Recombinant plasmid, recombinant system and construction method and application thereof

Abstract

The invention belongs to the technical field of gene engineering, and relates to a recombinant plasmid, a recombinant system and a construction method and application thereof. The plasmid expresses an operon under the induction of a promoter, and the operon is composed of a CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-associated 9) system and a Red / ET recombinase system derived from pseudoalteromonas P. agarivorans Hao 2018. The recombinase system comprises a recombinase 2018Red [beta] and a recombinase 2018Red [alpha]; homologous arms with different lengths for recombination are inserted between the Cas9 protein and the recombinase 2018Red alpha beta. According to the invention, the homologous arm and the guide RNA sequence are constructed to the same site of the plasmid, and the construction can be completed by only one step of Gibson assembly, so that the workload is reduced, the working efficiency is improved, and the gene editing period can be completed in a relatively short time. The invention provides a new tool for gene editing, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and relates to a recombinant plasmid, a recombinant system, its construction method and application. Background Technology

[0002] Prokaryotes, especially bacteria, serve as core models in life science research and crucial foundational cells for industrial applications. Precise genome editing of prokaryotes is the cornerstone for understanding life mechanisms, optimizing metabolic pathways, and developing novel biotechnologies. In the early stages of technological development, prokaryotic genome modification primarily relied on homologous recombination and random mutation, which were inefficient, cumbersome, and time-consuming, severely hindering basic biological research in prokaryotes.

[0003] The emergence of the Red / ET recombination system, derived from λ phage, in the early 21st century represented a major leap forward in the field of prokaryotic genome editing. This system can efficiently mediate homologous recombination using relatively short homologous arms (generally less than 100 bp). The Red / ET recombination system mainly consists of recombinant proteins Redα / Redβ derived from λ phage and recombinant proteins RecE / RecT derived from Rac prophage. RecE and Redα possess exonuclease activity at their 5′ to 3′ ends, resulting in single-stranded 3′ ends, while RecT and Redβ are single-stranded annealing proteins that can stably expose the 3′ ends, mediating homologous recombination. The Red / ET recombination system has been widely used in genetic operations such as gene knockout, knock-in, and replacement, providing an effective tool and method for genome mining.

[0004] The CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated proteins) system is an acquired immune system widely found in bacteria and archaea. The engineered CRISPR-Cas9 system, requiring only simple spacer sequence design, allows the transcribed sgRNA (single guide RNA) to guide the Cas9 protein to cut the target sequence. The cell's own DNA damage repair mechanisms then produce insertion-deletion (indel) mutants in the target region. This method has been successfully applied to genome editing in mammalian cells, *Saccharomyces cerevisiae*, *Escherichia coli*, *Staphylococcus aureus*, and *Pseudomonas aeruginosa*. While this system is highly efficient, the results are often inaccurate insertions or deletions (indels). To achieve precise genome editing, it is usually combined with homologous recombination. Traditional homologous recombination is inefficient and difficult to obtain mutants without selection markers. Therefore, researchers often combine CRISPR-Cas9 with Red / ET to achieve precise genome editing. This system is currently in the exploratory stage and relies on electroconversion, which greatly limits its application.

[0005] *Pseudorabies* is a marine-endemic group. All known species of the genus *Pseudorabies* have been isolated from marine samples, and their distribution is extremely wide, ranging from the equator to the poles, and from coastal to deep sea. They are a dominant group in the marine environment, and their diverse habitats have resulted in rich metabolic pathways, making them highly valuable for research. Most *Pseudorabies* can be genetically manipulated, primarily relying on long homologous arm-mediated homologous recombination combined with reverse selection markers. Two rounds of selection are required to obtain the target mutant, which is time-consuming, labor-intensive, and inefficient. While Red / ET-mediated homologous recombination has been widely used in prokaryotes such as *Escherichia coli* and *Burkholderia*, it usually requires electroporation, making it difficult to apply to marine bacteria and Gram-positive bacteria, which are difficult to electroporate. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the purpose of this invention is to provide a pseudoalternating monoclonal recombinant system and its construction and application.

[0007] To achieve the above-mentioned objectives of the present invention, the present invention provides the following technical solution: A recombinant plasmid, under the inducible promoter, expresses an operon derived from the CRISPR-Cas9 system and *Pseudomonas pseudoalteromonas*.P. agarivorans The Red / ET recombinase system in Hao 2018 comprises recombinase 2018Redβ and recombinase 2018Redα; homologous arms of different lengths for recombination are inserted between the Cas9 protein and recombinase 2018Redαβ.

[0008] Preferably, the plasmid contains recombinase 2018. The expression of Redαβ and Cas9 proteins is regulated by the inducible tet promoter.

[0009] Preferably, in the plasmid, the expression of guide RNA is regulated by a constitutive tac promoter.

[0010] Preferably, the length of the homologous arm is 50 bp-200 bp.

[0011] Furthermore, the present invention provides a recombination system comprising the recombinant plasmid.

[0012] Furthermore, the present invention provides a method for constructing the recombinant plasmid, comprising: CRISPR-Cas9 and the Red / ET recombinase system derived from strain Hao 2018 were constructed onto the same plasmid to obtain plasmid pBBR1-km. R -oriT-2018 βα Cas9; Using Gibson assembly to assemble the spacer sequence of the target sequence sp Inserted into the plasmid to construct plasmid pBBR1-km R -oriT-2018 βα Cas9- sp ; Utilize the reserved Pvu The I restriction enzyme site inserts homologous arms (HA) of different lengths targeting the target sequence into the plasmid to obtain plasmid pBBR1-kmR-oriT-2018. βα Cas9- sp -PvuI-HA.

[0013] Preferably, the homologous arm and the guide RNA sequence are constructed into the same site on the plasmid to obtain plasmid pBBR1-kmR-oriT-2018. βα Cas9-HAsp.

[0014] Furthermore, the present invention also provides the application of the recombinant plasmid and the recombinant system in gene editing.

[0015] This invention constructs the homologous arm and guide RNA sequence at the same site on the plasmid. The recombinant plasmid can be assembled in a single Gibson step, greatly reducing workload and improving efficiency. This allows the gene editing cycle, including plasmid construction, binding transfer, induction of systemic expression, and colony PCR screening of target mutants, to be completed in a shorter time. This invention provides a new tool for gene editing and has broad application prospects. Attached Figure Description

[0016] Figure 1 The Red / ET recombinase system derived from *Pseudomonas aeruginosa* is used in this embodiment of the invention. Figure 2 For the construction and efficiency of RECC system; Figure 3 The efficiency of the CRISPR-Cas9 system with redundant bases in the 5′ spacer sequence; Figure 4 To improve the efficiency of the RECC system; Figure 5 The gene clusters 1-4 are shown, along with their predicted product structures and the activation of these gene clusters using a promoter substitution strategy. Detailed Implementation

[0017] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Unless otherwise specified, the methods in the embodiments adopt conventional methods, and the reagents used are conventional commercially available reagents or reagents prepared according to conventional methods unless otherwise specified.

[0018] Reagents and instruments: In this embodiment, *Pseudomonas alterniflora* DSM 14401 was purchased from DSMZ. The reagents were mainly molecular biology experimental reagents. Single-stranded nucleotides were synthesized from Shanghai Sangon Biotech Co., Ltd., and restriction endonucleases and DNA polymerases were purchased from New England Biolabs.

[0019] Culture medium: 2216E: 5 g peptone, 1 g yeast extract, 15 g agar powder (solid culture medium), 1 L seawater, pH 7.2.

[0020] LB: 10 g tryptone, 5 g yeast extract, 1 g NaCl, 12 g agar powder (solid culture medium), 1 L distilled water, pH natural.

[0021] MLB: Add 50 mM MgSO4•7H2O to LB medium.

[0022] Resistance screening: Freshly cultured pseudoalteromonas DSM 14401 (hereinafter referred to as DSM 14401) was spread on 2216E plates containing different antibiotics and incubated overnight at 30 °C. Its growth was then observed.

[0023] Promoter selection: This invention utilizes firefly luciferase reporter gene screening to select inducible promoters (controlling the expression of recombination systems) and constitutive strong promoters (for activating silent gene clusters) that can work in DSM 14401. The plasmid construction employs Gibson assembly.

[0024] The specific methods for promoter screening are as follows: (1) The constructed plasmid was transferred into DSM 14401 via conjugation transfer mediated by Escherichia coli WM 3064; (2) For inducible promoters, the transformant should first be transferred to 1 mL of MLB liquid medium containing kanamycin and cultured at 30 ℃ and 950 rpm for 12 h. Then, 40 µL of seed culture should be transferred to 1 mL of MLB liquid medium containing kanamycin and cultured at 30 ℃ and 950 rpm for 2 h. After adding the corresponding inducer, culture for another 1 h. For constitutive promoters, the transformant should be transferred to 1 mL of MLB liquid medium containing kanamycin and cultured at 30 ℃ and 950 rpm for 18 h. (3) Take 100 μL of the above culture and mix it with fresh MLB medium to dissolve the OD. 600 The values ​​are uniformized to 1; (4) Take 90 uL of homogenized bacterial culture, add 10 uL of phosphate buffer (pH 7.8) and place it in a -80 ℃ refrigerator for 15 min; (5) Place the frozen sample in a room temperature water bath until the sample is completely thawed, then add 100 uL of lysozyme solution and 200 uL of 2× cell lysis buffer (add 5 mg / mL of BSA before use), and incubate at room temperature for 10 min. (6) Add 20 uL of cell lysis buffer and 100 uL of luciferase substrate to a black 96-well plate, measure the signal intensity, and calculate the mean and standard deviation of the three parallel experiments.

[0025] Example 1: Using BLAST to identify the Red / ET recombinase system derived from Pseudoalteromonas. The Red / ET recombinase system has been successfully implemented in Escherichia coli, Pseudomonas, and Burkholderia, greatly facilitating gene manipulation in these bacterial groups. However, the Red / ET recombinase system exhibits strong species specificity. Therefore, this invention utilizes known Red / ET recombinase systems to search for homologous proteins of Pseudomonas pseudoalteromonas in a database, ultimately obtaining three Red / ET recombinase systems, such as... Figure 1 As shown. Derived from *Pseudomonas aeruginosa*. P. sp. DL6 and P. shioyasakiensis The HI0053 recombination system possesses a single-strand binding protein (SSB), which shares 46.6% similarity with the known SSB protein, which enhances recombination efficiency. Redβ, however, shares 46.4% similarity with known recombination systems, while Redα shares only 26.3%. (Source: [Original Source Name]) P. agarivorans The Hao 2018 Red / ET recombination system consists of Redβ and Redα, with similarities of 50.5% and 25.6% respectively to known recombination systems; it originated from *Pseudomonas pseudoalteromonas*. P. nigrifaciens Although the FME53 recombinant system also consists of Redβ and Redα, its Redβ is shorter and its Redα is longer, and the Redα shows low similarity to known recombinant systems. Since all three recombinant systems originate from non-type strains, obtaining these strains is difficult. To obtain these recombinant systems, this invention uses gene synthesis to synthesize two of them (DL6...). sβα and Hao2018 βα To conduct subsequent experiments.

[0026] Example 2: Construction and functional verification of the Pseudoalteromonas Recombinant System (RECC System) This invention constructs CRISPR-Cas9 and the Red / ET recombinase system derived from strain Hao 2018 onto the same plasmid, creating the plasmid pBBR1-km. R -oriT-2018 βα Cas9, in which the tetracycline promoter Ptet regulates the expression of recombinase 2018Redαβ and Cas9 proteins, while the promoter portion of Ptac initiates the expression of guide RNA ( Figure 2 Then, using Gibson assembly, the spacer sequence of the slr gene, which targets the marker protein encoding DSM14401, was inserted into the plasmid to construct the plasmid pBBR1-km. R -oriT-2018 βα Cas9- slr Reuse the reserved PvuI restriction enzyme sites were used to insert homologous arms of different lengths into the plasmid to obtain plasmid pBBR1-kmR-oriT-2018. βα Cas9-slr-PvuI-200bp, pBBR1-kmR-oriT-2018 βα Cas9-slr-PvuI-100bp and pBBR1-kmR-oriT-2018 βα Cas9-slr-PvuI-50bp was transduced into DSM 14401 and pre-cultured for 6 hours. After induction with anhydrous tetracycline for 18 hours, the culture was plated on MLB plates containing kanamycin, and single colonies were picked for validation. It was found that 100bp could effectively mediate recombination, with a recombination efficiency of about 20%.

[0027] Besides liquid induction, solid plate induction has also been reported. Solid plate induction occurs after conjugation transfer, where the conjugated bacterial colony is directly spread onto plates containing inducers and resistance for induction and screening, eliminating the need for transfer induction and significantly reducing the experimental cycle. This invention also attempted solid plate induction, finding that while relatively high recombination efficiency was achieved with a 200bp homologous arm, the number of clones growing on each induction plate varied greatly, sometimes even failing to produce a single clone. Therefore, this invention utilizes a 100bp homologous arm combined with liquid induction for subsequent research.

[0028] Example 3: Optimization of the RECC System In the initial experiments, this invention constructed the spacer sequence and homologous arm from the RECC system at different sites on the plasmid. This required the plasmid construction to be performed in two steps, and the repetitive steps of plasmid extraction, enzyme digestion, gel recovery, and Gibson assembly significantly impacted experimental efficiency. To overcome this problem, this invention constructed the homologous arm and spacer sequence at the same site on the plasmid. This invention constructed the plasmid pBBR1-kmR-oriT-Cas9-100bp-Slr, with the spacer sequence and a 100 bp homologous arm together, and transformed it into DSM 14401 to test whether adding a 200 bp redundant base to the 5′ end of the spacer sequence would affect the efficiency of CRISPR-Cas9. The results are as follows: Figure 3 As shown, the addition of redundant bases did not affect the operation of CRISPR-Cas9, and no marker proteins were found in any of the selected clones. To further confirm this result, five clones were randomly selected for sequencing, and the results showed... slr The spacer sequence in the gene did indeed undergo single-base insertion or deletion mutations. This finding lays the foundation for simplifying the construction of RECC system plasmids.

[0029] After confirming that redundant bases would not affect the operation of the CRISPR-Cas9 system, this invention constructed a series of RECC system recombinant plasmids with homologous arms and spacer sequences, mainly including pBBR1-kmR-oriT-2018. βα Cas9-100bp-slr, pBBR1-kmR-oriT-2018 βα Cas9-50bp-slr and pBBR1-kmR-oriT-2018 βα Cas9-25bp-slr ( Figure 4 They were then transferred into DSM 14401, and the accuracy of monoclonal colonies grown on the plates after induction was measured. The results were similar to those before. Figure 4 A 100 bp homologous arm can effectively mediate homologous grouping, achieving a cloning accuracy of over 20%. RECC recombinant plasmids with homologous arms and spacer sequences can be assembled in a single Gibson step, and the entire RECC system has a consistent plasmid backbone; only the spacer and homologous arm sequences need to be modified according to the gene editing objective. This improvement significantly reduces workload and increases efficiency, allowing the gene editing cycle—including plasmid construction, conjugation transfer, induction of RECC system expression, and colony PCR screening of target mutants—to be completed within 5 days.

[0030] Thus, this invention has initially constructed a gene editing system that can work efficiently in Pseudoalteromonas. The biggest advantage of this system is that it does not rely on electroporation, which also provides ideas for the development of gene editing methods for other groups with low electroporation efficiency.

[0031] Example 4: Activation of silent gene clusters in DSM 14401 using the RECC system After constructing the recombination system, this invention attempts to activate silent gene clusters in DSM 14401 using the newly constructed RECC system combined with promoter insertion. The expression of silent gene clusters 1-4 in DSM 14401 is activated using the strong promoter P18. Gene clusters 1-4 are a PKS-NRPS heterozygous cluster. Using bioinformatics analysis, this invention predicts the possible structures of its products (…). Figure 5The constructed promoter insertion plasmid pBBR1-kmR-oriT-2018βαCas9-P18-BGC1-4 was transformed into DSM 14401, and the promoter replacement was completed using the RECC gene editing system, successfully obtaining the mutant strain DSM14401::P18_pfl. Simultaneously, the gene cluster was inactivated using CRISPR-Cas9, obtaining the mutant strain DSM14401Δpfl. Fermentation, crude extract preparation, and HPLC-MS detection were then performed on the mutant strain and the wild-type strain. The results showed that two distinct specific ion peaks with mass-to-charge ratios of 984 (compound 6) and 970 (compound 7) appeared in the fermentation broth of the P18 promoter insertion mutant strain. These two peaks were not found or had extremely low abundance in the original strain or the gene cluster-inactivated mutant strain, indicating that they are products of gene cluster activation. Figure 5 ).

Claims

1. A recombinant plasmid, characterized in that: This plasmid, under the inducible promoter, expresses an operon derived from the CRISPR-Cas9 system and *Pseudomonas pseudoalteromonas*. P. agarivorans The Red / ET recombinase system in Hao 2018 comprises recombinase 2018Redβ and recombinase 2018Redα; homologous arms of different lengths for recombination are inserted between the Cas9 protein and recombinase 2018Redαβ.

2. The recombinant plasmid according to claim 1, characterized in that: The expression of recombinase 2018Redαβ and Cas9 proteins is regulated by an inducible promoter.

3. The recombinant plasmid according to claim 1, characterized in that: The expression of guide RNA is regulated by a constitutive promoter.

4. The recombinant plasmid according to claim 1, characterized in that: The length of the homologous arm is 50 bp-200 bp.

5. A recombinant system comprising the recombinant plasmid according to any one of claims 1-4.

6. The method for constructing the recombinant plasmid according to any one of claims 1-4, characterized in that, include: CRISPR-Cas9 and the Red / ET recombinase system derived from strain Hao 2018 were constructed onto the same plasmid to obtain plasmid pBBR1-km. R -oriT-2018 βα Cas9; Using Gibson assembly to assemble the spacer sequence of the target sequence sp Inserted into the plasmid to construct plasmid pBBR1-km R -oriT-2018 βα Cas9- sp ; Utilize the reserved Pvu The I restriction enzyme site inserts homologous arms (HA) of different lengths targeting the target sequence into the plasmid to obtain plasmid pBBR1-kmR-oriT-2018. βα Cas9- sp -PvuI-HA.

7. The construction method according to claim 6, characterized in that: Homologous arms and guide RNA sequences were constructed at the same site on the plasmid to obtain plasmid pBBR1-kmR-oriT-2018. βα Cas9-HAsp.

8. The application of the recombinant plasmid according to any one of claims 1-4 and the recombinant system according to claim 5 in gene editing.

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