Miniature DNA nuclease as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of bacterial plasmid editing, and particularly relates to miniature DNA nuclease as well as a preparation method and application thereof. The preparation method of the micro DNA nucleotidase comprises the following steps: carrying out expression on a p15A-IscB-2. 0 plasmid, so as to obtain the micro DNA nucleotidase. Wherein the nucleotide sequence of the p15A-IscB-2 plasmid is shown as SEQ ID NO.2, and the p15A-IscB-2 plasmid is obtained by performing synonymous mutation on a 5 '-AACNNNNNNGYGC-3' sequence in an IscB gene in p15A-IscB. The p15A-IscB-2 plasmid disclosed by the invention is non-toxic to escherichia coli, can express an IscB protein, and is high in conversion efficiency, so that the p15A-IscB-2 plasmid can be directly used for gene editing of escherichia coli.

Description

Technical Field

[0001] This invention belongs to the field of bacterial plasmid editing technology, specifically relating to a miniature DNA nuclease, its preparation method, and its application. Background Technology

[0002] Escherichia coli (E. coli) is widely found in the intestines of humans and many animals, and is the most abundant bacterial species. Among them, the E. coli MG1655 strain has become a focus of current industrial host research due to its extensive metabolic network, rapid growth rate, and strong resilience. Research on E. coli often requires highly efficient gene-editing tools.

[0003] Currently, RNA-guided DNA nucleases are used to cut bacterial genomic DNA, creating double-strand breaks (DSBs). Bacteria then complete gene editing by repairing these DSBs. Double-strand breaks are abbreviated as DSBs. Generally, bacteria initiate two main endogenous repair mechanisms for DSBs: First, non-homologous end joining (NHEJ) directly joins the broken ends, usually leading to insertion or deletion mutations, thus achieving gene knockout. Second, introducing an exogenous donor DNA template carrying homologous arms allows for precise gene insertion, substitution, or base correction through homology-directed repair (HDR). For *E. coli*, only the HDR repair pathway exists.

[0004] Recent studies have revealed that the IscB protein, an RNA-guided DNA nuclease encoded by the IS200 / IS605 superfamily transposons, is small, consisting of only 496 amino acids—one-third the size of the more widely reported DNA nuclease Cas9—and is speculated to be an ancestor of Cas9. Similar to Cas9, IscB contains HNH and RuvC domains, enabling it to bind to OMEGA RNA to form a ribonucleoprotein complex. The IscB system recognizes a target-adjacent motif, TAM, with a 5'-CAGGAA-3' sequence and a target sequence length of 16 bp, enabling cleavage of the target DNA. This gives IscB the potential to be developed into a novel gene-editing tool. However, the application of IscB in *E. coli* MG1655 is severely limited, as expression plasmids containing IscB cannot be successfully introduced into MG1655, thus hindering gene editing in MG1655.

[0005] Therefore, existing IscB plasmids cannot be directly used for gene editing of Escherichia coli MG1655, and it is necessary to develop IscB plasmids that can be directly used for gene editing of Escherichia coli. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a miniature DNA nuclease, its preparation method, and its applications. The miniature DNA nuclease is the IscB-2.0 protein described in the specification.

[0007] The purpose of this invention is to provide a method for preparing a miniature DNA nuclease, comprising the following steps: expressing the p15A-IscB-2.0 plasmid to obtain a miniature DNA nuclease.

[0008] The nucleotide sequence of the p15A-IscB-2.0 plasmid is shown in SEQ ID NO. 2. It is obtained by synonymous mutation of the 5'-AACNNNNNNGYGC-3' sequence in the IscB gene of p15A-IscB.

[0009] The nucleotide sequence of the IscB-2.0 gene obtained after the IscB gene mutation is shown in SEQ ID NO. 1.

[0010] The nucleotide sequence of SEQ ID NO. 1 is as follows:

[0011] Preferably, the expression refers to the transformation of the p15A-IscB-2.0 plasmid into engineered bacteria for expression.

[0012] Preferably, the engineered bacteria is Escherichia coli.

[0013] Preferably, the p15A-IscB-2.0 plasmid includes the IscB-2.0 gene expressing the IscB protein, the λ-red recombination system, the araC operon, the p15A replicon, and the chloramphenicol resistance gene CmR.

[0014] Preferably, the IscB gene fragment contains two 5'-AACNNNNNNGYGC-3' segments.

[0015] Preferably, the construction of the p15A-IscB-2.0 plasmid includes the following steps: Using p15A-IscB as a template, loop PCR reaction one was performed using two pairs of primers, is-mut-1-up and is-mut-1-dn, to obtain PCR product one. The PCR product one was sequenced using the verf-iscb-ce-1 primer, and p15A-IscB-1 with a single base mutation that showed the target mutation was recovered.

[0016] Using p15A-IscB-1 as a template, loop PCR reaction II was performed using two pairs of primers, is-mut-2-up and is-mut-2-dn, to obtain PCR product II. PCR product II was sequenced using the verf-iscb-ce-2 primer. After the target mutation was found, the template was digested with DpnI digestive enzyme and purified to obtain p15A-IscB-2.0 plasmid.

[0017] The is-mut-1-up primer has a 5'-aggcacgtccttatttctgctttcgatctggg-3' nucleotide sequence. The is-mut-1-dn primer has a 5'-cgaaaccagaaataaggacgtgcctaagct-3' nucleotide sequence. The verf-iscb-ce-1 primer has a 5'-aaaagaattttgtgttataatc-3' nucleotide sequence. The is-mut-2-up primer has a 5'-ccgctgcaccttgggattgttcatggccatgaag-3' nucleotide sequence. The is-mut-2-dn primer has a 5'-tcatggccatgaacaatcccaaggtgcagcggtg-3' nucleotide sequence. The verf-iscb-ce-2 primer has a 5'-taaaggcagcgtggaagag-3' nucleotide sequence.

[0018] Miniature DNA nuclease prepared by any of the preparation methods described herein.

[0019] A second objective of this invention is to provide an application of a miniature DNA nuclease in bacterial gene editing.

[0020] Preferably, the bacteria are Escherichia coli MG1655 and Escherichia coli ATCC9637.

[0021] Preferably, the gene editing is gene knockout.

[0022] Compared with the prior art, the present invention has the following beneficial effects: 1. The method for preparing the miniature DNA nucleotidase of the present invention includes the following steps: expressing the p15A-IscB-2.0 plasmid to obtain the miniature DNA nucleotidase. The nucleotide sequence of the p15A-IscB-2.0 plasmid is shown in SEQ ID NO. 2, which is obtained by synonymous mutation of the 5'-AACNNNNNNGYGC-3' sequence in the IscB gene of p15A-IscB. The nucleotide sequence of the IscB-2.0 gene obtained by synonymous mutation of the 5'-AACNNNNNNGYGC-3' sequence in the IscB gene of the p15A-IscB plasmid is shown in SEQ ID NO. 1. The p15A-IscB-2.0 plasmid of the present invention can be directly used for gene editing of *Escherichia coli* MG1655, and has high transformation efficiency. This invention utilizes the miniature DNA nuclease IscB as a programmable endonuclease, successfully constructing a mutant IscB-2.0 through mutation, and for the first time successfully establishing a genome editing tool based on IscB-2.0 in Escherichia coli MG1655 and Escherichia coli ATCC9637, achieving successful editing.

[0023] 2. The preparation method of the p15A-IscB-2.0 plasmid of the present invention includes the following steps: Using p15A-IscB as a template, a circular PCR reaction one is performed using two pairs of primers, is-mut-1-up and is-mut-1-dn, to obtain PCR product one. The PCR product one is sequenced using the verf-iscb-ce-1 primer. If the target mutation is found, the PCR product is purified and recovered, and then the template is digested with DpnI digestive enzyme to obtain p15A-IscB-2.0 with a single-base mutation. Using p15A-IscB-2.0 with a single-base mutation as a template, a circular PCR reaction two is performed using two pairs of primers, is-mut-2-up and is-mut-2-dn, to obtain PCR product two. The PCR product two is sequenced using the verf-iscb-ce-2 primer. If the target mutation is found, the template is digested with DpnI digestive enzyme, and the p15A-IscB-2.0 plasmid is purified.

[0024] Based on the research results of this invention, the original IscB gene expression plasmid was transformed into *E. coli* MG1655, plated on LB agar plates, and the number of surviving colonies on the plates was counted to calculate the transformation efficiency. It was found that there were no colonies on the plates, indicating extremely low transformation efficiency. Furthermore, this invention discovered that the IscB gene sequence contains two "5'-AACNNNNNNGYGC-3'" sequences, which are restriction enzyme sites recognized by enzymes expressed by the type I restriction modification system of *E. coli* MG1655. When the plasmid expressing the IscB gene is introduced into the MG1655 strain, the plasmid is cleaved by the restriction enzymes within the strain, resulting in low transformation efficiency and the inability to obtain transformants, ultimately hindering gene editing based on the IscB protein. Based on the above findings, this invention performs synonymous mutations on the two "5'-AACNNNNNNGYGC-3'" sequences in the IscB gene sequence to obtain the IscB-2.0 gene, avoiding plasmid cleavage by restriction enzymes within the strain and achieving high transformation efficiency.

[0025] 3. The p15A-IscB-2.0 plasmid of the present invention encodes IscB-2.0 and is assembled with guide RNA into an effector complex. This complex can specifically cleave the target sites of the genomes of Escherichia coli MG1655 and Escherichia coli ATCC9637. The homologous arms provided in plasmid form serve as templates for homologous recombination repair processes in cells, ultimately achieving precise editing of the target sites. Attached Figure Description

[0026] Figure 1 This is a comparison of the transformation efficiency of the IscB gene and the mutated IscB-2.0 gene in Escherichia coli MG1655.

[0027] Figure 2 This is a graph showing the transformation efficiency of the p15A-IscB-2.0 plasmid of the present invention in the genome of Escherichia coli MG1655.

[0028] Figure 3 This is a graph showing the efficiency of the IscB-2.0 protein in cleaving the genomic DNA of Escherichia coli MG1655 according to the present invention.

[0029] Figure 4 This is a graph showing the gene knockout efficiency of the p15A-IscB-2.0 plasmid of the present invention in Escherichia coli MG1655.

[0030] Figure 5 The image shows the gene knockout sequencing results of the p15A-IscB-2.0 plasmid of the present invention in Escherichia coli MG1655.

[0031] Figure 6 This is a graph showing the transformation efficiency of the p15A-IscB-2.0 plasmid of the present invention in the genome of Escherichia coli ATCC9637.

[0032] Figure 7 This is a graph showing the efficiency of the IscB-2.0 protein in cleaving the genomic DNA of Escherichia coli ATCC9637.

[0033] Figure 8 This is a graph showing the gene knockout efficiency of the p15A-IscB-2.0 plasmid of the present invention in Escherichia coli ATCC9637. Detailed Implementation

[0034] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the following detailed description, in conjunction with preferred embodiments and accompanying drawings, provides a clear and complete account of the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0035] It should be noted that all technical terms used in this invention are for the purpose of describing specific embodiments only and are not intended to limit the scope of protection of this invention. Unless otherwise specified, all raw materials, reagents, instruments and equipment used in the following embodiments of this invention can be purchased from the market or prepared by existing methods.

[0036] The main plasmids used in this invention are wild-type IscB plasmid, p15A-IscB plasmid, pωRNA-IscB plasmid, and pEcgRNA plasmid.

[0037] The p15A-IscB plasmid was purchased from the MolecularCloud platform, catalog number MC_0101511. The pωRNA-IscB plasmid was purchased from the Addgene platform, catalog number 205410 (enIscB plasmid). The pEcgRNA plasmid was purchased from the Addgene platform, catalog number 166581.

[0038] Example 1 A method for obtaining the p15A-IscB-2.0 plasmid includes the following steps: 1. Construct the p15A-IscB-2.0 plasmid expressing the IscB-2.0 protein. The specific construction procedure for the p15A-IscB-2.0 plasmid is as follows: Using the p15A-IscB plasmid as a template, a circular PCR reaction one is performed using two primer pairs, is-mut-1-up and is-mut-1-dn, to obtain PCR product one. This PCR product one is then sequenced using the verf-iscb-ce-1 primer. If the desired mutation is found, the PCR product is purified and recovered. The recovered product is the p15A-IscB plasmid with a single-base mutation. The template is then digested with DpnI digestive enzyme, and a circular PCR reaction two is performed using two primer pairs, is-mut-2-up and is-mut-2-dn, to obtain PCR product two. PCR product two is sequenced using the verf-iscb-ce-2 primer. If the result is correct, the template is digested with DpnI digestive enzyme, and the p15A-IscB-2.0 plasmid with the synonymous mutation is obtained.

[0039] Wild-type IscB plasmid and p15A-IscB-2.0 plasmid were transformed into E. coli MG1655, with TnpB plasmid as a control. The transformation efficiency was as follows: Figure 1 As shown in the figure. The wild-type IscB plasmid is designated as IscB, the p15A-IscB-2.0 plasmid as p15A-IscB-2.0, and the TnpB plasmid as the control. The results showed that the transformation efficiency of the control group was 4.8 × 10⁻⁶. 5 The transformation efficiency of the wild-type IscB plasmid was 2.3 × 10⁻⁶ CFU / μg plasmid DNA. 2 The transformation efficiency of the p15A-IscB-2.0 plasmid with CFU / μg DNA was 9.1 × 10⁻⁶. 4 CFU / μg. The experimental group and control group were 10... 5 The comparison of CFU / μg plasmid DNA indicates that the p15A-IscB-2.0 plasmid was not digested by enzymes after transformation into E. coli MG1655. Further sequencing verification of the mutant IscB-2.0 gene confirmed the successful construction of the p15A-IscB-2.0 plasmid.

[0040] 2. Construct the pωRNA-IscB-targetX and pωRNA-IscB-THR-targetX series of plasmids. In the pωRNA-IscB-targetX and pωRNA-IscB-THR-targetX series of plasmids, "targetX" represents the target gene. In this invention, the maeB gene is used as an example, corresponding to the pωRNA-IscB-maeB plasmid and the pωRNA-IscB-THR-maeB plasmid, respectively.

[0041] (1) Constructing the pωRNA-IscB plasmid: The pωRNA-IscB plasmid was constructed via homologous recombination. First, using primers pωRNA-IscB-F / pωRNA-IscB-R as a template, the ωRNA corresponding to the IscB gene was amplified by PCR. Then, using primers pωRNA-IscB-ptF / pωRNA-IscB-ptR as a template, the pωRNA vector was amplified by PCR. The ωRNA and pωRNA were then homologously recombinated to obtain the pωRNA-IscB plasmid.

[0042] (2) Constructing the pωRNA-IscB-maeB plasmid After constructing the pωRNA-IscB plasmid, a 20bp sequence with a GC content greater than 50% was selected upstream of the TAM sequence in its genome as the N of the pωRNA. 20 The region was then targeted. Subsequently, pωRNA suspension primers were designed targeting specific gene sites to construct pωRNA plasmids targeting the defined sites. Using pωRNA-IscB plasmid as a template, and pωRNA-IscB-maeB / pωRNA-dn-univers as suspension primers, overlap PCR was performed to replace the N terminus of the corresponding pωRNA-IscB plasmid. 20 The pωRNA-IscB-maeB plasmid was obtained from the region.

[0043] (3) Constructing the pωRNA-IscB-maeB-THR series of plasmids: After a gene is cut, it requires donor DNA, i.e., an exogenous repair template, to mediate homology repair. Therefore, donor DNA needs to be constructed in gene knockout experiments. First, the upstream sequence of the maeB gene is amplified using primers maeB-up-up and maeB-up-dn, and the downstream sequence is amplified using maeB-dn-dn. Next, PCR is performed using the same primers with the genome of *E. coli* MG1655 as a template, amplifying two 0.5kb fragments: one is the upstream sequence of maeB, and the other is the downstream sequence. Then, the two amplified fragments are mixed for overlap PCR. Here, because the ends of the maeB-up-dn primer and the front end of the maeB-dn-up primer are designed with complementary dangling sequences, the upstream and downstream fragments act as primers and templates for each other, extending and ligating through the overlapping region. After the fifth cycle of the overlap PCR reaction, maeB-up-up and maeB-up-dn primers are added to the system, and PCR is continued to obtain complete homologous arms. Then, the homologous arm was assembled with the linearized pωRNA-IscB-maeB plasmid vector through a recombination reaction to form the pωRNA-IscB-THR-maeB plasmid.

[0044] The specific process of the thermal shock conversion method in the above steps is as follows: (1) Remove the competent Escherichia coli DH5α cells from the -80℃ freezer and thaw them on ice for 10 min. The thawing time of the competent cells should not be too long, otherwise it will affect the transformation efficiency.

[0045] (2) Then add 10 μL of recombinant product or PCR product to 100 μL of competent cells, gently tap the tube wall to mix, and immediately incubate on ice for 30 min. The amount of recombinant product or PCR product added should not exceed 1 / 10 of the total volume of competent cells.

[0046] (3) After the competent cells have been allowed to stand, immediately place them in a water bath at 42°C for 45 seconds for heat shock, and then immediately cool them on ice for 2 to 3 minutes. During the ice-cooling process, avoid shaking, as it may affect the transformation rate.

[0047] After adding 900 μL of antibiotic-free LB liquid medium, the culture was incubated at 37°C and shaken at 220 rpm for 1 h.

[0048] (4) The resuscitation solution was then centrifuged at 3000 rpm for 3 min, 900 μL of supernatant was discarded, the bacterial cells were resuspended and spread on LB solid medium plates with the corresponding resistance, and cultured for 12 h until a single colony of suitable size appeared.

[0049] Table 1. Primers used in the strain construction process. Example 2 The application of p15A-IscB-2.0 plasmid in gene editing of Escherichia coli MG1655 includes the following steps: 1. Transform the p15A-IscB-2.0 plasmid into E. coli MG1655. 100 μL of competent *E. coli* cells were mixed with 300 ng of p15A-IscB-2.0 plasmid, with TnpB plasmid (10 kb in size) used as a control. TnpB plasmid was non-toxic to *E. coli* MG1655. Electroporation was performed in cooled 2 mm gene pulse cuvettes at 2.5 kV. Immediately after electroporation, the mixture was resuspended in fresh LB broth and incubated at 37°C for 1 h. Subsequently, the recovered cells were plated on LB agar plates containing chloramphenicol and incubated overnight at 37°C. Colony-forming units were calculated by counting colonies on the plates to determine plasmid transformation efficiency. Results are shown below. Figure 2 As shown in the figure. The TnpB plasmid used as a control is designated as the control. The transformation efficiency of the control group was 4.8 × 10⁻⁶. 5The transformation efficiency of p15A-IscB-2.0 plasmid in the experimental group was 9.1 × 10⁻⁶ CFU / μg plasmid DNA. 4 CFU / μg DNA, 10 CFU / μg DNA in experimental group and control group 5 The difference between CFU / μg plasmid DNA is on the order of magnitude. Therefore, further cleavage activity testing can be performed.

[0050] 2. The cleavage efficiency of IscB-2.0 protein on the genome of Escherichia coli MG1655 Currently, bacterial gene editing primarily relies on exogenous repair templates and homologous recombination to repair DSB (dysbiosis stem cell block). However, without exogenous repair templates, DSB caused by DNA nucleases typically cannot be repaired, leading to bacterial death. In E. coli, IscB-2.0 protein expression cannot bind to the target fragment, thus failing to cleave the E. coli genomic DNA, allowing the bacteria to survive normally. ωRNA, however, can carry the IscB-2.0 protein to the cleavage site of the target gene, enabling it to perform cleavage and causing E. coli death. Based on this principle, we tested the cleavage activity of IscB-2.0 protein on the genomic DNA of E. coli MG1655 without providing a repair homologous arm. 100 µL of competent E. coli MG1655 cells carrying the p15A-IscB-2.0 plasmid and expressing the IscB-2.0 protein were mixed with 100 ng of the pωRNA-IscB-2.0-maeB plasmid and the pCK2 plasmid, respectively. The pCK2 plasmid, used as a control, was 2.5 kb in size and did not target any genes in *E. coli* MG1655. The IscB-2.0-maeB-ωRNA produced by expressing the pωRNA-IscB-2.0-maeB plasmid could carry the IscB-2.0 protein targeting the maeB gene. Electroporation recovery fluid was obtained, plated, and incubated overnight at 37°C. Transformation efficiency was calculated based on the obtained CFU to determine the cleavage efficiency of the IscB-2.0 protein in *E. coli* MG1655. If the transformation efficiency of the pωRNA-IscB-2.0-maeB plasmid was comparable to that of the pCK2 plasmid, it indicated that the IscB-2.0 protein did not cleave the host genomic DNA. If the transformation efficiency of the pωRNA-IscB-2.0-maeB plasmid was significantly lower than that of the control plasmid by at least two orders of magnitude, it indicated that the IscB-2.0 protein could cleave the genomic DNA of *E. coli* MG1655.

[0051] The cleavage efficiency of IscB-2.0 protein in E. coli MG1655 is as follows: Figure 3 As shown in the figure. The results showed that the transformation efficiency for IscB-2.0 protein cleavage of the maeB gene was 499 CFU / μg, and the transformation efficiency was 10. 2The concentration of plasmid DNA was CFU / μg, and the difference compared to the control group was on the order of magnitude. This indicates that the IscB-2.0 protein has cleavage activity in E. coli MG1655.

[0052] 3. Gene editing efficiency of p15A-IscB-2.0 plasmid in Escherichia coli MG1655 After testing the cleavage activity of the IscB-2.0 system on Escherichia coli MG1655, the gene editing efficiency of the IscB-2.0 genome editing system in Escherichia coli MG1655 was further investigated. Therefore, the system was used to perform a knockout experiment on the maeB gene.

[0053] To test gene editing efficiency, L-arabinose at a final concentration of 10 mM was first added to *E. coli* MG1655 containing the p15A-IscB-2.0 plasmid and expressing IscB-2.0 to induce the expression of the λ-Red system, thereby improving gene editing efficiency. Subsequently, the pωRNA-THR-maeB plasmid targeting maeB was electroporated into *E. coli* MG1655 containing the p15A-IscB-2.0 plasmid and expressing IscB-2.0 protein, and cultured overnight until suitable-sized single colonies appeared. Ten colonies were randomly selected for colony PCR to verify the knockout results. Gel electrophoresis was used to determine whether the gene was knocked out. The results are as follows: Figure 4 As shown. Figure 4 In the diagram, WT represents the band without maeB gene knockout, and ΔmaeB represents the band after maeB gene knockout; the same applies below. The results indicate that the maeB gene was successfully deleted in the host cell, with a knockout efficiency of 40%. Further sequencing of the PCR products corresponding to the expected bands yielded the following results: Figure 5 As shown, the sequencing results further confirmed the successful gene knockout.

[0054] Example 3 The application of p15A-IscB-2.0 plasmid in gene editing of Escherichia coli ATCC9637 includes the following steps: 1. Transform the p15A-IscB-2.0 plasmid into E. coli ATCC9637. 100 μL of competent *E. coli* cells were mixed with 300 ng of p15A-IscB-2.0 plasmid, with TnpB plasmid used as a control. TnpB plasmid was non-toxic to *E. coli* ATCC9637. Electroporation was performed in cooled 2 mm gene pulse cuvettes at 2.5 kV. Immediately after electroporation, the mixture was resuspended in fresh LB broth and incubated at 37°C for 1 h. Subsequently, the recovered cells were plated on LB agar plates containing chloramphenicol and incubated overnight at 37°C. Colony-forming units were calculated by counting colonies on the plates, and plasmid transformation efficiency was calculated. The results are shown below. Figure 6 As shown in the figure. The results showed that the transformation efficiency of the TnpB plasmid in the control group was 5.93 × 10⁻⁶. 1 The transformation efficiency of p15A-IscB-2.0 plasmid in the experimental group was 6.87 × 10⁻⁶ CFU / μg plasmid DNA. 4 CFU / μg DNA, 10 CFU / μg DNA in experimental group and control group 6 The difference between CFU / μg plasmid DNA and other methods is on the order of three orders of magnitude. Furthermore, other researchers have confirmed that the IscB system can be used for gene editing in *E. coli* ATCC9637. Therefore, further cleavage activity testing can be performed.

[0055] 2. The cleavage efficiency of IscB-2.0 protein on the genome of Escherichia coli ATCC9637 100 µL of *E. coli* ATCC9637 competent cells carrying the p15A-IscB-2.0 plasmid and expressing the IscB-2.0 protein were mixed with 100 ng of the pωRNA-IscB-2.0-maeB plasmid, with the pCK2 plasmid used as a control. The pCK2 plasmid does not target any gene in *E. coli* MG1655. The IscB-2.0-maeB-ωRNA produced by expression of the pωRNA-IscB-2.0-maeB plasmid can carry the IscB-2.0 protein and target the maeB gene. Electroporation was performed to obtain electroporation recovery fluid, which was then plated and incubated overnight at 37°C. The transformation efficiency was calculated based on the obtained CFU to determine the cleavage efficiency of the IscB-2.0 protein in *E. coli* MG1655. If the transformation efficiency of the pωRNA-IscB-2.0-maeB plasmid was comparable to that of the pCK2 plasmid, it indicates that the protein did not cleave the host genomic DNA.

[0056] The cleavage efficiency of IscB-2.0 protein in E. coli ATCC9637 is as follows: Figure 7 As shown in the figure. The results showed that the transformation efficiency of the control group was 339 CFU / μg plasmid DNA, while the transformation efficiency obtained by targeting the maeB gene with pωRNA-IscB-2.0-maeB plasmid was 10. 2CFU / μg plasmid DNA. The difference compared to the control group indicates that the IscB-2.0 protein has cleavage activity in E. coli ATCC9637.

[0057] 3. Gene editing efficiency of p15A-IscB-2.0 in Escherichia coli ATCC9637 After testing the cleavage activity of the IscB-2.0 system on Escherichia coli ATCC9637, the gene editing efficiency of the p15A-IscB-2.0 plasmid-based genome editing system in Escherichia coli ATCC9637 was further investigated. Therefore, the system was used to perform a knockout experiment on the maeB gene.

[0058] First, L-arabinose at a final concentration of 10 mM was added to *E. coli* ATCC9637 containing the IscB-2.0 protein to induce the expression of the λ-Red system, thereby improving gene editing efficiency. Then, the pωRNA-THR-maeB plasmid targeting the maeB gene was electroporated into *E. coli* ATCC9637 containing the IscB-2.0 protein and cultured overnight until suitable-sized single colonies appeared. Ten colonies were randomly selected for colony PCR to verify the knockout results. Gel electrophoresis was used to determine whether the gene was knocked out. The results are as follows: Figure 8 As shown in the figure. The results indicate that the maeB gene was successfully deleted in the host cells, with a knockout efficiency of 10%. Further sequencing of the PCR products corresponding to the expected bands confirmed the successful gene knockout.

[0059] The specific steps of the electroconversion process described above are as follows: (1) Take 10 μL of Escherichia coli culture and inoculate it into 5 mL of antibiotic-free LB medium. Incubate overnight at 37°C and 220 rpm.

[0060] (2) On the second day, inoculate 1% into 50mL of antibiotic-free LB medium. When the bacterial culture grows to an OD600 value of 0.6~0.8, transfer it to a 50mL sterile centrifuge tube and incubate on ice for 30min.

[0061] (3) Then, centrifuge the bacterial solution at 4000 rpm and 4°C for 10 min, discard the supernatant, add pre-cooled sterile water or 10% glycerol solution, and blow the precipitated bacteria evenly on ice. Repeat step (3) three times.

[0062] (4) Based on the number of transformed samples, add pre-cooled sterile water to the centrifuge tube at a rate of 100 μL of competent cells per sample, and mix the bacterial solution by blowing and blowing.

[0063] (5) Then add plasmids of no more than 1 / 10 of the total volume to 100 μL of competent cells, and add the well mixed bacterial solution to a 1 mm electric rotary cup that has been pre-cooled on ice.

[0064] (6) After carefully drying the metal electrode on the outside of the electroporation cup containing the mixture, place it in the electroporator and electroporate it at 1.8kV and 25μF. Immediately after electroporation, add 900μL of antibiotic-free LB medium, mix well, and transfer to a 1.5mL centrifuge tube.

[0065] (7) The mixture was then shaken and recovered at 37°C and 220 rpm for 1 h, and then spread on LB medium plates containing chloramphenicol and incubated overnight at 37°C.

[0066] (8) The next day, transformants were cut and transferred to liquid LB medium containing chloramphenicol and cultured overnight at 37°C and 220 rpm.

[0067] (9) On the second day, inoculate 1% of the culture into 50 mL of LB medium and add L-arabinose to a final concentration of 10 mM to induce the expression of the λ-Red system. When the bacterial culture reaches an OD600 value of 0.6-0.8, transfer it to a 50 mL sterile centrifuge tube and incubate on ice for 30 min.

[0068] (10) The bacterial culture was then centrifuged at 4000 rpm and 4°C for 10 min. After discarding the supernatant, pre-cooled sterile water or 10% glycerol solution was added, and the culture was blown evenly on ice. Step (10) was repeated three times.

[0069] (11) Based on the number of transformed samples, add pre-cooled sterile water to the centrifuge tube at a rate of 100 μL of competent cells per sample, and mix the bacterial solution by blowing and blowing.

[0070] (12) Then add pωRNA-THR-maeB plasmid with a total volume not exceeding 1 / 10 of 100 μL of competent cells, and add the well mixed bacterial solution to a 1 mm electric rotary cup that has been pre-cooled on ice.

[0071] (13) The electroporation vessel containing the bacterial culture and plasmid mixture was electroporated at 1.8 kV and 25 μF. Immediately after electroporation, the mixture was transferred to 900 μL of antibiotic-free LB medium. Finally, the bacterial culture was shaken and recovered at 37°C for 1 h, and then spread on LB medium plates containing chloramphenicol and spectinomycin and incubated overnight at 37°C.

[0072] in conclusion This invention obtains the IscB-2.0 gene by synonymous mutation of the IscB gene. The IscB-2.0 gene ensures that it is not recognized by the restriction modification system of *E. coli*, while still expressing the correct IscB protein, and has no toxic effect on *E. coli*. The p15A-IscB-2.0 plasmid of this invention achieves a 40% knockout efficiency of the maeB gene in *E. coli* ATCC9637. The p15A-IscB-2.0 plasmid achieves a 10% knockout efficiency of the maeB gene in *E. coli* ATCC9637.

[0073] Existing wild-type IscB gene expression plasmids cannot be transformed into *E. coli* MG1655. This invention provides a mutated IscB-2.0 gene and a method for constructing the p15A-IscB-2.0 plasmid. A method for constructing the p15A-IscB-2.0 plasmid constitutively expressing the IscB protein is provided. This invention provides a gene editing process in *E. coli* MG1655 and *E. coli* ATCC9637 using a gene editing vector system based on the mini nuclease IscB-2.0 protein. Specifically, the p15A-IscB-2.0 plasmid encodes the IscB-2.0 protein, which is then assembled with guide RNA to form an effector complex. This complex can specifically cleave target sites in the *E. coli* genome. The homologous arms provided in plasmid form serve as templates for intracellular homologous recombination repair processes, ultimately achieving precise editing of the target sites.

[0074] In summary, this invention successfully constructed an upgraded mutant IscB-2.0 gene by using the miniature DNA nuclease IscB and through mutation. For the first time, a genome editing tool based on the IscB-2.0 gene was successfully established in Escherichia coli MG1655 and Escherichia coli ATCC9637, and editing was successfully achieved.

[0075] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described in this invention to avoid redundancy. Although preferred embodiments of this invention have been described, those skilled in the art, once they understand the inventive concept of this invention, can make other changes and modifications to these embodiments, and all such changes and modifications fall within the scope of this invention.

[0076] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. If such modifications and variations fall within the scope of equivalents of this invention, then this invention also intends to include these modifications and variations.

Claims

1. A method for preparing a miniature DNA nucleotidase, characterized in that, The process includes the following steps: expressing the p15A-IscB-2.0 plasmid to obtain a miniature DNA nuclease; The nucleotide sequence of the p15A-IscB-2.0 plasmid is shown in SEQ ID NO.

2. It is obtained by synonymous mutation of the 5'-AACNNNNNNGYGC-3' sequence in the IscB gene of p15A-IscB.

2. The method for preparing a miniature DNA nucleotidase according to claim 1, characterized in that, The expression process refers to the transformation of the p15A-IscB-2.0 plasmid into engineered bacteria for expression.

3. The method for preparing a miniature DNA nucleotidase according to claim 2, characterized in that, The engineered bacteria is Escherichia coli.

4. The method for preparing a miniature DNA nuclease according to claim 1, characterized in that, The construction of the p15A-IscB-2.0 plasmid includes the following steps: Using p15A-IscB as a template, loop PCR reaction one was performed using two pairs of primers, is-mut-1-up and is-mut-1-dn, to obtain PCR product one; PCR product one was sequenced using the verf-iscb-ce-1 primer, and p15A-IscB-1 with a single base mutation containing the target mutation was recovered. Using p15A-IscB-1 as a template, two pairs of primers, is-mut-2-up and is-mut-2-dn, were used to perform loop PCR reaction II to obtain PCR product II. PCR product II was sequenced using the verf-iscb-ce-2 primer. After the target mutation was found, the template was digested with DpnI digestive enzyme and purified to obtain p15A-IscB-2.0 plasmid. The is-mut-1-up primer has a 5'-aggcacgtccttatttctgctttcgatctggg-3' nucleotide sequence; the is-mut-1-dn primer has a 5'-cgaaaccagaaataaggacgtgcctaagct-3' nucleotide sequence; the verf-iscb-ce-1 primer has a 5'-aaaagaattttgtgttataatc-3' nucleotide sequence; the is-mut-2-up primer has a 5'-ccgctgcaccttgggattgttcatggccatgaag-3' nucleotide sequence; the is-mut-2-dn primer has a 5'-tcatggccatgaacaatcccaaggtgcagcggtg-3' nucleotide sequence; and the verf-iscb-ce-2 primer has a 5'-taaaggcagcgtggaagag-3' nucleotide sequence.

5. A miniature DNA nuclease prepared by the preparation method according to any one of claims 1 to 4.

6. The application of a miniature DNA nuclease according to claim 5 in bacterial gene editing.

7. The application of a miniature DNA nuclease according to claim 6 in bacterial gene editing, characterized in that, The bacteria are Escherichia coli MG1655 and Escherichia coli ATCC9637.

8. The application of a miniature DNA nuclease according to claim 6 in bacterial gene editing, characterized in that, The gene editing referred to is gene knockout.

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