CRISPR-Cas9-based pig pasteurella multocida gene editing vector as well as construction method and application thereof

Abstract

The invention belongs to the technical field of gene engineering, and particularly discloses a pig pasteurella multocida gene editing vector based on CRISPR-Cas9 as well as a construction method and application of the pig pasteurella multocida gene editing vector based on CRISPR-Cas9, escherichia coli-pasteurella multocida shuttle plasmid pMI28 is taken as a skeleton, plasmids pCas9, psodc-tpia and the like are taken as templates, and pasteurella multocida conjugational transfer type gene editing plasmid pSCTcas9 is constructed; the CRISPR-Cas9 gene editing system constructed by the invention can efficiently construct the pasteurella multocida gene deletion strain, and compared with the traditional method, the operation is simpler and more convenient, the consumed time is shorter, the in-depth study on the gene function of the pasteurella multocida is facilitated, and a foundation is laid for revealing the pathogenic mechanism and the drug resistance mechanism of the pasteurella multocida; through targeted elimination of drug-resistant genes and direct sterilization, the drug resistance of the pasteurella multocida can be reduced, the sensitivity of the pasteurella multocida to antibiotics can be recovered, a new strategy is provided for clinical treatment of pasteurella multocida infection, and the current antibiotic resistance crisis can be relieved.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a CRISPR-Cas9-based gene editing vector for Pasteurella multocida in pigs, its construction method, and its application. Background Technology

[0002] Since the discovery of antibiotics in the mid-19th century, they have been widely used to treat diseases caused by various pathogens, achieving excellent results. However, due to the irrational use and abuse of antibiotics, some bacteria have developed resistance, leading to multidrug resistance, including Pasteurella multocida. In recent years, the prevalence of multidrug-resistant bacteria in animals and humans, as well as the difficulty in treating bacterial infections, have increased.

[0003] Pasteurella multocida is a Gram-negative zoonotic bacterium that can cause diseases such as swine pneumonia and atrophic rhinitis, resulting in significant economic losses to the livestock industry. Currently, the clinical prevention and treatment of Pasteurella multocida infection mainly relies on antibiotics. However, with the widespread use of antibiotics, bacterial resistance is becoming increasingly serious, and multidrug resistance is common, posing a significant challenge to the effectiveness of existing treatment methods.

[0004] In the study of gene function in *Pasteurella multocida*, traditional methods, such as suicide plasmid-mediated homologous recombination, suffer from drawbacks such as long processing time and high workload, making it difficult to meet the demand for rapid and efficient gene function research. The CRISPR-Cas9 system, as a novel gene editing technology, has the advantages of simple operation and high gene editing efficiency, and has been applied in various organisms, but its application in *Pasteurella multocida* is relatively limited. Therefore, developing an efficient and convenient gene editing method for *Pasteurella multocida* and applying it to fields such as drug resistance control is of significant practical importance.

[0005] The application of the traditional CRISPR-Cas9 system in Pasteurella multocida is limited by problems such as low vector delivery efficiency and poor host adaptability. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a CRISPR-Cas9-based gene editing vector for Pasteurella multocida in pigs, its construction method, and its application.

[0007] To achieve the above objectives, the present invention is implemented according to the following technical solution: The first technical solution provided by this invention is a method for constructing a CRISPR-Cas9-based gene editing vector for Pasteurella multocida in porcine bacteria, comprising the following steps: S1. Using the Escherichia coli-Pasteurella multocida shuttle plasmid pMI28 as a template, the pan-host replicon ori and the kanamycin resistance gene KanR were amplified. S2. Using psodc-tpia plasmid as a template, amplify the promoters psodc and ptpia; using pCas9 plasmid as a template, amplify the gRNA scaffold; using pCasSA plasmid as a template, amplify the cas9 gene; splice psodc, gRNA scaffold, and ptpia by overlap PCR, and perform seamless cloning with the cas9 gene and pMI28 to obtain the intermediate vector pSTcas9. S3. Using the suicide plasmid pRE112 as a template, the conjugation transfer sequence was amplified, digested, and ligated into the intermediate vector pSTcas9 to construct the vector pSCTcas9.

[0008] Further, in step S1, the primers used to amplify the pan-host replicon ori and the kanamycin resistance gene KanR are p28fx, including: F: 5'-TCTGGACTCAGATCTCGAGCTCA-3', and R: 5'-GGTATATCTCCTTCGCATGCCT-3'.

[0009] Further, in step S2, the primers used to amplify the gRNA scaffold are sg, including: gRNA scaffold forward primer: 5'-TCAGCTTTATCCACGTCGCACCGACTCGGTGCCAC-3', and gRNA scaffold reverse primer: 5'-AGGAGGAAACGAGACCATTGGTCTCAGTT-3'; The primers used to amplify the Cas9 gene are tycas9, including: CAS9 forward primer: GCATGCGAAGGAGATATACCTCAGTCACCTCCTAGCTGACTCAA-3', and CAS9 reverse primer: 5'-CGGAGAAAATTATGGATAAGAAATACTCAATAGGCTTAGA-3'.

[0010] Further, in step S3, the primers used to amplify the conjugation transfer sequence are 112-ta9, comprising: F: 5'-TGACTGCAGCTGGATGTCGACCCGTCACAGGTATTAGGGCCC-3', and R: 5'-TGAACATCAACCCAGGTCGACCGTTTCGTGATTGTCACGCTC-3'.

[0011] The second technical solution provided by the present invention is a CRISPR-Cas9-based gene editing vector for Pasteurella multocida in pigs constructed using the above method.

[0012] The third technical solution provided by this invention is the application of the above-mentioned CRISPR-Cas9-based *Pasteurella multocida* gene editing vector in constructing *Pasteurella multocida* gene deletion strains, including the following steps: 1) Design upstream and downstream homologous arms Lupp and Rupp and target sequence BDupp for the target gene upp; The Lupp primer sequences include: F:5'-CGGTGGAGCCCGGGCCCGCGGATTCGCTAATGAAAAAAATTGTTGTT-3', and R: 5'-GTTGGGATTACATGGTAGGCTCCTTTTGGGT-3'; The Rupp primer sequences include: F: 5'-GCCTACCATGTAATCCCAACACAAGCGGCA-3', and R:5'-CTGCGGTAGACGGCACCGCGGAGCTTCTAATTTAAATTCAGGGGTAG-3'; BDupp target sequences include: F: 5'-GAAAAGGCAGTTTATTAACATACG-3', and R: 5'-AAACCGTATGTTAATAAACTGCCT-3'; 2) Connect the homologous arms and target sequences to the vector pSCTcas9 to construct the pSCTcas9-ULR-bu plasmid; 3) Gene-deleted strain CVCC434-Δupp was obtained by conjugation of Escherichia coli WM3064 to Pasteurella multocida.

[0013] The fourth technical solution provided by this invention is the application of the above-mentioned CRISPR-Cas9-based Pasteurella multocida gene-editing vector in targeted sterilization, including the following steps: 1) Design the target sequence BDkmt1 of the chromosome gene kmt1 and construct the pSCTcas9-bk plasmid; The BDkmt1 target sequence includes: F: 5'-AAACCACTGGGTAAATAGCGGATA-3', and R: 5'-GAAATATCCGCTATTTACCCAGTG-3'; 2) By introducing Pasteurella multocida through conjugation transfer, the bacteria are targeted to cut chromosomes, leading to bacterial death and achieving specific sterilization.

[0014] The fifth technical solution provided by this invention is the application of the above-mentioned CRISPR-Cas9-based Pasteurella multocida gene editing vector in eliminating drug-resistant plasmids, comprising the following steps: 1) Design the target sequence BDflor against the drug resistance gene floR and construct the pSCTcas9-bf plasmid; The BDflor target sequence includes: F: 5'-GAAACCGTGTGCTCATAGGCCAAG-3', and R: 5'-AAACCTTGGCCTATGAGCACACGG-3'; 2) The drug resistance gene floR was eliminated by introducing the clinical strain GDA22P84P containing the drug resistance plasmid through conjugation transfer.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1) This invention uses the Escherichia coli-Pasteurella multocida shuttle plasmid pMI28 as the backbone and plasmids pCas9 and psodc-tpia as templates to construct the Pasteurella multocida conjugation transfer gene editing plasmid pSCTcas9. The CRISPR-Cas9 gene editing system constructed in this invention can efficiently construct Pasteurella multocida gene deletion strains. Compared with traditional methods, it is simpler to operate and less time-consuming, which helps to conduct in-depth research on the gene function of Pasteurella multocida and lays the foundation for revealing its pathogenic mechanism and drug resistance mechanism.

[0016] 2) This invention reduces the drug resistance of Pasteurella multocida by targeting and eliminating drug-resistant genes and directly killing bacteria, restoring its sensitivity to antibiotics. This provides a new strategy for the clinical treatment of Pasteurella multocida infection and helps alleviate the current antibiotic resistance crisis.

[0017] 3) It has enriched the research methods for Pasteurella multocida, provided technical support for the development of new vaccines and diagnostic methods, promoted the healthy development of animal husbandry, and protected the health of animals and humans. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the CRISPR-Cas9 system's operation.

[0019] Figure 2 This is a map of the conjugation transfer plasmid pSCTcas9.

[0020] Figure 3 Gel electrophoresis image used to verify the deletion of the upp gene in Pasteurella multocida.

[0021] Figure 4 Comparison of single colony growth on resistance plates without and with the kmt1 gene target: a) Growth of Pasteurella multocida containing the kmt1 gene target plasmid pSCTcas9-bk; b) Growth of Pasteurella multocida containing the original plasmid pSCTcas9.

[0022] Figure 5 Gel electrophoresis image used to verify the elimination of Pasteurella multocida floR. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0024] The materials used in the following embodiments are as follows: Experimental samples, strains and plasmids: Pig lung samples were collected from a slaughterhouse in Guangdong Province between 2020 and 2022; the strains used included Escherichia coli ATCC25922, Escherichia coli DH5α, Escherichia coli WM3064, and Pasteurella multocida CVCC434; the plasmids used included pCas9, pCasSA, pMI28, psodc-tpia, etc., and some plasmids were constructed for this experiment.

[0025] Reagents and culture media: Prepare various reagents such as anhydrous magnesium sulfate, tris(hydroxymethyl)aminomethane, and EDTA disodium salt dihydrate; and various culture media such as LB broth, LB nutrient agar, and hydrolyzed casein broth. For detailed preparation methods, please refer to the instruction manual.

[0026] Experimental reagents: Prepare a variety of experimental reagents such as diaminobutyric acid, kanamycin sulfate, and ceftiofur, and determine their purity and source.

[0027] Main instruments and equipment: A variety of instruments and equipment are used, including an SW-CJ-2F double-sided vertical clean bench and an HZC-280 constant temperature shaking incubator, to ensure stable and accurate experimental conditions. Example 1: Construction of a CRISPR-Cas9-based gene editing vector for Pasteurella multocida (conjugation transfer plasmid pSCTcas9) like Figure 1The diagram shown illustrates the working principle of the CRISPR-Cas9 system in its three stages of adaptation, expression, and interference, which helps to understand the molecular mechanism of gene editing in this invention. The specific process is as follows: 1) The pan-host replicon ori and the kanamycin resistance gene KanR were amplified using the Escherichia coli-Pasteurella multocida shuttle plasmid pMI28 as a template; the primers used for amplifying the pan-host replicon ori and the kanamycin resistance gene KanR were p28fx, including: F: 5'-TCTGGACTCAGATCTCGAGCTCA-3', and R: 5'-GGTATATCTCCTTCGCATGCCT-3'; 2) Using psodc-tpia plasmid as a template, amplify the promoters psodc and ptpia; using pCas9 plasmid as a template, amplify the gRNA scaffold; using pCasSA plasmid as a template, amplify the Cas9 gene; splice psodc, the gRNA scaffold, and ptpia using overlap PCR, and perform seamless cloning with the Cas9 gene and pMI28 to obtain the intermediate vector pSTcas9; the primers used for amplifying the gRNA scaffold are SG, including: gRNA scaffold forward primer: 5'-TCAGCTTTATCCACGTCGCACCGACTCGGTGCCAC-3', and gRNA scaffold reverse primer: 5'-AGGAGGAAACGAGACCATTGGTCTCAGTT-3'; The primers used to amplify the Cas9 gene are tycas9, including: CAS9 forward primer: GCATGCGAAGGAGATATACCTCAGTCACCTCCTAGCTGACTCAA-3', and CAS9 reverse primer: 5'-CGGAGAAAATTATGGATAAGAAATACTCAATAGGCTTAGA-3'; 3) Using the suicide plasmid pRE112 as a template, the conjugation transfer sequence was amplified, digested, and ligated into the intermediate vector pSTcas9 to construct the vector pSCTcas9. The primers used for amplifying the conjugation transfer sequence were 112-ta9, including: F: 5'-TGACTGCAGCTGGATGTCGACCCGTCACAGGTATTAGGGCCC-3', and R: 5'-TGAACATCAACCCAGGTCGACCGTTTCGTGATTGTCACGCTC-3'.

[0028] The above method enables the construction of the CRISPR-Cas9-based gene-editing vector pSCTcas9 for Pasteurella multocida in porcine species, and its plasmid map is shown below. Figure 2 As shown, the structure and component composition of the pSCTcas9 plasmid are illustrated, providing intuitive information for understanding plasmid function and gene editing operations.

[0029] Example 2: Application of vector pSCTcas9 in constructing Pasteurella multocida gene-deleted strains 1) Design upstream and downstream homologous arms Lupp and Rupp and target sequence BDupp for the target gene upp; The Lupp primer sequences include: F:5'-CGGTGGAGCCCGGGCCCGCGGATTCGCTAATGAAAAAAATTGTTGTT-3', and R: 5'-GTTGGGATTACATGGTAGGCTCCTTTTGGGT-3'; The Rupp primer sequences include: F: 5'-GCCTACCATGTAATCCCAACACAAGCGGCA-3', and R:5'-CTGCGGTAGACGGCACCGCGGAGCTTCTAATTTAAATTCAGGGGTAG-3'; BDupp target sequences include: F: 5'-GAAAAGGCAGTTTATTAACATACG-3', and R: 5'-AAACCGTATGTTAATAAACTGCCT-3'; 2) Connect the homologous arms and target sequences to the vector pSCTcas9 to construct the pSCTcas9-ULR-bu plasmid; 3) The *E. coli* WM3064 conjugation transfer was performed into *Pasteurella multocida*, and the gene-deleted strain CVCC434-Δupp was obtained through screening. The gel electrophoresis image verifying the *Pasteurella multocida* gene deletion upp is shown below. Figure 3 As shown, lane 1 is used to verify the construction of the gene-deleted strain (upp deletion strain). Lane 2 is for verifying the gene-deleted strain. M is the DL2000 DNA Marker. Figure 3The image clearly shows the difference in electrophoretic bands before and after gene deletion, indicating that the gene-deleted strain CVCC434-Δupp was successfully constructed in this embodiment. Compared with traditional methods (which require multiple rounds of screening and take several weeks), this method is simpler to operate, reduces the time by more than 50%, and significantly improves editing efficiency, providing an efficient tool for the study of Pasteurella multocida gene function.

[0030] Example 3: Application of the pSCTcas9 carrier in targeted sterilization 1) Design the target sequence BDkmt1 of the chromosome gene kmt1 and construct the pSCTcas9-bk plasmid; The BDkmt1 target sequence includes: F: 5'-AAACCACTGGGTAAATAGCGGATA-3', and R: 5'-GAAATATCCGCTATTTACCCAGTG-3'; 2) By introducing Pasteurella multocida through conjugation transfer, the bacteria are targeted to cut chromosomes, leading to bacterial death and achieving specific sterilization.

[0031] The comparison of single colony growth on resistance plates with and without the KMT1 gene target is shown in the figure below. Figure 4 As shown, Figure 4 The bactericidal effect of pSCTcas9-bk was clearly demonstrated, providing an intuitive basis for evaluating the gene editing effect.

[0032] Example 3: Application of the vector pSCTcas9 in eliminating drug-resistant plasmids Epidemiological survey of *Pasteurella multocida*: Porcine lung tissue samples were collected, processed in a clean bench, and cultured on TSA agar. Bacterial DNA was purified and extracted, and PCR identification was performed using specific primers. Minimum inhibitory concentrations (MICs) of *Pasteurella multocida* against 16 antimicrobial agents were determined according to CLSI standards to assess drug resistance. Genomic DNA was extracted, primers were designed for PCR amplification, and capsular and lipopolysaccharide (LPS) strains were identified. A total of 96 *Pasteurella multocida* strains were isolated, with an isolation rate of 7.7%. Tetracycline resistance was the highest, reaching 89.58%, indicating severe multidrug resistance. A, D, F, and unknown capsular types were identified, with type F being the most prevalent; L1, L3, and L6 LPS strains were identified, with type L3 being the most prevalent.

[0033] Whole-genome sequencing and analysis of multidrug-resistant Pasteurella multocida**: Forty strains of multidrug-resistant Pasteurella multocida were selected, genomic DNA was extracted and sequenced, resistance genes and MLST serotypes were analyzed using the CGE network database, virulence genes were screened using the VFDB database, a heatmap of resistance genes and virulence genes was drawn, and CRISPR sequences in the genome were searched. Results of whole-genome sequencing and analysis of multidrug-resistant Pasteurella multocida: Four MLST serotypes were identified, mainly ST12; the strains carried nine resistance genes and four virulence genes, with F:L3:ST12 being the predominant circulating serotype. The genome contains two CRISPR-Cas systems: the type I F subtype system and the type II C subtype system.

[0034] 1) Then, the target sequence BDflor was designed for the drug resistance gene floR, and the pSCTcas9-bf plasmid was constructed; The BDflor target sequence includes: F: 5'-GAAACCGTGTGCTCATAGGCCAAG-3', and R: 5'-AAACCTTGGCCTATGAGCACACGG-3'; 2) The clinical strain GDA22P84P containing the drug-resistant plasmid was introduced via conjugation transfer to eliminate the drug-resistant gene floR. The gel electrophoresis image verifying the elimination of floR in Pasteurella multocida is shown below. Figure 5 As shown, Figure 5 Lanes 1-4 of the middle electrophoresis apparatus are for verifying Pasteurella multocida, verifying the knockout plasmid, amplifying the knockout strain GDA22P84P-ΔfloR, and amplifying the original strain GDA22P84P, respectively. M is the DL2000 DNA Marker. This is used to verify the elimination of the drug resistance gene floR. The gene editing results are presented visually through changes in electrophoretic bands.

[0035] In summary, this invention successfully constructed a CRISPR-Cas9 gene editing system for Pasteurella multocida, which can be used to construct gene-deleted strains, target bacteria, and eliminate drug-resistant plasmids. It provides an effective method for studying the gene function of Pasteurella multocida and controlling drug resistance, and has significant application value.

[0036] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A method for constructing a CRISPR-Cas9-based gene-editing vector for Pasteurella multocida in porcines, characterized in that, Includes the following steps: S1. Using the Escherichia coli-Pasteurella multocida shuttle plasmid pMI28 as a template, the pan-host replicon ori and the kanamycin resistance gene KanR were amplified. S2. Using psodc-tpia plasmid as a template, amplify the promoters psodc and ptpia; using pCas9 plasmid as a template, amplify the gRNA scaffold; using pCasSA plasmid as a template, amplify the cas9 gene; splice psodc, gRNA scaffold, and ptpia by overlap PCR, and perform seamless cloning with the cas9 gene and pMI28 to obtain the intermediate vector pSTcas9. S3. Using the suicide plasmid pRE112 as a template, the conjugation transfer sequence was amplified, digested, and ligated into the intermediate vector pSTcas9 to construct the vector pSCTcas9.

2. The method for constructing a CRISPR-Cas9-based gene-editing vector for Pasteurella multocida in porcine species according to claim 1, characterized in that, In step S1, the primers used to amplify the pan-host replicon ori and the kanamycin resistance gene KanR are p28fx, including: F: 5'-TCTGGACTCAGATCTCGAGCTCA-3', and R: 5'-GGTATATCTCCTTCGCATGCCT-3'.

3. The method for constructing a CRISPR-Cas9-based gene-editing vector for Pasteurella multocida in porcine species according to claim 1, characterized in that, In step S2, the primers used to amplify the gRNA scaffold are sg, including: gRNA scaffold forward primer: 5'-TCAGCTTTATCCACGTCGCACCGACTCGGTGCCAC-3', and gRNA scaffold reverse primer: 5'-AGGAGGAAACGAGACCATTGGTCTCAGTT-3'; The primers used to amplify the Cas9 gene are tycas9, including: CAS9 forward primer: GCATGCGAAGGAGATATACCTCAGTCACCTCCTAGCTGACTCAA-3', and CAS9 reverse primer: 5'-CGGAGAAAATTATGGATAAGAAATACTCAATAGGCTTAGA-3'.

4. The method for constructing a CRISPR-Cas9-based gene-editing vector for Pasteurella multocida in porcine species according to claim 1, characterized in that, In step S3, the primers used to amplify the conjugation transfer sequence are 112-ta9, including: F: 5'-TGACTGCAGCTGGATGTCGACCCGTCACAGGTATTAGGGCCC-3', and R: 5'-TGAACATCAACCCAGGTCGACCGTTTCGTGATTGTCACGCTC-3'.

5. A CRISPR-Cas9-based gene editing vector for Pasteurella multocida constructed according to any one of claims 1-4.

6. The application of the CRISPR-Cas9-based *Pasteurella multocida* gene-editing vector as described in claim 5 in the construction of *Pasteurella multocida* gene-deleted strains, characterized in that... Includes the following steps: 1) Design upstream and downstream homologous arms Lupp and Rupp and target sequence BDupp for the target gene upp; The Lupp primer sequences include: F:5'-CGGTGGAGCCCGGGCCCGCGGATTCGCTAATGAAAAAAATTGTTGTT-3', and R: 5'-GTTGGGATTACATGGTAGGCTCCTTTTGGGT-3'; The Rupp primer sequences include: F: 5'-GCCTACCATGTAATCCCAACACAAGCGGCA-3', and R:5'-CTGCGGTAGACGGCACCGCGGAGCTTCTAATTTAAATTCAGGGGTAG-3'; BDupp target sequences include: F: 5'-GAAAAGGCAGTTTATTAACATACG-3', and R: 5'-AAACCGTATGTTAATAAACTGCCT-3'; 2) Connect the homologous arms and target sequences to the vector pSCTcas9 to construct the pSCTcas9-ULR-bu plasmid; 3) Gene-deleted strain CVCC434-Δupp was obtained by conjugation of Escherichia coli WM3064 to Pasteurella multocida.

7. The application of the CRISPR-Cas9-based Pasteurella multocida gene-editing vector as described in claim 5 in targeted sterilization, characterized in that, Includes the following steps: 1) Design the target sequence BDkmt1 of the chromosome gene kmt1 and construct the pSCTcas9-bk plasmid; The BDkmt1 target sequence includes: F: 5'-AAACCACTGGGTAAATAGCGGATA-3', and R: 5'-GAAATATCCGCTATTTACCCAGTG-3'; 2) By introducing Pasteurella multocida through conjugation transfer, the bacteria are targeted to cut chromosomes, leading to bacterial death and achieving specific sterilization.

8. The application of the CRISPR-Cas9-based Pasteurella multocida gene-editing vector as described in claim 5 in eliminating drug-resistant plasmids, characterized in that, Includes the following steps: 1) Design the target sequence BDflor against the drug resistance gene floR and construct the pSCTcas9-bf plasmid; The BDflor target sequence includes: F: 5'-GAAACCGTGTGCTCATAGGCCAAG-3', and R: 5'-AAACCTTGGCCTATGAGCACACGG-3'; 2) The drug resistance gene floR was eliminated by introducing the clinical strain GDA22P84P containing the drug resistance plasmid through conjugation transfer.

Images