Method for seamless editing of double copy target genes in bacterial artificial chromosome

Abstract

This invention provides a method for seamless editing of a double-copy target gene in a bacterial artificial chromosome. The method includes: first, constructing a bacterial artificial chromosome containing a double-copy target gene and introducing it into *E. coli* SW102 to obtain a first recombinant bacterium; second, constructing a target fragment including a chloramphenicol resistance gene, a gentamicin resistance gene, and a galactokinase encoding gene, contacting this target fragment with the first recombinant bacterium, and obtaining a second recombinant bacterium through a first homologous recombination; finally, constructing a target fragment for editing the target gene and obtaining a third recombinant bacterium through a second homologous recombination, thus completing the seamless editing of the double-copy gene. The method provided by this invention can simultaneously recombine and modify a double-copy target gene in a bacterial artificial chromosome, avoiding the problems of low double-site knock-in rate and high screening difficulty associated with single galactokinase encoding gene marker screening, and achieving seamless editing of the double-copy target gene.

Description

Technical Field

[0001] This invention relates to the field of gene editing technology, and in particular to a method for seamless editing of a double-copy target gene in a bacterial artificial chromosome. Background Technology

[0002] The development of bacterial artificial chromosomes (BACs) represents a major breakthrough in the research of DNA viral gene structure, function, and vaccines. Using bacterial artificial chromosomes for cloning, viral genomes can replicate stably within bacterial cells and undergo homologous recombination. Furthermore, any required gene recombination or mutation can be rapidly and easily achieved within bacterial artificial chromosomes. Therefore, the establishment of bacterial artificial chromosomes has greatly facilitated the construction of recombinant viruses and the study of viral gene function.

[0003] Herpes simplex virus HSV-1 has been studied as an oncolytic virus and viral vector for a long time. Using BAC as a vector, gene knockout or mutants of HSV-1 can be constructed rapidly. This not only allows for the study of viral gene function or the construction of attenuated viruses, but also enables the exogenous insertion of foreign genes (such as fluorescent proteins and immunomodulatory factors) into the viral genome through the BAC platform. This has led to the development of oncolytic viral vectors for cancer treatment and has broad application prospects.

[0004] ICP34.5 is a neurotoxic factor encoded by the HSV-1 ICP34.5 gene. It mediates the dephosphorylation of the translation initiation factor eIF2α by specifically binding to the host cell phosphatase PP1, thereby regulating viral replication and assembly and the host immune response. The ICP34.5 gene locus is located in the inverted repeat regions of the IRL and TRL in the HSV-1 genome, and there are two copies. Modifying the ICP34.5 gene is a crucial step in constructing oncolytic viruses, attenuating the virus's ability to kill normal cells while retaining or enhancing its selective lysis ability against tumor cells.

[0005] The galactokinase (galk) encoding gene serves as a positive and negative selection marker. When the galk gene is inserted into the genome of the HSV BAC vector, the recombinant bacteria can grow in a medium with galactose as the sole carbon source. When the galk gene is missing from the genome, the recombinant bacteria can grow in a medium containing 2-deoxy-galactose (DOG). This allows for the rapid screening of recombinant bacteria to achieve traceless modification of the target gene.

[0006] However, when using only galk as a selection marker for galK knock-in positive screening, insertion at any ICP34.5 gene site can result in a positive result, leading to a low rate of dual-site knock-in of the ICP34.5 gene and requiring extensive screening operations. Therefore, how to perform positive screening for simultaneous knock-in of dual-copy genes is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0007] This invention provides a method for seamless editing of double-copy target genes in bacterial artificial chromosomes.

[0008] In a first aspect, the present invention provides a method for seamless editing of a double-copy target gene in a bacterial artificial chromosome, comprising:

[0009] S1. Construct a bacterial artificial chromosome containing two copies of the target gene, and introduce the bacterial artificial chromosome into Escherichia coli SW102 to obtain the first recombinant bacterium; S2. Construct a first plasmid and a second plasmid for the first homologous recombination; the first plasmid includes an upstream homologous arm of the double-copy target gene, a chloramphenicol resistance gene expression cassette, a galactokinase gene expression cassette, and a downstream homologous arm of the double-copy target gene; the second plasmid includes an upstream homologous arm of the double-copy target gene, a gentamicin resistance gene expression cassette, a galactokinase gene expression cassette, and a downstream homologous arm of the double-copy target gene; S3. Amplify the target fragment from the first plasmid and the second plasmid, contact the target fragment from the first plasmid and the second plasmid with the first recombinant bacteria, and replace the fragment between the upstream and downstream homologous arms of the double-copy target gene in the bacterial artificial chromosome with the target fragment from the first plasmid and the second plasmid through homologous recombination, and screen for recombinant bacteria that are resistant to both chloramphenicol and gentamicin to obtain the second recombinant bacteria. S4. Construct a third plasmid for the second homologous recombination, wherein the third plasmid includes an upstream homologous arm of a double-copy target gene, a DNA fragment to be replaced, and a downstream homologous arm of a double-copy target gene; S5. Amplify the target fragment from the third plasmid, contact the target fragment from the third plasmid with the second recombinant bacteria, and replace the fragment between the upstream and downstream homologous arms of the double-copy target gene in the second recombinant bacteria with the target fragment from the third plasmid through a second homologous recombination. Screen for recombinant bacteria that can grow on a medium containing 2-deoxy-galactose to obtain the third recombinant bacteria, thus completing the seamless editing of the double-copy gene.

[0010] In one specific embodiment, the double-copy gene is the ICP34.5 gene derived from herpes simplex virus. The bacterial artificial chromosome can be constructed using conventional techniques in the art, and the bacterial artificial chromosome includes at least the double-copy ICP34.5 gene derived from herpes simplex virus. In one specific embodiment, the nucleotide sequence of the upstream homologous arm of the ICP34.5 gene in the bacterial artificial chromosome is SEQ ID NO:1 or its reverse complementary sequence, and the nucleotide sequence of the downstream homologous arm is SEQ ID NO:2 or its reverse complementary sequence.

[0011] As described above, the *E. coli* SW102 is primarily used for BAC recombination technology. Its genome retains the complete gene editing toolkit (λ-Red phage system), but the metabolic gene galK has been deleted to allow for bidirectional screening. *E. coli* SW102 needs to be cultured at a temperature not exceeding 32°C to avoid unnecessarily expressing the three λ-Red encoded genes (exo, bet, and gam recombinases).

[0012] In the method described above, the nucleotide sequence of the chloramphenicol resistance gene is the nucleotide sequence shown at positions 3657-4316 of SEQ ID NO:3 or its reverse complementary sequence; and / or, the nucleotide sequence of the galactokinase gene is the nucleotide sequence shown at positions 4607-5755 of SEQ ID NO:3 or its reverse complementary sequence; and / or, the nucleotide sequence of the gentamicin resistance gene is the nucleotide sequence shown at positions 3682-4215 of SEQ ID NO:4 or its reverse complementary sequence.

[0013] Furthermore, the chloramphenicol resistance gene expression cassette, gentamicin resistance gene expression cassette, and galactokinase gene expression cassette, in addition to including the corresponding resistance gene and galactokinase gene, also include corresponding promoters. In one specific embodiment, the chloramphenicol resistance gene expression cassette includes a cat promoter, the nucleotide sequence of which is the nucleotide sequence described in positions 4317-4419 of SEQ ID NO:3; the galactokinase gene expression cassette also includes an EM7 promoter, the nucleotide sequence of which is the nucleotide sequence described in positions 4528-4575 of SEQ ID NO:3; and the gentamicin resistance gene expression cassette includes a Pc promoter, the nucleotide sequence of which is the nucleotide sequence described in positions 4404-4432 of SEQ ID NO:4.

[0014] Furthermore, the nucleotide sequence of the target fragment containing the chloramphenicol resistance gene expression cassette and the galactokinase gene expression cassette in the first plasmid is the nucleotide sequence shown at positions 3522-5897 of SEQ ID NO:3.

[0015] Furthermore, the nucleotide sequence of the target fragment containing the gentamicin resistance gene expression cassette and the galactokinase gene expression cassette in the second plasmid is the nucleotide sequence shown in SEQ ID NO:4, numbers 3522-5941.

[0016] As described above, the seamless editing includes the deletion or replacement of a double-copy gene. In one specific embodiment, seamless editing of the ICP34.5 gene includes deleting the ICP34.5 gene from the bacterial artificial chromosome and replacing the ICP34.5 gene with a DNA fragment (named aPD-1::aVEGFA) that includes an anti-PD-1 antibody encoding gene and an anti-VEGFA antibody encoding gene. The anti-PD-1 antibody encoding gene expresses anti-PD-1 antibodies, blocking the binding of PD-1 receptors on the surface of T cells to PD-L1 on tumor cells, thereby activating the immune system to attack the tumor. The anti-VEGFA antibody encoding gene expresses anti-VEGFA antibodies, inhibiting vascular endothelial growth factor (VEGFA), cutting off the tumor's blood supply, and simultaneously reducing immunosuppressive factors in the tumor microenvironment, improving the penetration of drugs and viruses. Replacing the ICP34.5 gene with aPD-1::aVEGFA not only helps reduce the pathogenicity of HSV virus to healthy tissues but also enhances the immune response and inhibits tumor growth. Furthermore, the nucleotide sequence of the anti-PD-1 antibody encoding gene is the nucleotide sequence shown in positions 594-2099 of SEQ ID NO:5, and the nucleotide sequence of the anti-VEGFA antibody encoding gene is the nucleotide sequence shown in positions 2517-3311 of SEQ ID NO:5.

[0017] Furthermore, when the third plasmid is used for ICP34.5 gene knockout, the target fragment derived from the third plasmid includes the upstream homologous arm and the downstream homologous arm of the ICP34.5 gene, and the nucleotide sequence of the target fragment is the nucleotide sequence shown in positions 121-744 of SEQ ID NO:6.

[0018] Furthermore, when the third plasmid is used for aPD-1::aVEGFA gene insertion, the nucleotide sequence of the target fragment derived from the third plasmid is the nucleotide sequence shown in positions 121-3804 of SEQ ID NO:5.

[0019] In a second aspect, the present invention provides a biomaterial, said biomaterial being selected from at least one of A1)-A9): A1) DNA molecules containing chloramphenicol resistance gene expression cassettes and galactokinase gene expression cassettes; A2) DNA molecules containing gentamicin resistance gene expression cassettes and galactokinase gene expression cassettes; A3) A DNA molecule containing an upstream homologous arm of a double-copy target gene, a chloramphenicol resistance gene expression cassette, a galactokinase gene expression cassette, and a downstream homologous arm of a double-copy target gene; A4) A DNA molecule containing an upstream homologous arm of a double-copy target gene, a gentamicin resistance gene expression cassette, a galactokinase gene expression cassette, and a downstream homologous arm of a double-copy target gene; A5) A DNA molecule containing an upstream homologous arm of a double-copy target gene, a DNA fragment to be replaced, and a downstream homologous arm of a double-copy target gene; A6) An expression vector containing any of the DNA molecules described in A1)-A5); A7) Recombinant bacteria containing any of the DNA molecules described in A1)-A5); A8) Primers used to amplify the target fragment from the expression vector described in A6); A9) Primers used to detect the recombinant bacteria described in A7).

[0020] In the biological material described above (A6), the expression vector is selected from at least one of a first plasmid, a second plasmid, and a third plasmid, wherein the nucleotide sequence of the first plasmid is SEQ ID NO:3; the nucleotide sequence of the second plasmid is SEQ ID NO:4; and the nucleotide sequence of the third plasmid is any one of SEQ ID NO:5-6.

[0021] In the biological materials described above, A8)-A9), the primers are selected from at least one of SEQ ID NO:7-14.

[0022] Thirdly, the present invention provides a kit comprising any of the biological materials described above.

[0023] Fourthly, the present invention provides the application of any of the above-described biological materials or the above-described kits in the seamless editing of double-copy target genes in bacterial artificial chromosomes.

[0024] This invention uses chloramphenicol resistance genes and gentamicin resistance genes in combination with the galactokinase encoding gene galK to form a dual-marker screening method. This method can simultaneously perform recombination and modification on dual-copy target genes in bacterial artificial chromosomes, avoiding the problems of low dual-site knock-in rate and high screening difficulty in single galk marker screening, and achieving seamless editing of dual-copy target genes. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the process for modifying the ICP34.5 gene according to the present invention; Figure 2 The image is of the donor of the first plasmid A7-Cmr-galk-34.5; Figure 3 The image is of the donor of the second plasmid A8-Gmr-galk-34.5; Figure 4 The spectrum of the third plasmid A9-pGRB-Survivin-aPD1-TERT-aVEGFA; Figure 5 The spectrum of the fourth plasmid A10_pGRB-Survivin-BiTE-2_34.5 del donor; Figure 6 Example of electrophoresis of PCR products of HSV-BAC DNA (BAC-B4) plasmid positive clones; where 1#-6# represent the PCR products of six positive clones; Figure 7 This is an example diagram of PCR amplification of CmR-galK and GmR-galK gene cassettes. The amplified DNA fragment is about 3kb in size and contains flanking homologous sequences at both ends of the insertion site. Figure 8 Example diagram of PCR identification for CmR-galK and GmR-galK insertion into HSV-BAC (BAC-B6); among them, clone 25# contains both 3.4kb and 1.5kb fragments, indicating a unilateral insertion; clone 35# contains only a 3.4kb fragment, indicating simultaneous insertion at the ICP34.5 double-copy gene site; clones 36-37# amplify only a 1.5kb fragment, indicating a negative result and no correct insertion; Figure 9 Example images of PCR amplification products from donors with ICP34.5 deletion and aPD-1::aVEGFA gene insertion; where clone 1# has a PCR product fragment of 3706bp with aPD-1::aVEGFA gene cassette insertion; clone 2# has a PCR product fragment of 646bp with ICP34.5 deletion. Figure 10 Example diagrams for identifying PCR clones with ICP34.5 deletion and aPD-1::aVEGFA gene insertion; In A, the PCR product of clone 13 is approximately 1071 bp, indicating a positive clone for ICP34.5 gene deletion, while the others are non-recombinant clones; In B, the PCR products of clones 1, 2, 3, 4, and 6 are approximately 4131 bp, indicating positive clones for aPD-1::aVEGFA gene insertion; the PCR products of clones 5 and 8 are approximately 3447 bp, indicating non-recombinant clones; the PCR product of clone 7 contains both 4131 bp and 3447 bp fragments, indicating a single-copy recombinant clone of the aPD-1::aVEGFA gene. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, embodiments of this invention, and should not be construed as limiting the invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention. In the description of this invention, it should be understood that the terminology used is for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0028] The composition and preparation method of the culture medium involved in the following examples are as follows: Low-salt LB medium: Dissolve 10 g peptone (purchased from Bacto), 5 g yeast extract and 5 g NaCl in 1 L ddH2O, and autoclave the mixture at 121°C for 20 minutes.

[0029] Low-salt LB agar plates: Dissolve 10 g peptone (purchased from Bacto), 5 g yeast extract, 5 g NaCl, and 15 g agar powder in 1 L of ddH2O. Autoclave the mixture at 121°C for 20 minutes. After cooling to 50°C, add the corresponding antibiotic for resistance screening, and then plate the plates.

[0030] 10× M9 medium: Dissolve 60 g Na2HPO4, 30 g KH2PO4, 10 g NH4Cl and 5 g NaCl in 1 L ddH2O, and autoclave the mixture at 121℃ for 20 minutes.

[0031] 5× M63 buffer: 10 g (NH4)2SO4, 68 g KH2PO4, 2.5 mg FeSO4 Dissolve 7H2O in 1 LddH2O, adjust the pH to 7.0 with 10 N KOH, and autoclave.

[0032] M63 basal culture plate supplements: D-biotin (0.2 mg / mL, sterile filtered); D-galactose (20%, autoclaved); 2-deoxygalactose (DOG, 20%, freshly prepared before use, sterile filtered); glycerol (20%, autoclaved); L-leucine (10 mg / mL, dissolved by heating, then cooled and sterile filtered); MgSO4 7H2O (1 M, autoclaved); chloramphenicol (20 mg / mL, soluble in ethanol).

[0033] M63 basal medium plates: Autoclave 15 g agar (purchased from Bacto, the agar contains no carbon source to allow for strict selection of galK-negative clones) in 800 mL ddH2O and cool to 50°C; add 200 mL 5×M63 buffer and 1 mL 1 M MgSO4. Add 7H2O; if necessary, adjust the volume to 1L with sterile ddH2O. Add 5mL biotin (1 mg), 4.5mL leucine (45 mg), and 500μL chloramphenicol (25 μg / mL working concentration). Add carbon source and selective compounds to complete the preparation: for galactose-based medium plates, add 10 mL 20% D-galactose; for DOG-based medium plates, add 10 mL 20% glycerol and 10 mL 20% 2-deoxy-galactose.

[0034] 50×TAE buffer (1 L): 242 g Tris base, 57.1 mL glacial acetic acid, 100 mL 0.5 M EDTA (pH 8.0). Working dilution: 1× or 0.5×.

[0035] The first plasmid A7-Cmr-galk-34.5 donor, the second plasmid A8-Gmr-galk-34.5 donor, the third plasmid A9-pGRB-Survivin-aPD1-TERT-aVEGFA, and the fourth plasmid A10_pGRB-Survivin-BiTE-2_34.5 del donor involved in the following examples were all constructed by the applicant, and their nucleotide sequences are SEQ ID NO:3-6, and their maps are as follows: Figures 2-5The first plasmid, A7-Cmr-galk-34.5 donor, and the second plasmid, A8-Gmr-galk-34.5 donor, serve as donor amplification templates for galK knock-in, including the CmR-galK gene cassette, the GmR-galK gene cassette, and flanking homologous sequences at the ICP34.5 gene locus. The third plasmid, A9-pGRB-Survivin-aPD1-TERT-aVEGFA, and the fourth plasmid, A10_pGRB-Survivin-BiTE-2_34.5 del donor, serve as donor amplification templates for heterologous aPD-1::aVEGFA sequence insertion and ICP34.5 deletion, respectively containing the human aPD-1::aVEGFA gene cassette, the ICP34.5 gene deletion donor, and flanking homologous sequences at the ICP34.5 gene locus. Figures 2-5 In this context, LA represents the upstream homologous sequence of the ICP34.5 gene locus, and RA represents the downstream homologous sequence of the ICP34.5 gene locus.

[0036] The nucleotide sequences of the primers involved in the following examples are shown in Table 1.

[0037] Table 1. Nucleotide sequences of primers involved in the embodiments of the present invention

[0038] Example 1: Modification of the HSV-1 ICP34.5 gene using chloramphenicol and gentamicin combined with galK double-label recombination engineering. like Figure 1 As shown, in this embodiment, heterologous PD-1 & VEGFA antibody-targeted insertion or ICP34.5 deletion is performed on the ICP34.5 gene locus in the inverted repeat sequence region at both ends of the UL gene region of the HSV-1 genome using a combination of chloramphenicol and gentamicin with the galK selection marker.

[0039] 1. Construction of HSV-BAC DNA (named BAC-B4) A bacterial artificial chromosome comprising a double-copy ICP34.5 gene derived from herpes simplex virus (HSV-1) was constructed. In the bacterial artificial chromosome, the upstream homologous arm sequence of the ICP34.5 gene is SEQ ID NO:1 or its reverse complementary sequence, and the downstream homologous arm sequence of the ICP34.5 gene is SEQ ID NO:2 or its reverse complementary sequence. Primers as shown in Table 1 were designed based on the nucleotide sequence of the bacterial artificial chromosome.

[0040] 2. Electroporate HSV-BAC (BAC-B4) into SW102 bacterial strain. Escherichia coli SW102 is a strain derived from DH10B used to carry and amplify HSV-BAC, allowing the use of galK positive / negative selection to modify the bacterial artificial chromosome (BAC). Escherichia coli SW102 carries a λ prophage encoding a recombinase and has the galactokinase gene (galK) deleted from the galactose operon.

[0041] The specific steps include: Take 10 μL of SW102 glycerol bacterial stock solution and add it to 1 mL of low-salt LB medium, then incubate at 30°C for 1 hour. Dilute 1 μL of the bacterial solution to 10 mL of LB medium. -3 10 -4 10 -5 50 μL of the diluted bacterial culture was plated onto tetracycline-resistant low-salt LB agar plates and incubated at 30°C for 48 h for single-clonal growth. A single colony of SW102 was picked from the plate and transferred to 5 mL of low-salt LB agar (containing 12.5 μg / mL tetracycline) for overnight culture at 30°C. The overnight culture was then scaled up to 100 mL at a 1:50 ratio and placed in a 500 mL culture flask. The flask was incubated at 30°C in a shaker for approximately 3-4 hours, with bacterial density measured periodically. During bacterial growth, an ice-water mixture was prepared, and 1 L of sterile ddH2O, 50 mL centrifuge tubes, 1.5 m centrifuge tubes, and a 0.1 cm electroporation cup were pre-chilled. When the SW102 bacterial concentration (OD600) approached 0.5-0.6, the flask containing the bacteria was cooled in the ice-water mixture at 0°C for 5 minutes, and then transferred to the pre-chilled 50 mL centrifuge tubes. Centrifuge at 4000 × g for 10 min in a pre-chilled (0°C) centrifuge to precipitate the bacteria. Discard all supernatant, invert the tube onto absorbent paper to drain any remaining liquid, and then add 5 mL of pre-chilled ddH2O at 0°C, while keeping the tube with the bacterial precipitate in an ice-water mixture. Disperse the precipitate by gently agitating the centrifuge tube in the ice-water mixture until the cells are completely resuspended. Then, fill the tube to 50 mL with pre-chilled ddH2O, invert to mix, and centrifuge at 0°C, 4000 × g for 10 min in a cold centrifuge. Perform a second wash as described above, and then remove all supernatant by inverting the tube onto a paper towel. Add 1 mL of pre-chilled ddH2O at 0°C to disperse the bacterial precipitate, and then store the prepared competent cells on ice for later use.

[0042] Transfer 100 μL of freshly prepared competent cells to a pre-chilled 1.5 mL centrifuge tube and add 500 ng - 2 μg of HSV-BAC DNA (BAC-B4) to be transformed. Gently aspirate twice to mix and transfer to a pre-chilled 0.1 cm electroporation cuvette. Perform electroporation (200 Ω, 25 μF, 1.8 kV) and immediately add 1 mL of ice-cold pre-chilled low-salt LB medium to the cuvette. Transfer the bacteria to a 1.5 mL centrifuge tube and incubate at 30°C in a shaker for approximately 1 hour. Spread the transformed bacteria onto low-salt LB agar plates containing carbenicillin (50 μg / mL) to obtain single colonies. Incubate at 30°C for 1–2 days.

[0043] Select 10-50 single clones and disperse them into sterile 96-well plates containing 10 μL of low-salt LB medium. Use primer pairs 34.5-F and 34.5-R to identify SW102 positive clones containing the HSV-BAC DNA (BAC-B4) plasmid by PCR. This primer pair can accurately amplify the 34.5 gene and its flanking sequences in the IRL and TRL regions of the HSV-BAC DNA (BAC-B4) genome. The amplification process specifically includes: mixing 10 μL of Taq 2X Master Mix (purchased from neb), 0.4 μL of 10 μM forward and reverse primers (final concentration 0.25 μM), and 8.2 μL of ddH2O to obtain the PCR mixture; as a template, add 1 μL of bacterial culture directly dissolved in the PCR mixture. Amplification conditions: Initial denaturation at 95℃ for 3 min, followed by 30 cycles: 95℃ for 30 s, 64℃ for 30 s, 68℃ for 30 s, and a final extension at 68℃ for 2 min. After PCR, a 1% nucleic acid electrophoresis gel was prepared, and electrophoresis was performed at 150V for 25 min. The size of the amplified bands of single clones was detected, and the results are as follows. Figure 6 As shown in the figure, the PCR amplification fragment includes two DNA fragments of 201 bp and 292 bp, indicating that the plasmid was successfully introduced.

[0044] Select 3-5 colonies whose amplification products match the size of the positive band in the remaining mixture, and dilute to 10⁻⁶ using ddH₂O. -5 50 μL of the diluted solution was plated onto a new low-salt LB solid medium plate containing carbapenem (50 μg / mL) for purification. After three generations of purification, a single SW102 positive clone containing HSV-BAC DNA was obtained.

[0045] 3. CmR / GmR and galK dual-marker combined gene recombination The GmR-galK gene cassette expressing the gentamicin resistance gene and the galactokinase gene, and the CmR-galK gene cassette expressing the chloramphenicol resistance gene and the galactokinase gene, were simultaneously inserted into the desired positions on the HSV-BAC DNA to achieve galK knock-in. Recombinant clones exhibiting resistance to both chloramphenicol and gentamicin were then positively selected through solid-state culture with chloramphenicol and gentamicin resistance. The specific steps include: 3.1 Acquisition of the CmR-galK gene cassette Using the first plasmid, A7-Cmr-galk-34.5 donor, as a template, PCR amplification was performed using primers Donor1-CX-F and SEP-21. Specifically: 10 μL of 5× Q5 Reaction Buffer, 10 μL of 5X Q5 High GC Enhancer, 1 μL of 10 mM dNTPs, 2.5 μL of 10 μM forward and reverse primers (final concentration 0.5 μM), 0.5 μL of Q5 High-Fidelity DNA Polymerase (purchased from NEB), and 23 μL of ddH2O were mixed, and 1 μL of 2 ng / μL of the first plasmid, A7-Cmr-galk-34.5 donor, was added as a template. Amplification conditions: initial denaturation and hot start at 98℃ for 1 min, followed by 30 cycles: 98℃ for 10 s, 58℃ for 30 s, 72℃ for 90 s, and a final extension at 72℃ for 2 min. Take 5 μL of PCR product and electrophoresis it on a 0.8% agarose gel at 150 V for 30 min. The electrophoresis results of the PCR amplification products are as follows. Figure 7 As shown, a band of approximately 3.1 kbp appears, indicating successful amplification.

[0046] 3.2 Acquisition of the GmR-galK gene cassette Using the second plasmid A8-Gmr-galk-34.5 donor as a template, PCR amplification was performed using the same primers Donor1-CX-F and SEP-21 as in 3-1, and the PCR amplification method was used. The amplification products were detected by gel electrophoresis, and the results are as follows. Figure 7 As shown, a band of approximately 3.1 kbp also appears, proving that the amplification was successful.

[0047] 50 μL of PCR product was digested with 1 μL (40 U) of DpnI restriction endonuclease at 37°C for 1 hour to remove methylated A7 and A8 plasmid template. The DpnI digestion product was then electrophoresed again on a 0.8% agarose gel (150 V, 30 min), and the CmR-galK and GmR-galK DNA bands were purified by gel extraction centrifugation. The DNA fragments were eluted in 30 μL of nuclease-free ddH2O (without salt buffer to avoid interference with subsequent electroporation). The concentration of recovered DNA was measured using a UV spectrophotometer and diluted with ddH2O to a final concentration of 400 ng / μL. (Higher donor template concentrations are beneficial for improving homologous recombination efficiency.) 3.3 First Homologous Recombination A single SW102 colony containing HSV-BAC (BAC-B4) was inoculated into 5 mL of low-salt LB medium containing carbenicillin (50 μg / mL) and incubated overnight at 30°C with shaking. 2 mL of the overnight culture was diluted into 100 mL of low-salt LB medium containing carbenicillin (25 μg / mL) and incubated at 30°C with shaking until the OD600 reached between 0.55 and 0.65. Simultaneously, 1 L of sterile ddH2O, 50 mL centrifuge tubes, 1.5 mL centrifuge tubes, and a 0.1 cm electroporation cup were pre-chilled in an ice-water mixture at 0°C. The culture was aliquoted into two 250 mL bottles, 50 mL per bottle. One bottle was incubated in a 42°C water bath with shaking for 15 min (carefully check the temperature of the shaking water bath; lower temperatures may reduce induction efficiency and cause a sharp decrease in recombination frequency) to induce λ prophage recombinase (induction sample), while the other bottle was kept at 30°C (uninduced control). Both cultures (induced and uninduced control samples) were cooled on ice for 5 minutes, transferred to 50 mL pre-chilled centrifuge tubes, and centrifuged at 4000 × g for 10 minutes at 0°C. All supernatant was discarded, and 5 mL of sterile, ice-chilled ddH2O was added. The precipitate was gently resuspended by vortexing in an ice-water slurry. The centrifuge tubes were filled to 50 mL with sterile, ice-chilled ddH2O, inverted to mix, and centrifuged at 4000 × g for 10 minutes at 0°C to precipitate the bacterial cells. The above steps were repeated, discarding all supernatant, inverting the tubes on absorbent paper to completely remove the supernatant, and gently resuspending the bacterial precipitate in the small amount of ddH2O remaining in the tubes by vortexing in an ice-water slurry. After adding 500 μL of ddH2O, the competent cells were placed on ice for later use.

[0048] In a pre-chilled 1.5 mL centrifuge tube, mix 100 μL of competent cells, 5 μL of CmR-galK, and 5 μL of the CmR-galK fragment (concentration 400 ng / μL). Gently aspirate the DNA-cell mixture twice and transfer it to a pre-chilled 0.1 cm electroporation cuvette at 25 μF, 1.8 kV, and 200 Ω. Add 1 mL of ice-cold low-salt LB medium and incubate at 30 °C in a shaker for 2 hours to revive the bacteria. Wash the bacteria with 1 × M9 medium, centrifuge approximately 1 mL of the culture at 14,000 × g for 15 seconds, and carefully remove the supernatant with a micropipette. Resuspend the pellet in 1 mL of 1 × M9 medium. Repeat the above steps. Spread the culture onto low-salt LB agar plates containing three antibiotics: carbenicillin (50 μg / mL), chloramphenicol (25 μg / mL), and gentamicin (50 μg / mL). Incubate the plates at 30°C for 2 days. To ensure that positive clones are obtained in one run, select approximately 50-200 single clones and add them to sterile 96-well plates containing 10 μL of low-salt LB. Identify positive clones of the inserted fragment by PCR.

[0049] The PCR process specifically included: mixing 7.5 μL of 2×Phanta Flash Master Mix (Dye Plus) (purchased from Nanjing Novizan Biotechnology, catalog number P520), 0.6 μL of 10 μM primer pair 79 and 82 (final concentration 0.5 μM), and 5.3 μL of ddH2O to obtain the PCR mixture; then adding 1 μL of bacterial culture directly to the PCR mixture. Amplification conditions: initial denaturation at 98℃ for 2 minutes, followed by 30 cycles: 98℃ for 20 seconds, 64℃ for 10 seconds, 72℃ for 35 seconds, and a final extension at 72℃ for 2 minutes. After PCR, a 1% nucleic acid electrophoresis gel was prepared, and electrophoresis was performed at 150V for 30 minutes to detect the size of the amplified bands of single clones. The inserted CmR-galK and GmR-galK positive fragments are approximately 3.4 kb in size, while the PCR product of a negative clone without insertion is approximately 1.5 kb in size. A single insertion of the ICP34.5 double-copy gene contains both 3.4 kb and 1.5 kb fragments. Figure 8 As shown.

[0050] Select 3-5 colonies whose amplification products match the size of the positive band in the remaining mixture, and dilute to 10⁻⁶ using ddH₂O. -550 μL of the diluted solution was plated onto new low-salt LB agar plates containing three types of antibiotics: carbenicillin (50 μg / mL), chloramphenicol (25 μg / mL), and gentamicin (50 μg / mL) for P2 generation single-clone purification. After single colony growth for 2 days, 6-10 clones were picked from each plate of 3-5 selected positive P2 clones and subjected to PCR detection and electrophoresis verification under the same conditions. The P3 generation purification was then performed. The P3 purified single clones were sequenced using PCR products and the bacterial culture was preserved. The HSV BAC with GmR / CmR-galK insertion was named BAC-B6.

[0051] 3.4 Second Homologous Recombination The ICP34.5 gene was deleted or the aPD-1::aVEGFA gene was inserted into the GmR-galK and CmR-galK DNA sequences in the HSV-BAC (BAC-B6) clone. Negative selection of galK was performed using 2-deoxy-galactose (2-DOG) (2-DOG is toxic when phosphorylated by the galK gene product) to ensure the identification of recombinant clones.

[0052] The donor DNA fragment designed for insertion into the aPD-1::aVEGFA gene was amplified using the third plasmid A9-pGRB-Survivin-aPD1-TERT-aVEGFA as a template, with primers 95 and 96. The amplification process included the following steps: 25 μL of 2×Phanta Flash Master Mix (purchased from Nanjing Novizan Biotechnology, catalog number P520), 2.5 μL of 10 μM forward and reverse primers (final concentration 0.5 μM), and 19 μL of ddH2O were mixed to obtain the PCR mixture. 1 μL of 50 ng / μL of the third plasmid A9-pGRB-Survivin-aPD1-TERT-aVEGFA was added as a template. The amplification conditions were: initial denaturation and hot start at 98℃ for 2 minutes, followed by 30 cycles: 98℃ for 20 seconds, 64℃ for 10 seconds, and 72℃ for 40 seconds. Examine the PCR products on a 0.8% agarose gel (150 V, 40 min). Figure 9 As shown, the amplified aPD-1::aVEGFA gene insertion fragment of 3706 bp can be confirmed.

[0053] Donor DNA fragment designed for ICP34.5 gene deletion: Using plasmid A10_pGRB-Survivin-BiTE-2_34.5 del donor as a template, PCR amplification was performed using primers 95 and 96 with the same PCR amplification method as described above, and the amplification products were detected by electrophoresis. Results are as follows: Figure 9As shown, it can be confirmed that the amplified product is 646bp.

[0054] 50 μL of PCR product was digested with 1 μL (40 U) of DpnI restriction endonuclease and reacted at 37 °C for 1 hour to remove methylated template. The DpnI digestion product was electrophoresed on a 0.8% agarose gel (150 V, 30 min), and the target band was purified by gel extraction and centrifugation. The fragment was eluted in 30 μL of nucleic acid-free ddH2O. The transgenic fragment was quantified using a UV spectrophotometer and diluted to a final concentration of 500 ng / μL with ddH2O.

[0055] HSV-BAC (BAC-B6) positive clones were inoculated into 5 mL of low-salt LB medium containing carbenicillin (50 μg / mL), gentamicin (50 μg / mL), and chloramphenicol (25 μg / mL). Competent cells were prepared according to the method for preparing competent cells in the first homologous recombination in section 3.3. 100 μL of competent cells and 10 μL of gene fragment (500 ng / μL) were mixed in a pre-chilled 1.5 mL centrifuge tube. The DNA-cell mixture was transferred to a pre-chilled 0.1 cm electroporation cuvette with parameters of 25 μF, 1.8 kV, and 200 Ω. After electroporation, 1 mL of ice-chilled low-salt LB medium was immediately added, followed by transfer to a 50 mL centrifuge tube containing 9 mL of room-temperature low-salt LB medium. The tube cap was loosened, and the bacteria were incubated at 30°C in a shaker for 4.5 hours. 2 mL of the culture was centrifuged at 13000 g for 30 s to precipitate the bacterial cells, and the cells were washed twice with 1×M9 salt. After washing, the bacterial pellet was resuspended in 500 μL of 1× M9 salt and spread onto M63 DOG basal medium containing carbenicillin (50 μg / mL) for negative selection against galk. The plates were incubated at 30°C for 4 days.

[0056] 200 / 300 single colonies were selected from the induction sample plates and added to sterile 96-well plates containing 10 μL of low-salt LB medium. PCR amplification was performed using primer pairs 79 and 82 to detect the success of gene modification. The PCR process included mixing 7.5 μL of 2×Phanta Flash Master Mix (Dye Plus) (purchased from Nanjing Novizan Biotechnology, catalog number P520), 0.6 μL of 10 μM forward and reverse primers (final concentration 0.5 μM), and 5.3 μL of ddH2O to obtain the PCR mixture. 1 μL of bacterial culture was added directly to the PCR mixture to dissolve it. Amplification conditions: initial denaturation at 98℃ for 2 minutes, followed by 30 cycles: 98℃ for 20 seconds, 64℃ for 10 seconds, 72℃ for 35 seconds, and a final extension at 72℃ for 2 minutes. After PCR amplification, a 0.8% nucleic acid electrophoresis gel was prepared, and electrophoresis was performed at 150V for 30 minutes to detect the size of the amplified bands of the single clones. The results are as follows Figure 10 As shown in Figure A, lanes 1-12 had no amplification products, indicating non-recombinant clones, while lane 13 had an amplification product of 1071 bp, indicating a positive clone for ICP34.5 gene deletion. In Figure B, the PCR products of lanes 1, 2, 3, 4, and 6 were approximately 4131 bp, indicating a positive clone for aPD-1::aVEGFA gene insertion; the PCR products of lanes 5 and 8 were approximately 3447 bp, indicating non-recombinant clones; and the PCR product of lane 7 contained both 4131 bp and 3447 bp fragments, indicating a single-copy recombinant clone of the aPD-1::aVEGFA gene.

[0057] Select 3-5 colonies whose amplification products match the size of the positive band in the remaining mixture, and dilute to 10⁻⁶ using ddH₂O. -5 50 μL of the diluted solution was plated onto a new low-salt LB agar plate containing carbenicillin (50 μg / mL) resistance for P2 generation single-clone purification. After single colonies had grown for 2 days, 6-10 clones were picked from each plate of 3-5 selected positive P2 clones for PCR verification, followed by P3 generation purification. The single clones obtained from the P3 generation purification were then subjected to PCR product sequencing verification and bacterial culture preservation.

[0058] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for seamless editing of a double-copy target gene in a bacterial artificial chromosome, characterized in that, include: S1. Construct a bacterial artificial chromosome containing two copies of the target gene, and introduce the bacterial artificial chromosome into Escherichia coli SW102 to obtain the first recombinant bacterium; S2. Construct a first plasmid and a second plasmid for the first homologous recombination; the first plasmid includes an upstream homologous arm of the double-copy target gene, a chloramphenicol resistance gene expression cassette, a galactokinase gene expression cassette, and a downstream homologous arm of the double-copy target gene; the second plasmid includes an upstream homologous arm of the double-copy target gene, a gentamicin resistance gene expression cassette, a galactokinase gene expression cassette, and a downstream homologous arm of the double-copy target gene; S3. Amplify the target fragment from the first plasmid and the second plasmid, contact the target fragment from the first plasmid and the second plasmid with the first recombinant bacteria, and replace the fragment between the upstream and downstream homologous arms of the double-copy target gene in the bacterial artificial chromosome with the target fragment from the first plasmid and the second plasmid through homologous recombination, and screen for recombinant bacteria that are resistant to both chloramphenicol and gentamicin to obtain the second recombinant bacteria. S4. Construct a third plasmid for the second homologous recombination, wherein the third plasmid includes an upstream homologous arm of a double-copy target gene, a DNA fragment to be replaced, and a downstream homologous arm of a double-copy target gene; S5. Amplify the target fragment from the third plasmid, contact the target fragment from the third plasmid with the second recombinant bacteria, and replace the fragment between the upstream and downstream homologous arms of the double-copy target gene in the second recombinant bacteria with the target fragment from the third plasmid through a second homologous recombination. Screen for recombinant bacteria that can grow on a medium containing 2-deoxy-galactose to obtain the third recombinant bacteria, thus completing the seamless editing of the double-copy gene.

2. The method according to claim 1, characterized in that, The seamless editing includes the deletion or replacement of double-copy genes.

3. The method according to claim 1, characterized in that, The double-copy target gene is the ICP34.5 gene derived from herpes simplex virus.

4. The method according to any one of claims 1-3, characterized in that, The nucleotide sequence of the chloramphenicol resistance gene is the nucleotide sequence shown at positions 3657-4316 of SEQ ID NO:3 or its reverse complementary sequence; and / or, the nucleotide sequence of the galactokinase gene is the nucleotide sequence shown at positions 4607-5755 of SEQ ID NO:3 or its reverse complementary sequence; and / or, the nucleotide sequence of the gentamicin resistance gene is the nucleotide sequence shown at positions 3682-4215 of SEQ ID NO:4 or its reverse complementary sequence.

5. The method according to any one of claims 1-3, characterized in that, The nucleotide sequence of the target fragment containing the chloramphenicol resistance gene expression cassette and the galactokinase gene expression cassette in the first plasmid is the nucleotide sequence shown in positions 3522-5897 of SEQ ID NO:3; and / or, the nucleotide sequence of the target fragment containing the gentamicin resistance gene expression cassette and the galactokinase gene expression cassette in the second plasmid is the nucleotide sequence shown in positions 3522-5941 of SEQ ID NO:

4.

6. A biomaterial, characterized in that, The biomaterial is selected from at least one of A1)-A9): A1) DNA molecules containing chloramphenicol resistance gene expression cassettes and galactokinase gene expression cassettes; A2) DNA molecules containing gentamicin resistance gene expression cassettes and galactokinase gene expression cassettes; A3) A DNA molecule containing an upstream homologous arm of a double-copy target gene, a chloramphenicol resistance gene expression cassette, a galactokinase gene expression cassette, and a downstream homologous arm of a double-copy target gene; A4) A DNA molecule containing an upstream homologous arm of a double-copy target gene, a gentamicin resistance gene expression cassette, a galactokinase gene expression cassette, and a downstream homologous arm of a double-copy target gene; A5) A DNA molecule containing an upstream homologous arm of a double-copy target gene, a DNA fragment to be replaced, and a downstream homologous arm of a double-copy target gene; A6) An expression vector containing any of the DNA molecules described in A1)-A5); A7) Recombinant bacteria containing any of the DNA molecules described in A1)-A5); A8) Primers used to amplify the target fragment from the expression vector described in A6); A9) is a primer used to detect the recombinant bacteria described in A7).

7. The biomaterial according to claim 6, characterized in that, In A6), the expression vector is selected from at least one of a first plasmid, a second plasmid, and a third plasmid, wherein the nucleotide sequence of the first plasmid is SEQ ID NO:3; the nucleotide sequence of the second plasmid is SEQ ID NO:4; and the nucleotide sequence of the third plasmid is any one of SEQ ID NO:5-6.

8. The biomaterial according to claim 6, characterized in that, In A8)-A9), the primers are selected from at least one of SEQ ID NO:7-14.

9. A reagent kit, characterized in that, Includes the biomaterials described in any one of claims 6-8.

10. The use of the biomaterial according to any one of claims 6-8 or the kit according to claim 9 in the seamless editing of a double-copy target gene in a bacterial artificial chromosome.

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