_{101}ts, SEQ ID No.8: MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKERTVSFTYNQYVQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFSSEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANIEISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQVELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLRNGLRKTLHDALTAKIQLTSFEAKFLSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI。

[0066] Amino acid sequence of RepA101, SEQ ID No.9: MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKERTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFSSEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANIEISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQVELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLRNGLRKTLHDALTAKIQLTSFEAKFLSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI。

Claims

1. A helper plasmid for dual sgRNA expression, characterized in that: The plasmid pTargetF serves as the backbone, carrying sgRNA-1 and sgRNA-2; sgRNA-1 targets the cas9 gene, and sgRNA-2 targets the wild-type replicon pSC101.

2. The helper plasmid for dual sgRNA expression according to claim 1, characterized in that: The nucleotide sequence of sgRNA-1 is shown in SEQ ID No. 1, and the nucleotide sequence of sgRNA-2 is shown in SEQ ID No.

2.

3. A helper plasmid for dual sgRNA expression according to claim 1 or 2, characterized in that: The plasmid pTargetF also carries the sacB gene.

4. The method for constructing the auxiliary plasmid according to any one of claims 1-3, characterized in that, Includes the following steps: Add one copy of sgRNA to plasmid pTargetF to construct plasmid pTargetDouble; We designed sgRNA-1 and sgRNA-2, sgRNA-1 targeting the cas9 gene of plasmid pCasM, and sgRNA-2 targeting the wild-type replicon pSC101 of plasmid pCasM, and constructed a helper plasmid for dual sgRNA expression.

5. The construction method according to claim 4, characterized in that, The sacB gene was introduced into the helper plasmid.

6. A CRISPR-Cas9 system, characterized in that, Includes the auxiliary plasmid as described in any one of claims 1-3.

7. A CRISPR-Cas9 system according to claim 6, characterized in that, The CRISPR-Cas9 system also includes the plasmid pCasM, which contains the cas9 gene, the wild-type replicon pSC101, and the arabinose-induced λ-Red system.

8. The use of the helper plasmid according to any one of claims 1-3 or the CRISPR-Cas9 system according to claim 6 or 7 in the gene editing plasmid pCasM.

9. A method for dropping plasmid pCasM in gene editing using the CRISPR-Cas9 system as described in claim 6, characterized in that, Includes the following steps: Strains containing plasmid pCasM and with plasmid pTargetF-X excised were screened, induced with arabinose, transformed with the helper plasmid, plated on azithromycin plates, and pCasM was cut to verify whether pCasM was successfully excised.

10. The method according to claim 9, characterized in that, Single colonies of bacteria from which plasmid pCasM was successfully removed were cultured at 37°C without antibiotics and streaked onto antibiotic-free plates containing sucrose. Then, the removal of the auxiliary plasmids was verified to be successful, thus completing the removal of all plasmids.

Images