CRISPR / Cas9 (clustered regularly interspaced short palindromic repeats / CRISPR / Cas9) system for specific targeted traceless elimination of blaNDM-1 gene and IncN plasmid and application

Abstract

The invention belongs to the technical field of biological genetic engineering, and discloses a CRISPR / Cas9 (clustered regularly interspaced short palindromic repeats / associated protein 9) system for specific targeted traceless elimination of a blaNDM-1 gene and an IncN plasmid and application of the CRISPR / Cas9 system. According to the system, sgRNA of an original point conserved sequence is copied by designing an IncN type plasmid carrying a blaNDM-1 gene in a targeting manner, so that the replication capability of the plasmid is destroyed while a drug-resistant gene is eliminated; the carrier delivery efficiency is greatly improved by conjugational transfer to recipient bacteria; the expression of the Cas9 gene is controlled by adopting an arabinose inducible promoter, so that the constitutive toxicity of Cas9 protein is reduced, and the system stability and the editing success rate are improved; the sucrose-induced carrier self-elimination gene carried by the carrier realizes controllable self-elimination of the carrier. The system is not simply superposed, but forms an operation process with a controllable time sequence: efficient delivery is firstly carried out through joint, then cutting is controlled through induction, and finally self-elimination is induced.

Description

A specific targeted scarless elimination method NDM-1 CRISPR / Cas9 system for genes and IncN plasmids and its applications Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a method for specifically targeting and eliminating bladder without scarring. NDM-1 CRISPR / Cas9 system for genes and IncN plasmids and its applications. Background Technology

[0002] Currently, clinical treatments target New Delhi metallo-β-lactamase-1 (BLA). NDM-1 Treatment options for drug-resistant bacterial infections have limitations, making it difficult to fundamentally block the spread of resistance. Current treatments mainly rely on antibiotics such as polymyxin, tigecycline, and fosfomycin. Among these, polymyxin has a high incidence of nephrotoxicity and neurotoxicity; tigecycline has low concentrations in urine and cerebrospinal fluid, making it ineffective against urinary tract and central nervous system infections; and fosfomycin easily induces heterogeneous bacterial resistance. In recent years, combination therapies of β-lactamase inhibitors (such as ceftazidime-avibactam and meropenem-faborabactam) have been marketed to combat drug resistance in bladder infections. NDM-1 Ineffective because NDM-1 is a metalloenzyme, and its active site differs from that of serine enzymes; existing inhibitors cannot bind to it. Hospital-level infection control and antibiotic management measures cannot eliminate bacillus already present in the environment or within the host. NDM-1 The gene and the plasmid carrying the gene. Current methods can only temporarily control the infection and cannot remove the drug resistance gene. Once the pressure of antibiotics is reduced, the remaining drug resistance plasmids can cause drug resistance to recur through horizontal transfer.

[0003] The CRISPR / Cas9 system offers a novel strategy for targeting and eliminating drug-resistant genes. It works by designing gRNA to guide the Cas9 nuclease to cleave specific DNA sequences, thereby inactivating the target gene. In the field of combating drug-resistant bacteria, this strategy involves designing gRNA to target specific DNA sequences. NDM-1 On the one hand, gRNAs in gene or plasmid replicons can disable the ability of drug-resistant bacteria to produce NDM-1 enzymes, reversing their sensitivity to β-lactam antibiotics and restoring the therapeutic effect of traditional antibiotics. On the other hand, by designing gRNAs that target the origin of plasmid backbone replication, they can simultaneously disrupt the replication of bacteria carrying the NDM-1 enzyme. NDM-1 Plasmids of the gene fundamentally block the horizontal transfer of drug resistance genes, thus breaking the chain of drug resistance transmission. Furthermore, the components of the CRISPR / Cas9 system (gRNA, Cas9) can be modified through genetic engineering to adapt to different bacterial hosts (e.g., signal peptide optimization for Gram-negative bacteria, vector adaptation), and can be based on bla... NDM-1 Types of gene mutations (e.g., bla) NDM-2 bla NDM-5(e.g., subtypes) adjust the gRNA sequence to ensure coverage of variant drug-resistant genes. However, existing CRISPR / Cas9 anti-drug-resistant bacteria technologies have significant drawbacks: 1) Single target: Most studies only target the drug-resistant gene itself, rather than simultaneously targeting the essential functional regions of the plasmid (such as the origin of replication), resulting in a high plasmid residual rate and the risk of horizontal transfer.

[0004] 2) Low delivery efficiency: It relies heavily on physical delivery methods such as electroporation, which has low transformation efficiency for many clinical isolates, making it difficult to achieve widespread application.

[0005] 3) Biosafety risks: The vector lacks a self-elimination mechanism, and foreign genes (such as Cas9 and antibiotic resistance genes) may remain or spread in the bacterial community, posing environmental biosafety risks.

[0006] Therefore, developing a safe delivery system that can simultaneously and efficiently eliminate drug resistance genes and their plasmids, and has self-elimination capabilities, has clear demand and research value. Summary of the Invention

[0007] To address the aforementioned technical problems, the purpose of this invention is to provide a specific targeted, scarless CRISPR / Cas9 removal system. This system is a multifunctional, integrated, efficient, and safe system that includes features targeting bla NDM-1 The system integrates a series of functionalities: a specific, targeted, and traceless sgRNA sequence for gene and IncN plasmid elimination; an arabinose-inducible promoter for controllable expression of the Cas9 nuclease gene; a conjugation transfer initiation sequence for enhanced transformation of clinically resistant recipient bacteria; and a sucrose-inducible vector self-elimination gene for the self-elimination of exogenous gene residues. This comprehensive solution, combining all these features, provides a closed-loop solution for the simultaneous elimination of genes and plasmids. It not only improves delivery efficiency and the thoroughness of targeted clearance but, more importantly, overcomes biosafety bottlenecks, laying the foundation for subsequent clinical translation.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: a specific targeted scarless elimination method for bla NDM-1 A CRISPR / Cas9 system for a gene and IncN plasmid, the system being integrated into a suicide plasmid vector, the system comprising: 1) a specific targeting module: containing at least one sgRNA sequence whose target site is located on the gene carrying the IncN plasmid. NDM-1The sgRNA sequence contains a conserved origin of replication sequence for the gene and the IncN plasmid, used to simultaneously disrupt the replication ability of the drug resistance gene and its plasmid; the nucleotide sequence of the sgRNA sequence is shown in any one of SEQ ID NO. 1-3; 2) Controllable expression module: containing the Cas9 nuclease gene, whose expression is regulated by the arabinose-inducible promoter and the araC gene; 3) Highly efficient delivery module: containing the conjugation transfer initiation sequence, used to mediate the conjugation transfer of the suicide plasmid vector from the donor bacteria to the clinically drug-resistant recipient bacteria; 4) Self-elimination safety module: containing a sucrose-inducible vector self-elimination gene, used to eliminate the vector itself by inducing the toxicity of the expression product after the system completes targeted cleavage.

[0009] A suicide plasmid vector for implementing the system, the vector being named pCasCure-apr-oriT, the nucleotide sequence of which is shown in SEQ ID NO.4; the pCasCure-apr-oriT suicide plasmid vector includes an sgRNA sequence, a Cas9 nuclease gene, an araC gene, an arabinose inducible promoter, a conjugation transfer initiation site sequence, and a sucrose inducible vector self-elimination gene.

[0010] A method for constructing the system includes the following steps: (1) annealing the sgRNA as described to obtain a double-stranded DNA sequence; (2) linearizing the suicide plasmid vector as described, and then performing homologous recombination ligation with the double-stranded DNA sequence to obtain the system.

[0011] One of the systems described above is used in the preparation of drugs that block the drug resistance gene bla in Klebsiella pneumoniae. NDM-1 Its application in horizontal transfer reagents via IncN plasmids.

[0012] One of the systems described above improves the blavin content of clinical strains. NDM-1 Application of gene and IncN plasmid elimination in transformation efficiency.

[0013] A method for non-therapeutic purposes of eliminating bacterial resistance without leaving a trace using the system described above, comprising the following steps: S1. Conjugation delivery: combining donor bacteria containing the system with bacteria carrying the system. NDM-1 The recipient bacteria containing the IncN plasmid were co-cultured, and conjugation transfer was mediated by the conjugation origin sequence to introduce the system into the recipient bacteria; S2. Induction cleavage: arabinose was added to the recipient bacteria as an inducer to activate the arabinose-inducible promoter to express Cas9 protein, which specifically cleaved the origin of replication and bla of the IncN plasmid under the guidance of sgRNA. NDM-1The gene leads to plasmid loss and drug resistance elimination; S3. Induced self-elimination: After confirming drug resistance elimination, sucrose is added to the bacterial culture as an inducer to activate the expression of the sucrose-induced vector self-elimination gene, producing toxicity, selectively killing strains still carrying the system or causing vector loss, and obtaining trace-eliminating strains without any foreign genes.

[0014] The method for constructing the suicide plasmid vector includes the following steps: S1, designing primer pairs, performing PCR amplification using plasmid pCas as a template, recovering the target fragment, ligating it to the suicide plasmid backbone for homologous recombination, plate-coating, sequencing identification, plasmid extraction, and obtaining the suicide plasmid vector; S2, transforming the suicide plasmid vector into competent WM3064 bacteria by electroporation and screening to obtain WM3064 donor bacteria containing the vector.

[0015] Beneficial effects: This invention provides a specific targeted, scarless CRISPR / Cas9 elimination system. This system, through the synergistic action of four modules, constitutes a time-controlled operational flow of "binding delivery - induced cleavage - induced self-elimination," achieving bla NDM-1 The system achieves seamless removal of genes and IncN plasmids without leaving any foreign gene residue. It is a multifunctional, integrated, efficient, and secure system.

[0016] The system integrates the following four key functional components through modular design to solve the defects of the above-mentioned existing technologies: (1) Targeting module: significantly improves the thoroughness of drug resistance plasmid removal and effectively blocks the horizontal transfer of drug resistance genes.

[0017] By designing targeted carriers of Bla NDM-1 The sgRNA of the conserved sequence of the IncN-type plasmid replication origin (ori) eliminates the drug resistance gene while simultaneously disrupting the plasmid's replication ability. After treatment of clinical isolates (such as strains 253 and 254), the plasmid elimination rate was significantly higher than that of traditional methods that only target the drug resistance gene, fundamentally reducing the risk of drug resistance spreading horizontally via plasmids. This strategy aims to eliminate the drug resistance phenotype and disrupt the propagation vector (plasmid) of the drug resistance gene with a single intervention, thereby significantly improving the thoroughness of eradication and blocking horizontal transfer. This is fundamentally different from most existing technologies that only target a single drug resistance gene, significantly improving the thoroughness of eradication and blocking horizontal transfer.

[0018] (2) Controllable expression module: Reduces the constitutive toxicity of Cas9 protein, improves system stability and editing success rate.

[0019] Using arabinose-inducible promoters (P BADThe expression of the Cas9 gene is controlled. In the uninduced state, Cas9 is not expressed or expressed at very low levels, reducing its metabolic burden and potential toxicity to the host bacteria, which is beneficial for maintaining vector stability. When needed, Cas9 expression is induced by adding arabinose, thus initiating the targeted cleavage function. This temporal control strategy improves the stability of the entire system within bacteria and the controllability and efficiency of gene editing operations.

[0020] (3) High-efficiency delivery module: greatly improves vector delivery efficiency and overcomes the bottleneck of difficult transformation of clinical strains.

[0021] This method utilizes conjugative transfer to transfer vectors from donor bacteria (such as WM3064) to a variety of clinically resistant recipient bacteria via bacterial conjugation. The delivery efficiency for clinical isolates that are difficult to transform via electroporation is several orders of magnitude higher than that of electroporation, enabling the technology to be practically applied to a wide range of clinically resistant strains. This design leverages the natural bacterial conjugative transfer mechanism to achieve efficient and active delivery of vectors from specific donor bacteria to a variety of clinically resistant recipient bacteria, effectively overcoming the limitations of physical transformation methods (such as electroporation) which are inefficient or ineffective for many important clinical pathogens, thus expanding the application scope of the technology.

[0022] (4) Self-elimination module: realizes the controllable self-elimination of the vector and solves the biosafety problem of exogenous gene residue.

[0023] The vector carries a sucrose-inducible vector self-elimination gene (sacB). After CRISPR / Cas9 cleavage, the addition of sucrose induces the expression of this gene, and its product has a toxic effect on the bacteria, thus efficiently eliminating the vector itself. Ultimately, a strain free of any foreign genes (such as Cas9 or antibiotic resistance markers) can be obtained, avoiding the risk of persistent foreign genes in the environment or bacterial community. This module allows for the specific elimination of the vector itself by adding sucrose, a harmless inducer, after the predetermined gene editing function is completed, ultimately obtaining a "clean" strain free of any foreign DNA. This design directly addresses the biosafety issues of potential foreign gene residue and diffusion in CRISPR technology applications.

[0024] This system integrates conjugation transfer delivery (oriT) and sucrose-induced self-elimination (sacB) into a single CRISPR / Cas9 system, forming a closed-loop solution through strategies targeting plasmid replicons and controlling inducible Cas9 expression. This not only improves delivery efficiency and the thoroughness of targeted clearance but, more importantly, overcomes biosafety bottlenecks, laying the foundation for subsequent clinical translation. This integration is not a simple superposition but rather constitutes a time-controlled operational flow: efficient delivery via conjugation, controlled cleavage via induction, and finally, induced self-elimination. This multifunctional, modular, and controllable system-level design constitutes the core innovation of this invention. Attached Figure Description

[0025] Figure 1 is a schematic diagram of the functional modules of the pCasCure-apr-oriT plasmid; Figure 2 is a schematic diagram of the bla NDM-1 Figure 3 shows the PCR verification results of gene elimination; Figure 4 shows the PCR verification results of IncN plasmid elimination; Figure 5 shows the PCR verification results of KB drug susceptibility test; Figure 6 shows the PCR verification results of vector self-elimination. Detailed Implementation

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the present invention will be briefly introduced below in conjunction with the accompanying drawings and descriptions of the embodiments or the prior art. Obviously, the following description of the structure of the accompanying drawings is only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. It should be noted that the description of these embodiments is for the purpose of helping to understand the present invention, but does not constitute a limitation of the present invention.

[0027] Example 1: Construction of pCasCure-apr-oriT suicide plasmid vector. This invention constructs a self-eliminating CRISPR / Cas9 vector system called pCasCure-apr-oriT.

[0028] 1.1 Homologous recombination to construct the pCasCure-apr-oriT suicide plasmid vector: The apramycin resistance gene apr was amplified from the pCasKp-apr plasmid using primers Apr-R and Apr-F. The Cas9 nuclease gene, araC gene, and arabinose gene inducible promoter P were amplified from the p15A-ParaB-Cas9-PT5-Redγβα plasmid using primers Cas9-F and Cas9-R. BADThe sacB gene was amplified from the pSGKP-km plasmid using primers sacB-F and sacBR (sacB-1-R, sacB-2-R, sacB-3-R). The sgRNA gene containing the N20 sequence was amplified from the pSGKp plasmid using primers N20F (N20-1-F, N20-2-F, N20-3-F) and N20-R. The OriT conjugation transfer region was amplified from the pCVD442-GmR plasmid using primers oriT-F and oriT-R. After amplification, the target fragment was recovered, homologous recombination was performed, transformation was performed, the sample was plated, positive clones were identified, and plasmids were extracted.

[0029] The nucleotides of the conserved sgRNA sequence are shown in SEQ ID NO.1-3.

[0030] Figure 1 shows a schematic diagram of the functional module of the pCasCure-apr-oriT plasmid. Its nucleotide sequence is shown in SEQ ID NO. 4.

[0031] 1.1.1 Primers, PCR amplification system and conditions

[0032] PCR amplification system

[0033] PCR amplification conditions

[0034] 1.1.2 Gel recovery product 1) Column equilibration: Add 500 µL of equilibration solution BL to the adsorption column CB2 (the adsorption column is placed in the collection tube), centrifuge at 12000 rpm for 1 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0035] 2) Cut the single DNA band from the agarose gel, place it in a clean centrifuge tube, and weigh it.

[0036] 3) Add an equal volume of PC solution to the gel block (if the gel weighs 0.1 g, its volume can be regarded as 100 µL, then add 100 µL of PC solution), and place in a 50 ℃ water bath for about 10 min, during which time the centrifuge tube is gently turned up and down continuously to ensure that the gel block is fully dissolved.

[0037] 4) Add the solution obtained in the previous step to an adsorption column CB2 (place the adsorption column in the collection tube), centrifuge at 12000 rpm for 1 min, discard the waste liquid in the collection tube, and put the adsorption column CB2 back into the collection tube.

[0038] 5) Add 600 µL of wash buffer PW to the adsorption column CB2, let stand for 5 min, centrifuge at 12000 rpm for 1 min, discard the waste liquid in the collection tube, and put the adsorption column CB2 back into the collection tube. Repeat this step once.

[0039] 6) Place the adsorption column CB2 into the collection tube and centrifuge at 12,000 rpm for 2 minutes to remove as much of the washing solution as possible. Let the adsorption column air dry completely at room temperature for several minutes.

[0040] 7) Place the adsorption column CB2 into a clean centrifuge tube, add an appropriate amount of ddH2O dropwise to the middle of the adsorption membrane, let it stand at room temperature for 2 min, centrifuge at 12000 rpm for 2 min, and collect the DNA solution.

[0041] 1.1.3 Homologous recombination preparation of pCasCure-apr-oriT: The recycled gel products were homologously recombined and linked according to the system in the table below.

[0042] Preparation of Tri-U6-CRISPR / Cas12a-Zeo linking system and linking conditions by homologous recombination

[0043] 1.1.4 Transform the ligation product into DH5α cells 1) Take 100 µL of DH5α competent cells thawed in an ice bath, add 10 µL of the ligated homologous recombination product, gently pipette to mix, and incubate on ice for 30 min.

[0044] 2) Heat shock in a 42℃ water bath for 45 seconds, then let stand in an ice bath for 3 minutes.

[0045] 3) Add 890 µL of sterile LB medium (antibiotic-free) to the centrifuge tube and incubate at 37 °C and 200 rpm for 1 hour.

[0046] 4) Spread 100 µL of bacterial culture onto an LB solid medium plate containing apramycin antibiotic, invert the plate, and incubate overnight at 37°C.

[0047] 1.1.5 Identification of positive colony clones 1) Pick single colonies from overnight culture plates and culture them in 1 mL LB liquid medium containing apramycin antibiotic at 37 ℃ and 200 rpm for 5 hours.

[0048] 2) Take 2 µL of bacterial culture for PCR identification. The identification primers are N20-R and N20F (N20-1-F, N20-2-F, N20-3-F). Refer to step 1.1.1 for the PCR reaction system and conditions.

[0049] 3) Send the amplified products to Qingke for sequencing and identification.

[0050] 1.1.6 Plasmid Extraction 1) Column Equilibration Step: Add 2.5 mL of equilibration solution BL to the adsorption column CP6 (place the adsorption column in a 50 mL collection tube), centrifuge at 8000 rpm for 2 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0051] 2) Take 100 mL of overnight cultured bacterial solution and add it to a centrifuge tube. Centrifuge at 8000 rpm for 3 min at room temperature to collect the bacteria, and remove as much of the supernatant as possible.

[0052] 3) Add 8 mL of solution P1 to the centrifuge tube containing bacterial precipitate, and use a vortex mixer to suspend the bacterial cell precipitate at the bottom.

[0053] 4) Add 8 mL of solution P2 to the centrifuge tube, and immediately and gently invert it 6-8 times to fully lyse the bacteria. Let it stand at room temperature for 5 minutes.

[0054] 5) Add 8 mL of solution P4 to the centrifuge tube, and immediately and gently invert it 6-8 times to mix thoroughly until a white, dispersed flocculent precipitate appears. Let it stand at room temperature for 10 min, then centrifuge at 8000 rpm for 10 min to allow the white precipitate to settle to the bottom of the tube. Carefully pour the entire solution into filter CS1, and slowly push the push handle to filter. Collect the filtrate in a clean 50 mL tube.

[0055] 6) Add 0.3 times the volume of isopropanol to the filtrate, mix by inverting the container, and then transfer it to the adsorption column CP6.

[0056] 7) Centrifuge at 8000 rpm for 3 min at room temperature, discard the waste liquid in the collection tube, and put the adsorption column CP6 back into the collection tube.

[0057] 8) Add 10 mL of PW wash buffer to the adsorption column CP6, centrifuge at 8000 rpm for 2 min, discard the waste liquid in the collection tube, and put the adsorption column CP6 back into the collection tube. Repeat this operation once.

[0058] 9) Add 3 mL of anhydrous ethanol to the adsorption column CP6, centrifuge at 8000 rpm for 2 min at room temperature, and discard the waste liquid.

[0059] 10) Place the adsorption column CP6 back into the collection tube and centrifuge at 8000 rpm for 5 min to remove the residual washing solution in the adsorption column.

[0060] 11) Place the adsorption column CP6 in a clean 50 mL collection tube, add 1-2 mL of ddH2O dropwise to the middle of the adsorption membrane, let it stand at room temperature for 5 min, centrifuge at 8000 rpm for 2 min, and collect the plasmid solution.

[0061] Example 2: Transformation of pCasCure-apr-oriT plasmid into WM30642.1 via electroporation. Preparation of WM3064 electrocompetent cells: 1) Pre-culture: WM3064 strain was inoculated into 5 mL of LB broth containing 60 μg / mL DAP and cultured overnight at 37°C; 2) Expansion culture: 1 mL of overnight culture was added to 100 mL of LB broth containing 60 μg / mL DAP and cultured at 37°C with shaking at 180 rpm until OD500 was reached. 600 =0.4-0.6 (approximately 5 hours); 3) Competent cell treatment: Incubate the bacterial culture on ice for 30 minutes, centrifuge at 7200 rpm and 4°C for 5 minutes, and discard the supernatant; resuspend in 20 mL of ice-cold sterile ultrapure water, incubate on ice for 10 minutes, and centrifuge, repeat once; resuspend in 20 mL of ice-cold 10% glycerol, incubate on ice for 10 minutes, and centrifuge at 12000 rpm and 4°C for 15 minutes, and discard the supernatant; 4) Aliquoting and storage: Resuspend the cells in 0.5 mL of ice-cold 10% glycerol, aliquot into 100 μL / tube, and store at -80°C (valid for 6 months).

[0062] 2.2 Electroporation to transform pCasCure-apr-oriT plasmid into WM3064 1) Sample preparation: Take 100 μL of competent cells and thaw on ice for 5 min, add 400 ng of pCasCure-apr-oriT plasmid (volume ≤10 μL), incubate on ice for 30 min, and then transfer to a 0.2 cm pre-cooled electroporation cuvette; 2) Electroporation parameters: Use an electroporator, with parameters set to 1800 V, 200 Ω, and 25 μF; 3) Recovery and screening: Immediately after electroporation, add 1 mL of LB medium containing 60 μg / mL DAP, and recover at 37℃ and 180 rpm for 1.5 h; aspirate 100 μL of bacterial culture and spread it on a medium containing 50 μg / mL apramycin + 60 μg / mL... 4) Positive identification: Pick single clones and perform PCR identification using primers N20-R and N20F (N20-1-F, N20-2-F, N20-3-F) (same as 1.1.5). Positive bacterial cultures are stored in LB medium containing 25% glycerol at -80℃ for later use.

[0063] Example 3: Conjugation Transfer pCasCure-apr-oriT was transferred into the target strain. 3.1 Conjugation Transfer Procedure 1) Preparation of bacterial culture: The recipient bacteria (clinically isolated NDM-1 positive strains 253 and 254, Klebsiella pneumoniae) were inoculated into LB medium, and the donor bacteria (WM3064 / pCasCure-apr-oriT) were inoculated into LB medium containing 60 μg / mL DAP and cultured at 37°C and 180 rpm until OD600 = 0.5 (logarithmic growth phase); 2) Mixing of bacterial culture: 200 μL of donor bacteria and 200 μL of recipient bacteria (ratio 1:1) were respectively pipetted into 1.5 mL EP tubes and vortexed to mix; LB medium containing 60 μg / mL DAP was then inoculated into LB medium containing 60 μg / mL DAP. Place a 0.22 μm microporous membrane in the center of an LB agar plate containing DAP, drop 100 μL of mixed bacterial culture onto the membrane, let stand for 20 min, and then incubate at 37℃ for 18-24 h; 3) Elution and spreading: Use sterile forceps to transfer the membrane to a 50 mL tube containing 10 mL of LB liquid medium, and shake to elute the bacteria; spread 100 μL of the elution buffer onto a double-antibiotic LB agar plate (meropenem 4 μg / mL + 4) Control settings: The following controls were set up to verify successful conjugation: a. Recipient bacteria control: plated on bispecific antibody plate, meropenem antibody plate (4 μg / mL), and apramycin antibody plate (50 μg / mL); b. Donor bacteria control: plated on bispecific antibody plate, meropenem antibody plate, and apramycin antibody plate (containing 60 μg / mL LDAP); c. Criteria for successful conjugation: The colony morphology of the bacteria growing on the bispecific antibody plate is consistent with that of the recipient bacteria, and no colony growth is observed when the donor bacteria and recipient bacteria are plated separately on the bispecific antibody plate; the recipient bacteria grow on the meropenem antibody plate but not on the apramycin antibody plate; the donor bacteria grow on the apramycin antibody plate but not on the meropenem antibody plate.

[0064] 3.2 Calculation of Binding Transfer Frequency 1) Gradual Dilution: Dilute the eluent in a 10-fold gradient (10¹-10⁻¹⁰). 5 1) Apply 100 μL of each gradient to the double antibody plate and the meropenem antibody plate; 2) Colony counting: After overnight incubation at 37°C, count the number of conjugate colonies (C) on the double antibody plate and the number of recipient colonies (R) on the meropenem antibody plate; 3) Frequency calculation: Conjugation efficiency = (C / R) × 100%. The experiment was repeated 3 times, and the results are expressed as "mean ± standard deviation" (the conjugation efficiency of this invention is 1.2 × 10⁻⁶). -4 ±0.3×10 -4 ).

[0065] Example 4: pCasCure-apr-oriT targeted elimination of NDM-1 resistance gene and its plasmid 4.1 Induction cleavage and elimination efficiency detection 1) Induction treatment: Take 1 mL of conjugation transfer elution buffer, add 0.2% arabinose (to induce Cas9 expression) and 1 mM IPTG, and culture at 37℃ and 180 rpm for 2 h with shaking; 2) Plate plating: Take 100 μL of the induced bacterial culture and plate it on a double antibody plate (meropenem 4 μg / mL + apramycin 50 μg / mL) and an apramycin monoclonal antibody plate (50 μg / mL), and culture overnight at 37℃; 3) Elimination efficiency calculation: Elimination efficiency = (1 - number of colonies on double antibody plate / number of colonies on monoclonal antibody plate) × 100%, and the experiment was repeated 3 times.

[0066] The results showed that strains 253 and 254 had blavin content. NDM-1 The elimination rates of both the gene and the IncN plasmid reached 92.5% ± 2.1%.

[0067] 4.2 Molecular-level validation (PCR detection) 4.2.1 Primers, PCR amplification system and conditions

[0068] PCR amplification system

[0069] PCR amplification conditions

[0070] 4.2.2 Result Interpretation: If the NDM-F / NDM-R primers do not amplify an 813bp band, and the IncN-F / IncN-R primers do not amplify a 600bp band, the result is determined to be bla. NDM-1 The gene and IncN plasmid were successfully eliminated.

[0071] As shown in Figure 2, bla NDM-1 PCR validation results of gene elimination. M represents a 2000bp DNA marker; NC is the negative control (no template); PC is the positive control (original strain 253); the remaining lanes represent single colonies of strain 253 after elimination.

[0072] As shown in Figure 2, the results indicate the absence of the 813bp target band, proving that bla NDM-1 Gene elimination was successful.

[0073] Figure 3 shows the PCR validation results for the elimination of the IncN plasmid. M represents a 2000bp DNA marker; PC is the positive control (original strain 253); the remaining lanes represent single colonies of strain 253 after elimination.

[0074] As shown in Figure 3, there was no 600bp target band, proving that the IncN plasmid was successfully eliminated.

[0075] 4.3 Phenotypic Validation (KB Susceptibility Testing) was performed according to CLSI 2024 M100-S34 standards, with the following steps: 1) Preparation of bacterial culture: Select single colonies that have been successfully eliminated and inoculate them into LB medium containing 50 μg / mL apramycin, and incubate at 37°C until the logarithmic growth phase (OD50). 600 =0.5), adjust the bacterial concentration to ≈1×10 using a 0.5 McFarland turbidity standard. 8 CFU / mL; 2) Spreading and pasting of meropenem discs: Dip a sterile cotton swab in the bacterial solution, squeeze out excess liquid, and spread it evenly on the surface of an MHA (Mueller-Hinton Agar) plate (spread once every 60° rotation, for a total of 3 times); after the bacterial solution is absorbed, use sterile forceps to place meropenem discs (10μg / disc) onto the surface of the plate, and press gently to ensure contact; 3) Incubation and interpretation: Incubate overnight at 37℃ for 16-18h, and measure the diameter of the inhibition zone (including the diameter of the disc) with vernier calipers; according to CLSI standards, a meropenem inhibition zone diameter ≥23mm is considered sensitive (MIC≤1μg / mL).

[0076] Figure 4 shows the results of the KB drug susceptibility test. Control represents the original strains 253 and 254 (inhibition zone diameter 12 mm, resistant); △NDM+△IncN represents the results after eliminating NDM-1 and IncN plasmids (inhibition zone diameter 28 mm, sensitive).

[0077] The results, as shown in Figure 4, demonstrate that the inhibition zone diameter of the successfully eliminated strain reached 28±1.5 mm, restoring its sensitivity to meropenem.

[0078] 4.4 Vector self-elimination verification 1) Sucrose screening: Select single colonies that have been successfully eliminated and inoculate them on LB plates containing 10% sucrose. Incubate overnight at 37°C (the sacB gene produces toxic fructan in the presence of sucrose, which can only be grown by strains without the vector); 2) PCR verification: Select single colonies on the sucrose plate and perform PCR using Apr-F / Apr-R primers (same as 1.1.1).

[0079] If no 1419bpapr gene amplification band is found, the vector elimination is considered successful.

[0080] Figure 5 shows the PCR validation results of vector self-elimination. M represents a 2000bp DNA marker; PC is the positive control (containing pCasCure-apr-oriT at lane 253); the remaining lanes represent single colonies after sucrose selection.

[0081] The results, combined with Figure 5, show the absence of the 1419bp apr gene band, proving that the vector elimination was successful.

[0082] 3) Results: The carrier achieved a 100% self-elimination rate, realizing bla NDM-1 Scarless elimination of genes and IncN plasmids.

[0083] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A specific targeted, scarless elimination method for bla NDM-1 The CRISPR / Cas9 system for genes and IncN plasmids is characterized by, The system is integrated into a suicide plasmid vector, and the system includes: 1) a specific targeting module: containing at least one sgRNA sequence, the target site of which is located on the blavinocyte. NDM-1 The sgRNA sequence contains a conserved origin of replication sequence for the gene and the IncN plasmid, used to simultaneously disrupt the replication ability of the drug resistance gene and its plasmid; the nucleotide sequence of the sgRNA sequence is shown in any one of SEQ ID NO. 1-3; 2) Controllable expression module: containing the Cas9 nuclease gene, whose expression is regulated by the arabinose-inducible promoter and the araC gene; 3) Highly efficient delivery module: containing the conjugation transfer initiation sequence, used to mediate the conjugation transfer of the suicide plasmid vector from the donor bacteria to the clinically drug-resistant recipient bacteria; 4) Self-elimination safety module: containing a sucrose-inducible vector self-elimination gene, used to eliminate the vector itself by inducing toxicity of the expression product through sucrose after the system completes targeted cleavage.

2. A suicide plasmid vector for implementing the system as described in claim 1, characterized in that, The vector is named pCasCure-apr-oriT, and its nucleotide sequence is shown in SEQ ID NO.

4. The pCasCure-apr-oriT suicide plasmid vector includes an sgRNA sequence, a Cas9 nuclease gene, an araC gene, an arabinose inducible promoter, a conjugation transfer initiation site sequence, and a sucrose inducible vector self-elimination gene.

3. A method for constructing the system as described in claim 1, characterized in that, The procedure includes the following steps: (1) annealing the sgRNA as described in claim 1 to obtain a double-stranded DNA sequence; (2) linearizing the suicide plasmid vector as described in claim 2 and then performing homologous recombination ligation with the double-stranded DNA sequence to obtain the final product.

4. A system as described in claim 1 for preparing a drug resistance gene bla for blocking Klebsiella pneumoniae. NDM-1 Its application in horizontal transfer reagents via IncN plasmids.

5. A system as described in claim 1 for improving the efficacy of clinical strains in blavin. NDM-1 Application of gene and IncN plasmid elimination in transformation efficiency.

6. A method for non-therapeutic purposes that eliminates bacterial resistance without leaving a trace using the system as described in claim 1, characterized in that, The process includes the following steps: S1. Conjugation delivery: combining donor bacteria containing the system with bacteria carrying bla NDM-1 The recipient bacteria of the IncN type plasmid of the gene were co-cultured, and conjugation transfer was mediated by the conjugation transfer origin sequence to introduce the system into the recipient bacteria; S2. Induced cleavage: Adding arabinose as an inducer to the recipient bacteria activates the arabinose-inducible promoter to express the Cas9 protein, which, guided by sgRNA, specifically cleaves the origin of replication and bla of the IncN plasmid. NDM-1 The gene leads to plasmid loss and drug resistance elimination; S3. Induced self-elimination: After confirming drug resistance elimination, sucrose is added to the bacterial culture as an inducer to activate the expression of the sucrose-induced vector self-elimination gene, producing toxicity, selectively killing strains still carrying the system or causing vector loss, and obtaining trace-eliminating strains without any foreign genes.

Images