A gene editing system, a double-plasmid system, an engineered bacterium and application

Abstract

The application discloses a gene editing system, a double-plasmid system, engineering bacteria and application, and relates to the technical field of gene editing. The gene editing system comprises gRNA and a recombination system, and the recombination system comprises a nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3. The gene editing system provided by the application has good controllability, high gene editing efficiency, and can realize efficient gene editing under the condition of short homologous arms. The gene length of the recombination system is far less than that of a lambda-red system and a RecET system, and the recombination system does not depend on complex protein cooperation, is a kind of compact and efficient and broad-spectrum recombination system of gene cluster. In addition, the recombination system has good portability, good host compatibility, can realize efficient gene editing in host bacteria, has the modularization characteristics of 'plug and play', is convenient to operate, does not need to be passed through multiple generations, and does not need to be induced in advance.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of gene editing, in particular to a gene editing system, a double-plasmid system, an engineered bacterium and application. BACKGROUND

[0002] The genome editing technology mainly relies on the targeted cleavage effect of the CRISPR-Cas system to induce the recombination system for directional repair, and realizes the mutation, deletion or insertion of the target site according to the specific provided complementary homologous arm. The existing gene editing recombination system mainly comes from the lambda-red (i.e. λ-red) system of the lambda prophage of Escherichia coli and the RecET system of the prophage Rac. Among them, the λ-red system encodes three proteins, which are Gam protein, redα protein and redβ protein. The Gam protein has the effect of inhibiting the degradation of exogenous nucleic acid by the host RecBCD protein. The redα protein is a 5'-3' exonuclease that degrades one strand of double-stranded DNA (dsDNA) from the end of the dsDNA to generate a 3' single strand, providing a single-stranded DNA template for subsequent homologous recombination. In the gene knockout or knock-in experiment, the linear DNA donor is processed to expose the single-stranded region for pairing with the genomic target site. The redβ protein is a single-stranded annealing protein that binds to the single-stranded DNA (ssDNA) generated by the redα protein to protect it from nuclease degradation and promote the pairing of the single-stranded DNA with the homologous region of the genome to mediate homologous recombination. The RecET system only encodes RecE protein and RecT protein, wherein the RecE protein has a function similar to that of the redα protein, and the RecT protein has a function similar to that of the redβ protein.

[0003] However, both the λ-red system and the RecET system have a large gene cluster and rely on complex protein cooperation, have potential off-target effects, and have host compatibility problems. Specifically, the λ-red system module is 1885 bp long, and the RecET system module is 3403 bp long, which is generally used in combination with the CRISPR-Cas system. The large recombination module can cause difficulty in transmission. The λ-red system relies on the complex cooperation of three effector proteins, and the proteins of the RecET system are larger, and the recombination efficiency is limited by the host environment or protein expression level. Homologous recombination relies on sequence matching, which can cause non-targeted integration. Existing studies have shown that the λ-red system alone can achieve bacterial genome editing, indicating that the system can cause non-specific recombination in repeated sequence regions in the genome. The activity of the current λ-red system and the RecET system in non-model bacteria needs to be optimized. In the process of recombination editing of the λ-red system and the RecET system, as the homologous arm shortens, the recombination efficiency decreases.

[0004] Therefore, the prior art still needs to be improved and developed. SUMMARY

[0005] Based on the deficiencies of the prior art, the purpose of the present application is to provide a gene editing system, a double-plasmid system, an engineering bacterium and an application, aiming to solve the problem that the gene cluster of the existing lambda-red system and RecET system is large and needs to rely on the synergistic action of complex proteins for gene editing.

[0006] The technical scheme of the present application is as follows:

[0007] In a first aspect of the present application, a gene editing system is provided, wherein the gene editing system comprises a gRNA and a recombination system, and the recombination system comprises a nucleotide sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.

[0008] Optionally, the recombination system further comprises a nucleotide sequence encoding a single-strand annealing protein.

[0009] Optionally, the recombination system comprises a nucleotide sequence as shown in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.

[0010] Optionally, the recombination system further comprises a nucleotide sequence encoding an exonuclease.

[0011] In a second aspect of the present application, a double-plasmid system is provided, wherein the double-plasmid system comprises a first plasmid and a second plasmid, the first plasmid comprises the gene editing system as described above in the present application; and the second plasmid comprises a nucleotide sequence encoding a Cas protein.

[0012] Optionally, the nucleotide sequence of the second plasmid is as shown in SEQ ID NO: 7.

[0013] Optionally, the first plasmid further comprises a nucleotide sequence encoding an arabinose operon and a promoter.

[0014] In a third aspect of the present application, an engineering bacterium is provided, wherein the engineering bacterium comprises the double-plasmid system as described above in the present application.

[0015] In a fourth aspect of the present application, the application of the gene editing system as described above in the present application, the double-plasmid system as described above in the present application or the engineering bacterium as described above in the present application in gene editing is provided.

[0016] Optionally, the gene editing is phage gene editing.

[0017] Beneficial effects: The gene editing system provided by the application has high gene editing efficiency, and can realize efficient gene editing under the condition of short homologous arms. When there is no cutting mediated by the CRISPR-Cas system, the gene editing system will not carry out gene editing, so the controllability of the gene editing system is good. The gene length of the recombination system is much smaller than that of the lambda-red system and the RecET system, and it does not depend on complex protein cooperation, so it is a more compact and efficient broad-spectrum recombination system of gene clusters. In addition, the recombination system has good portability, good host compatibility, can efficiently respond to DNA breaks, can efficiently edit genes in host bacteria, has the modular characteristics of "plug and play", is easy to operate, does not need to be passed through multiple generations, does not need to be induced in advance, and has high recombination efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Fig. a is a schematic diagram of the gene clusters of the lambda-red system, the RecET system and the Rec669 system, Fig. b is a phylogenetic analysis result diagram of the predicted SSAP protein of the Rec669 system and the single-strand annealing protein of the lambda-red system, Fig. c is a structure comparison result diagram of the predicted exonuclease of the Rec669 system and the exonuclease of the lambda-red system, Fig. d is a structure comparison result diagram of the predicted exonuclease of the Rec669 system and the exonuclease of the RecET system, Fig. e is a structure comparison result diagram of the predicted SSAP protein of the Rec669 system and the single-strand annealing protein of the lambda-red system, and Fig. f is a structure comparison result diagram of the predicted SSAP protein of the Rec669 system and the single-strand annealing protein of the RecET system.

[0019] Figure 2 Fig. is a phylogenetic analysis and distribution diagram of the Rec669 system in different bacteriophages.

[0020] Figure 3 Fig. is a result diagram of SSB protein containing DUF669 domain being divided into branches of infecting gram-negative and positive bacteria.

[0021] Figure 4 Fig. is a VConTACT network analysis result diagram of bacteriophages carrying the Rec669 system.

[0022] Figure 5 Fig. is a related information diagram of bacteriophage T7 gene editing in Example 3, wherein Fig. a is a schematic diagram of the bacteriophage T7 genome knockout site, and Fig. b is a schematic diagram of the gene clusters of different recombination systems.

[0023] Figure 6 Fig. is a schematic diagram of a kind of double-plasmid system in Example 3.

[0024] Figure 7 A schematic diagram of phage T7 gene editing for E. coli MG1655 containing a double plasmid system in Example 3.

[0025] Figure 8 A graph of the recombination editing efficiency of phage T7 genes by different systems in Example 3.

[0026] Figure 9 A graph of the band results after gene editing of phage T7 by different systems in Example 3.

[0027] Figure 10 A graph of the recombination editing efficiency of phage T7 genes by different systems in Example 4. DETAILED DESCRIPTION

[0028] The present application provides a gene editing system, a double plasmid system, an engineered bacterium and an application. In order to make the purpose, technical scheme and effect of the present application more clear and explicit, the present application is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present application and do not limit the present application.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0030] If the description of "first", "second" and the like is involved in the embodiments of the present application, the description of "first", "second" and the like is only for the purpose of description, and cannot be understood as indicating or implying the relative importance of the indicated technical features or implicitly indicating the number of the indicated technical features.

[0031] The embodiments of the present application provide a gene editing system, wherein the gene editing system comprises a gRNA (gRNA is a guide RNA for targeting a specific site that needs to be edited, which can be designed according to the target editing site to obtain) and a recombination system (named Rec669 system), and the Rec669 system comprises a nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.

[0032] In the present embodiment, the nucleotide sequence shown in SEQ ID NO: 1 encodes a single-stranded binding protein (SSB protein containing 280 amino acids, which contains a unique DUF669 domain), and the nucleotide sequence of the SSB protein is from Pseudomonas phage Φ47 (nomenclature classification as Pseudomonas phage Φ47, which is a bacteriophage of Pseudomonas aeruginosa). Pseudomonas phage, and was preserved in China General Microbiological Culture Collection Center, Beijing Chaoyang District, Beichen West Road No. 1, on June 27, 2025, with a preservation number of CGMCC No. 46497. The nucleotide sequence shown in SEQ ID NO: 2 encodes a truncated SSB protein (denoted as SSBs protein, containing 180 amino acids). The nucleotide sequence shown in SEQ ID NO: 3 encodes a truncated SSB protein (denoted as SSBss protein, containing 145 amino acids).

[0033] The inventors found through research that SSB protein can bind to R-loop ring generated by CRISPR-Cas, and then respond to DNA break caused by CRISPR-Cas (or Cas protein). The key domain of SSB protein binding to R-loop ring is in the N-terminal, and the C-terminal of SSB protein has bacterial toxicity. Therefore, the C-terminal of SSB protein is deleted to obtain truncated proteins, i.e. SSBs protein and SSBss protein.

[0034] In the gene editing system of the present embodiment, the gene length of the Rec669 system is 843 bp, 543 bp or 438 bp, which is much smaller than that of the lambda-red system (length of 1885 bp) and the RecET system (length of 3403 bp), and the Rec669 system only encodes one protein and does not depend on complex protein cooperation. It is a more compact and efficient and broad-spectrum recombination system. The Rec669 system can bind to the R-loop ring generated by CRISPR-Cas, and then quickly respond to the DNA break caused by CRISPR-Cas (or Cas). The Rec669 system can work together with gRNA and achieve efficient gene editing under the condition of short homologous arms.

[0035] In some embodiments, the Rec669 system further comprises a nucleotide sequence encoding a single-strand annealing protein (SSAP protein).

[0036] In some embodiments, the nucleotide sequence encoding the SSAP protein is from Pseudomonas phage Φ47 (see above for specific information). Therefore, the Rec669 system comprises the nucleotide sequence shown in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.

[0037] In this embodiment, the nucleotide sequence shown in SEQ ID NO: 4 encodes both the SSAP protein and the SSB protein. The nucleotide sequence shown in SEQ ID NO: 5 encodes both the SSAP protein and the SSBs protein. The nucleotide sequence shown in SEQ ID NO: 6 encodes both the SSAP protein and the SSBss protein. Specifically, the SSB protein (or the SSBs protein, or the SSBss protein) can bind the single-stranded DNA formed by the break, and then combine with the SSAP protein to perform homologous recombination repair (homologous recombination is a strand exchange between homologous sequences).

[0038] In this embodiment, the lengths of the Rec669 system are 1574 bp, 1274 bp and 1169 bp, respectively, which are much smaller than the length of the lambda-red system (1885 bp) and the length of the RecET system (3403 bp), and the Rec669 system only encodes two proteins and does not rely on complex protein cooperation, so it is a compact and efficient recombination system. The SSB protein (or the SSBs protein, or the SSBss protein) in the Rec669 system can bind the R-loop ring generated by CRISPR-Cas, and then quickly respond to the DNA break caused by CRISPR-Cas (or Cas), and the SSB protein (or the SSBs protein, or the SSBss protein) can bind the single-stranded DNA formed by the break, and then combine with the SSAP protein to perform homologous recombination repair. The Rec669 system can work with gRNA and achieve efficient gene editing under the condition of short homologous arms. Compared with the above-mentioned Rec669 system which only encodes the SSB protein (or the SSBs protein, or the SSBss protein), the Rec669 system which encodes two proteins in this embodiment has higher gene editing efficiency (up to 100%).

[0039] The gene editing system provided by the present application above does not perform gene editing when there is no cutting mediated by the CRISPR-Cas system, which indicates that the controllability of the system is good. In addition, the Rec669 system has good portability and good host compatibility, and can perform efficient gene editing in the host bacteria, which embodies the modular characteristics of the Rec669 system "plug and play". The Rec669 system efficiently responds to DNA break, which embodies the controllability of the Rec669 system. The Rec669 system is easy to operate, does not need to be passed multiple times, does not need to be induced in advance, and has high recombination efficiency.

[0040] In some embodiments, the Rec669 system further comprises a nucleotide sequence encoding an exonuclease.

[0041] For example, taking the Rec669 system which encodes both the SSAP protein and the SSB protein (containing the nucleotide sequence shown in SEQ ID NO: 4) as an example, as shown in Figure 1As shown, the Rec669 system differs from the mainstream lambda-red system and RecET system in components, amino acid sequences, structures, etc.

[0042] Specifically, the gene clusters of the lambda-red system, the RecET system and the Rec669 system are as shown in a of Figure 1 , wherein genes marked with the same color have the same function. The lambda-red system contains exonuclease gene red alpha , single-strand annealing protein gene red β and host nuclease inhibitor gene red y (that is, Gam ). The RecET system contains exonuclease gene recE and single-strand annealing protein gene recT . The Rec669 system contains single-strand annealing protein gene ssap , single-strand binding protein gene ssb and exonuclease gene exo . Among them, the exonuclease gene red alpha of the lambda-red system, the exonuclease gene recE of the RecET system and the exonuclease gene exo of the Rec669 system have the same function. The single-strand annealing protein gene red beta of the lambda-red system, the single-strand annealing protein gene recT of the RecET system and the single-strand annealing protein gene ssap of the Rec669 system have the same function. While the single-strand binding protein gene ssb of the Rec669 system has a different function from the genes in the lambda-red system and the RecET system. The phylogenetic analysis of the SSAP protein of the Rec669 system and the single-strand annealing protein (Pfam: PF03837.19) of the lambda-red system is shown in b of Figure 1 , indicating that the SSAP protein in the Rec669 system is evolutionarily distant from the single-strand annealing protein of the lambda-red system and has no amino acid similarity.

[0043] The alignment result of the predicted exonuclease of the Rec669 system and the structure of the exonuclease of the lambda-red system (PDB: 1AVQ) is shown in c of Figure 1 , and the root mean square deviation = 11.995 (covering 112 atoms). The alignment result of the predicted exonuclease of the Rec669 system and the structure of the exonuclease of the RecET system (PDB: 3H4R) is shown in d of Figure 1 , and the root mean square deviation = 15.969 (covering 105 atoms). The alignment result of the predicted SSAP protein of the Rec669 system and the predicted structure of the single-strand annealing protein of the lambda-red system is shown inFigure 1 As shown in 'e', ​​the root mean square deviation is 5.960 (covering 34 atoms). The alignment results of the predicted SSAP protein structure of the Rec669 system with the single-chain annealed protein structure of the RecET system (PDB: 7UB2) are as follows: Figure 1 As shown in f, the root mean square deviation is 12.237 (covering 64 atoms). This indicates that the exonuclease and single-chain annealing protein (i.e., SSAP protein) of the Rec669 system have no structural similarity to the exonuclease and single-chain annealing protein of the λ-red system and the RecET system.

[0044] Taking the Rec669 system encoding the SSB protein (containing the nucleotide sequence shown in SEQ ID NO: 1) as an example, such as Figure 2 , Figure 3 and Figure 4 As shown, phages carrying the SSB protein gene of the Rec669 system generally also carry the SSAP protein gene. The Rec669 system is widespread and conserved, and is present in a variety of phages that infect Gram-negative and Gram-positive bacteria. This suggests that homologous proteins encoded by the Rec669 system may also have recombination effects, and therefore, homologs of this system can also be used for gene editing.

[0045] Specifically, the phylogenetic analysis and distribution of the Rec669 system in different bacteriophages are as follows: Figure 2 As shown, this is a phylogenetic tree displaying the SSB proteins (containing the DUF669 domain) from 152 bacteriophages from the NCBI / refseq / viral database. Outgroups include those from thermophilic bacteria ( Thermus thermophilus The SSB proteins of *E. coli* phage T7 (GenBank ID: AAF27296.1), *E. coli* phage T4, and *E. coli* MG1655 were analyzed, indicating that the SSB proteins of the Rec669 system are evolutionarily distant from known bacterial and phage-derived SSB proteins. SSB proteins containing the DUF669 domain were divided into branches infecting Gram-negative and Gram-positive bacteria. Neighboring genes of the SSB proteins were annotated using the Pfam database and color-coded. The SSB and SSAP proteins of the Rec669 system were labeled, and the results are shown below. Figure 3 As shown, the results indicate that the Rec669 system, containing both SSAP and SSB proteins, is widely distributed in phages of Gram-negative and Gram-positive bacteria, and that the SSAP and SSB proteins are conserved. The results of VConTACT (viral contig classification and annotation tool) network analysis of phages carrying the Rec669 system are shown below. Figure 4 As shown, VConTACT network analysis revealed that these bacteriophages belong to six major genera, including Seuratvirus (Cerrovirus genus), Nipunavirus (Nipahvirus) , Moineauvirus (Mojo virus) , Septimatrevirus (Sepultiviruses), Rosemountvirus (Rosellavirus) and Psavirus (Pisuvirus).

[0046] The embodiment of the present application also provides a double-plasmid system, wherein the double-plasmid system comprises a first plasmid and a second plasmid, the first plasmid comprises the gene editing system as described in the embodiment of the present application; and the second plasmid comprises a nucleotide sequence encoding a Cas protein (such as a Cas9 protein, a Cas12 protein, etc.).

[0047] In some embodiments, the nucleotide sequence of the second plasmid is as shown in SEQ ID NO: 7.

[0048] In some embodiments, the first plasmid further comprises a nucleotide sequence encoding an arabinose operon and a promoter. Arabinose is used to induce the protein expression of the Rec669 system.

[0049] The embodiment of the present application also provides an engineered bacterium, wherein the engineered bacterium comprises the double-plasmid system as described in the embodiment of the present application. As an example, the engineered bacterium can be an engineered Escherichia coli, specifically, the double-plasmid system is introduced into the Escherichia coli to obtain the engineered Escherichia coli.

[0050] The embodiment of the present application also provides an application of the gene editing system as described in the embodiment of the present application, the double-plasmid system as described in the embodiment of the present application, or the engineered bacterium as described in the embodiment of the present application in gene editing.

[0051] In the process of gene editing, a homologous arm upstream and downstream of a target editing site needs to be used, and the sequence thereof can be inserted into the first plasmid.

[0052] In some embodiments, the gene editing is phage gene editing.

[0053] The present application is further described below through specific examples.

[0054] The raw materials used in the following examples are commercially available products, unless otherwise specified.

[0055] Example 1

[0056] The bacteriophage Φ47 of Pseudomonas is isolated from municipal sewage, specifically comprising the following steps:

[0057] After filtering bacteria from 5 mL of municipal sewage sample through a 0.22 micron filter membrane, the sample is mixed with 10 mL of fresh 3xLB liquid medium, and overnight cultured Pseudomonas aeruginosa (ATCC 10145) is added. Pseudomonas aeruginosa) PA14 bacterial liquid (inoculation amount is 1:20), after 5 hours of shaking incubation at 37°C, centrifugation at 8000 rpm for 10 min, taking the supernatant to filter bacteria through a 0.22 micron filter membrane, and taking 5 mL of the filtrate to repeat the above steps twice to obtain the enrichment liquid of Pseudomonas bacteriophage Φ47. After 3 times of single spot purification of Pseudomonas bacteriophage Φ47, a single spot is taken and added to fresh LB medium, and fresh Pseudomonas aeruginosa (PA14) is added Pseudomonas aeruginosa ) PA14 bacterial liquid (inoculation amount is 1:50), 37°C shaking culture for 5 hours, and the supernatant after centrifugation is filtered to remove bacteria to obtain the purified Pseudomonas bacteriophage Φ47, which is taxonomically named Pseudomonas phage , and is preserved in the China General Microbiological Culture Collection Center, located at No. 1, Beichen West Road, Chaoyang District, Beijing, with a preservation date of June 27, 2025, and a preservation number of CGMCC No. 46497).

[0058] Example 2 Obtaining of Rec669 system

[0059] The gene sequence encoding SSB protein (as shown in SEQ ID NO: 1) and the gene sequence encoding SSAP protein and SSB protein (as shown in SEQ ID NO: 4) are amplified from the genome of Pseudomonas bacteriophage Φ47 (see the above for specific information), and the amplification procedure includes the following steps:

[0060] Initial denaturation: denaturation at 95°C for 3 minutes;

[0061] Cycle amplification: denaturation at 95°C for 15 seconds, annealing at 60°C for 15 seconds, extension at 72°C for 45 seconds, and repeating for 32 cycles;

[0062] Final extension: extension at 72°C for 5 minutes; and finally stored at 4°C.

[0063] The amplified product is recovered by ethanol precipitation method.

[0064] In addition, amino acid deletion is performed at the C-terminus of SSB protein to obtain the gene sequence encoding SSBs protein (as shown in SEQ ID NO: 2), and further amino acid deletion is performed at the C-terminus of SSAP protein to obtain the gene sequence encoding SSBss protein (as shown in SEQ ID NO: 3). At the same time, the gene sequence encoding SSAP protein and SSAP protein (as shown in SEQ ID NO: 5) is obtained, and the gene sequence encoding SSAP protein and SSBss protein (as shown in SEQ ID NO: 6) is obtained.

[0065] Therefore, 6 kinds of Rec669 systems are obtained, which are:

[0066] The Rec669 system (SSB) contains a gene sequence encoding the SSB protein (as shown in SEQ ID NO: 1).

[0067] The Rec669 system (SSBs) contains gene sequences encoding SSBs proteins (as shown in SEQ ID NO: 2).

[0068] The Rec669 system (SSBss) contains a gene sequence encoding the SSBss protein (as shown in SEQ ID NO: 3).

[0069] The Rec669 system (SSAP-SSB) contains gene sequences that encode both SSAP and SSB proteins (as shown in SEQ ID NO: 4).

[0070] The Rec669 system (SSAP-SSBs) contains gene sequences that encode both SSAP and SSB proteins (as shown in SEQ ID NO: 5).

[0071] The Rec669 system (SSAP-SSBss) contains gene sequences that encode both SSAP and SSBss proteins (SEQ ID NO: 6).

[0072] Example 3

[0073] Using the Rec669 system (SSB), Rec669 system (SSAP-SSB), and Rec669 system (SSAP-SSBs) from Example 2, targeting phage T7 gp17 (The gene encoding tail fibrin) and gp17.5 The non-coding region (58 bp) between genes encoding cleavage proteins was deleted (see the diagram of the phage T7 genome knockout site and the gene cluster diagram of each system as shown in the figure). Figure 5 (As shown), including the following steps:

[0074] A dual-plasmid system is provided, comprising a first plasmid and a second plasmid; the second plasmid encodes the Cas9 protein, and the nucleotide sequence of the second plasmid is shown in SEQ ID NO: 7; the method for constructing the first plasmid includes the following steps:

[0075] The nucleotide sequences of the arabinose operon and promoter, the gene sequence of the Rec669 system, and the pN20 plasmid backbone fragment were used for plasmid assembly. Simultaneously, based on the T7 phage... gp17 and gp17.5The gRNA (the nucleotide sequence of which is shown as SEQ ID NO: 8) was designed by using the non-coding region (58 bp) between the target editing site and the adjacent site, inserted into the gRNA region of the pN20 plasmid, and the sequences of 50 bp at both ends of the target editing site on the bacteriophage T7 genome (i.e. the upstream and downstream homologous arms) were added by using primer end (the nucleotide sequence of the upstream homologous arm is shown as SEQ ID NO: 9, and the nucleotide sequence of the downstream homologous arm is shown as SEQ ID NO: 10), to obtain a first plasmid;

[0076] The first plasmid was transformed into transT1 competent cells (i.e. a commonly used genetically engineered E. coli strain in molecular cloning) after 15 minutes at 50°C using the full-size gold basic assembly kit, and the successful construction of the plasmid was determined by sequencing; wherein the nucleotide sequence of the first plasmid containing the Rec669 system (SSAP-SSB) is shown as SEQ ID NO: 11.

[0077] The overnight culture of E. coli MG1655 was inoculated into 10 mL of fresh LB medium at a volume ratio of 1:50, and cultured at 37°C and 200 rpm for 4 hours. Then, the bacterial cells were collected by centrifugation at 4000 rpm and 4°C for 5 minutes, washed with sterile water, and then centrifuged again at 4000 rpm and 4°C for 5 minutes. After repeating the washing with sterile water once, the bacterial cells were resuspended with 0.2 mL of sterile water to obtain the E. coli MG1655 competent cells.

[0078] 500 ng of the first plasmid was added to 100 μL of the E. coli MG1655 competent cells, and then subjected to electroporation by adding a 2 mm electroporation cup and setting a voltage of 2.5 kV for 2 minutes of ice bath. Then, 0.9 mL of fresh LB medium was immediately added, and the mixture was incubated at 37°C for 1 hour. The bacterial cells were collected and plated on a solid LB medium containing 50 μg / mL of kanamycin to obtain the E. coli MG1655 carrying the first plasmid.

[0079] Then, the E. coli MG1655 carrying the first plasmid was used as the bacterial liquid for the preparation of competent cells (see the preparation of the E. coli MG1655 competent cells described above), and then the second plasmid was electroporated and plated on a solid LB medium containing 25 μg / mL of chloramphenicol and 50 μg / mL of kanamycin, and then incubated at 37°C overnight to obtain a monoclonal strain containing a double-plasmid system.

[0080] The monoclonal strain containing the double-plasmid system was inoculated into fresh LB medium and cultured aerobically at 37°C for 4 hours (OD 600The absorbance of the bacterial culture at 600 nm was approximately 0.6 (≈ 0.6). Phage T7 was diluted to a suitable titer (approximately 100 PFU / mL, ensuring at least 30 plaques per plate). One mL of monoclonal bacterial culture was mixed with the appropriate titer of phage T7 and a final concentration of 0.02% (w / v, mass / volume) of L-arabinose inducer, followed by the addition of 0.75% (w / v) LB semi-solid agar medium for plaque separation. After incubation at 37°C for 4 hours, 12 individual plaques were selected for site-specific PCR amplification and Sanger sequencing. Editing efficiency was quantified and recombinant editing efficiency was statistically analyzed by observing the PCR products on a 1% (w / v) agarose gel (prepared using TAE buffer, a buffer composed of tris(hydroxymethyl)aminomethane, acetic acid, and ethylenediaminetetraacetic acid).

[0081] When the Rec669 system contains gene sequences that simultaneously encode SSAP and SSB proteins, the dual plasmid system is as follows: Figure 6 As shown, this is a schematic diagram of E. coli MG155 containing a dual plasmid system used for phage T7 gene editing. Figure 7 As shown.

[0082] Simultaneously, gene editing of the aforementioned phage T7 was performed using green fluorescent protein (GFP protein, i.e., negative control), a dual plasmid system without Cas9 and gRNA, a dual plasmid system without upstream and downstream homologous arms, and other systems (λ-red system and RecET system). The recombination editing efficiency was calculated, and the recombination editing efficiency of the Rec669 system inhibited by 0.2% D-glucose (i.e., replacing the L-arabinose inducer with a final concentration of 0.02% (w / v) in the above text with a final concentration of 0.2% (w / v) D-glucose) was determined.

[0083] The results are as follows Figure 8 and Figure 9 As shown, Figure 8 (where n represents the number of individual phage plaques selected) In each group, each point represents the average recombination editing efficiency of 12 individual phage plaques (two-sample t-test, p<0.05). Figure 9Each small graph represents the recombination ratio in each group of experiments (12 single phage plaques as a group), and the short bar represents successful editing. It can be seen that the Rec669 system does not perform gene editing without Cas9-mediated cleavage, indicating that the Rec669 system has good controllability. However, after introducing Cas9, the Rec669 system (SSB), the Rec669 system (SSAP-SSB) and the Rec669 system (SSAP-SSBs) can all respond to Cas9 cleavage to perform efficient editing of the phage T7 genome, and the Rec669 system (SSAP-SSBs) has higher recombination editing efficiency than the Rec669 system (SSB) and the Rec669 system (SSAP-SSB). Specifically, the highest recombination editing efficiency of the Rec669 system (SSAP-SSB) mediated gene editing can reach 100%, and the average recombination editing efficiency can reach 85%; while the highest recombination editing efficiency of the Rec669 system (SSAP-SSBs) mediated gene editing can reach 100%, and the average recombination editing efficiency can reach 95%, which is significantly higher than the existing recombination system. In addition, the Rec669 system (SSB) can also achieve an average recombination editing efficiency of 60%, indicating that the recombination editing efficiency of the gene editing system provided by the present application is excellent, has good response ability to Cas cleavage, and has application value for promoting synthetic biology research.

[0084] Example 4

[0085] Using the Rec669 system (SSB), the Rec669 system (SSAP-SSB), the Rec669 system (SSBs), the Rec669 system (SSAP-SSBs), the Rec669 system (SSBss) and the Rec669 system (SSAP-SSBss) in Example 2 and the SSAP protein, the non-coding region (58bp) between and is deleted, and the specific steps are as described in Example 3, and the results are summarized in gp17 and gp17.5 It can be seen that the six systems and the SSAP protein all have recombination editing effect, and the Rec669 system (SSAP-SSBs) has the best recombination editing effect. Figure 10

[0086] It should be understood that the application of the present application is not limited to the above examples, and those skilled in the art can make improvements or changes according to the above description, and all these improvements and changes shall belong to the protection scope of the appended claims of the present application.​

Claims

1. A gene editing system, characterized in that, The gene editing system includes gRNA and a recombination system, wherein the recombination system includes the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO:

3.

2. The gene editing system according to claim 1, characterized in that, The recombinant system also includes a nucleotide sequence encoding a single-stranded annealing protein.

3. The gene editing system according to claim 2, characterized in that, The recombinant system includes the nucleotide sequence shown in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO:

6.

4. The gene editing system according to any one of claims 1-3, characterized in that, The recombinant system also includes a nucleotide sequence encoding an exonuclease.

5. A dual-plasmid system, characterized in that, The dual plasmid system includes a first plasmid and a second plasmid, wherein the first plasmid includes the gene editing system according to any one of claims 1-4; and the second plasmid includes a nucleotide sequence encoding a Cas protein.

6. The dual plasmid system according to claim 5, characterized in that, The nucleotide sequence of the second plasmid is shown in SEQ ID NO:

7.

7. The dual plasmid system according to claim 5, characterized in that, The first plasmid also includes nucleotide sequences encoding arabinose operons and promoters.

8. An engineered bacterium, characterized in that, The engineered bacteria include the dual plasmid system as described in any one of claims 5-7.

9. The use of the gene editing system according to any one of claims 1-4, the dual plasmid system according to any one of claims 5-7, or the engineered bacteria according to claim 8 in gene editing for purposes other than disease diagnosis and treatment.

10. The application according to claim 9, characterized in that, The gene editing referred to is bacteriophage gene editing.

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