Host cell which improves plasmid stability, and use thereof

Abstract

The present application relates to a host cell which improves plasmid stability, and the use thereof. The present application specifically relates to an Escherichia coli host cell capable of improving the stability of a nucleic acid molecule containing a hairpin structure sequence such as ITR or a simple sequence repeat such as a poly(A) sequence, wherein the sbcC and sbcD genes of the host cell are knocked out, and the gyrA gene expresses a GyrA variant or the gyrB gene expresses a GyrB variant.

Description

A host cell for enhancing plasmid stability and its applications

[0001] Cross-references to related applications

[0002] This application claims priority to Chinese patent application No. 202411435773.2, filed on October 14, 2024, the entire contents of which are incorporated herein by reference. Invention Field

[0003] This application relates to an engineered strain of *Escherichia coli* that enhances the stability (e.g., replication stability) of hairpin structure sequences such as inverted repeat sequences (ITRs) or simple repeat sequences such as poly(A) sequences in nucleic acid molecules, and its use, for example, in the preparation of nucleic acid molecules (e.g., plasmids) containing hairpin structure sequences or poly(A) sequences. Background Technology

[0004] Introducing a vector containing a target sequence into host cells and culturing the cells under appropriate conditions can amplify the vector and generate RNA and peptide / protein molecules. Some structures present in the target sequence, such as hairpin structures and poly(A) sequences, are prone to fragment loss during vector amplification. Additionally, some delivery vectors, such as adeno-associated virus (AAV) vectors, themselves contain hairpin structures, which can easily lead to fragment loss during vector amplification.

[0005] Recombinant adeno-associated virus (rAAV) is commonly used as a DNA delivery vector due to its high safety, low immunogenicity, wide applicability, and long duration of action. The AAV genome contains short palindromic / inverted terminal repeat (ITR) sequences at its ends, which can fold into hairpin structures and serve as the starting point for viral DNA replication. As shown in Figure 1, the outer 125 nt of the ITR (5'AB-B'-C-C'-A'3' or 5'AC-C'-B-B'-A'3') can form a unique self-complementary T-shaped palindromic hairpin structure, consisting of one longer palindromic arm (A-A' region) and two shorter palindromic arms (B-B' and C-C' regions). The remaining 20 nt is the D region, a single-stranded structure. The palindromic sequence of the AAV-ITR is highly stable and has a high GC content, making it prone to mutation or recombination loss during bacterial reproduction. However, as Jude Samulski, a pioneer in AAV gene therapy, emphasized in his review, the integrity of the ITR is crucial for efficient rAAV replication and packaging, suggesting that the ITR deletion should ideally be less than 20% (Flotte TR, Carter BJ. (1995) Adeno-associated virus vectors for gene therapy. Gene Ther. 2(6):357-62). The impact of ITR deletion on rAAV viral packaging and its application in gene therapy is highly complex and can produce unintended biological consequences through vector-host interactions. Using incomplete ITR sequences in the preclinical stage will reduce the reproducibility of experiments within and across laboratories, and may prevent the achievement of previous effects when using complete ITR sequences in clinical trials.

[0006] There is currently no good method to avoid ITR deletion during the construction of rAAV-ITR plasmids and amplification of E. coli. Some commercially available strains with recombination defects, such as JC8111, SURE2, Stabl3, and XL10-Gold, are often used for the construction and amplification of plasmids containing ITR structures, but they are not always effective. Compared with wild-type E. coli strains, these strains usually grow slowly and have poor resistance, resulting in low plasmid yield. Using these strains as host strains for plasmid cloning often leads to a prolonged production cycle. sbcC-sbcD nuclease is a chromosome structure maintenance (SMC) family protein that can cleave DNA hairpin structures (Connelly JC et al., (1998) The SbcCD nuclease of Escherichia coli is a structural maintenance of chromosomes (SMC) family protein that cleaves hairpin DNA. Proc Natl Acad Sci US A. 95(14):7969-74). Escherichia coli sbcC mutants allow stable propagation of DNA replicons containing a long palindrome (Chalker, Alison F. et al., (1988) Escherichia coli sbcC mutants permit stable propagation of DNA replicons containing a long palindrome. Gene 71(1):201-205). Palindromes such as shRNA or AAV ITR are more stable in sbcC knockout strains than in non-knockout strains. Knockout of the sbcC and sbcD genes significantly increased the number of recombinant clones containing plasmids with intact hairpin structures, but still could not achieve sufficiently high ITR integrity. Especially when the length of the inverted repeat sequence in the A-A' region exceeded 30 bp, significant deletions were observed even in the ΔsbcC-sbcD mutant strain.

[0007] In the vector production process, in addition to the hairpin structure, poly(A) or poly(T) sequences are also typical unstable sequences that are prone to deletion during vector replication.

[0008] The main sequence of mature mRNA is the coding region, with uncoding regions (UTRs) upstream and downstream of it. Eukaryotic mRNA molecules also have a 5' cap and a 3' poly(A) tail at both ends. The poly(A) tail plays a crucial role in maintaining mRNA stability, regulating translation efficiency, and maintaining mRNA transport. For mass mRNA synthesis, in vitro transcription (IVT) is currently the most efficient method. IVT primarily uses linear DNA as a template to prepare mRNA. The main process steps include transcribing linearized plasmid DNA into mRNA, chemical modification (e.g., adding a 5' cap and a 3' poly(A) tail), and purification. There are two main methods for adding the poly(A) tail during IVT mRNA synthesis. The first is enzymatic synthesis, where a poly(A) polymerase derived from *E. coli* is added after mRNA transcription. This method has the advantage of not requiring a template and is simple to operate, but it has the limitation of unstable tail length. The second method is co-transcription, which involves directly transcribing poly(A) sequences already present on template plasmid DNA or PCR products. Its advantage lies in eliminating the need for poly(A) polymerase, reducing process steps, and saving costs. However, it suffers from the problem of poly(A) sequence deletion. Large-scale plasmid production is typically carried out using E. coli fermentation. During amplification, plasmids carrying longer poly(A) sequences are inherently unstable, and the poly(A) tail coding sequence is prone to deletion during replication, resulting in plasmid impurity. To improve the stability of poly(A) sequences on plasmids, some scientists employ segmented poly(A) sequence co-transcription. Since each poly(A) binding protein PABP binds only about 30 A's, and a small number of A's act as spacers between two binding proteins, replacing these spacers with other non-A bases reduces the probability of recombination in the poly(A) tail coding sequence. Furthermore, the length and base composition of the spacer sequence can be optimized to further reduce the risk of poly(A) tail deletion. Even so, the risk of A deletion still exists, and the segmented preparation of poly(A) plasmids can affect the expression levels of downstream proteins. Furthermore, there are reports that low-temperature (30°C) fermentation can improve the integrity of the poly(A) tail coding sequence. However, low-temperature culture slows bacterial growth, reduces plasmid copy number, significantly decreases yield, and prolongs the production cycle, posing challenges to plasmid production.

[0009] Currently, there is no good method to avoid the deletion of the A base during the construction of poly(A) plasmids and the amplification of *E. coli*. Some recombination-deficient commercial strains, such as NEB stable and Stabl3, are often used for the construction and amplification of plasmids containing poly(A) structures, but they are not always effective. Therefore, finding more stable poly(A) fermentation strains that reduce the risk of poly(A) tail sequence deletion or impurity without affecting plasmid yield may become a new research direction. However, there are few reports on the impact of different strains on the stability of poly(A) tail sequences.

[0010] Therefore, there is an urgent need to develop a high-yield engineered strain of Escherichia coli that grows rapidly, has high transformation potential, and can be used to clone plasmids containing palindromic and inverted repeat sequences or poly(A) structures.

[0011] Helicases are type II DNA topoisomerases crucial for bacterial survival, participating in processes such as DNA replication, repair, recombination, and transcription (Huang WM. (1994) Type II DNA topoisomerase genes. Adv Pharmacol. 29A: 201-25). Helicases generally consist of two GyrA subunits and two GyrB subunits. The GyrA subunits are primarily responsible for DNA binding, creating gaps in the DNA double helix, and suturing these gaps. The GyrB subunits mainly mediate energy transfer and ATP hydrolysis. Specifically, helicases can unwind (+) supercoils caused by replication and transcription and can introduce (-) supercoils into genomic DNA.

[0012] Regardless of the detailed mechanisms governing DNA supercoiling, genetic studies of E. coli topoisomerase I mutant strains have highlighted the importance of balancing DNA supercoiling and relaxation activities. When gyrases are inhibited, the transcriptional activity of most genes decreases. For example, overexpression of GyrI, which inhibits gyrase activity, inhibits the growth of the strain (Martins LO et al., (2002) Molecular and biochemical characterization of a highly stable bacterial laccase that occurs as a structural component of the Bacillus subtilis endospore coat. J Biol Chem. 277(21):18849-59). Some antibacterial drugs, such as quinolone antibiotics and coumarin drugs, target gyrases, causing irreversible damage to bacterial DNA (Maxwell, A. (1993) The interaction between coumarin drugs and DNA gyrase Mol. Microbiol. 9(4):681–686; Maxwell, A. (1997) DNA gyrase as a drug target Trends Microbiol. 5(3):102–109). Some common commercial strains, such as Escherichia coli JM108, often contain the GyrA96 mutation to resist the quinolone nalidixic acid. Summary of the Invention

[0013] The inventors of this application have unexpectedly discovered that mutating GyrA or GyrB in host cells, particularly in *E. coli* host cells (i.e., expressing GyrA(H80A) or GyrB(R136C) variants), can increase the stability of hairpin sequences such as ITRs or UTRs, or simple repetitive sequences such as poly(A) or poly(T) sequences in plasmids, including replication stability and / or expression stability. Specifically, mutating GyrA (i.e., expressing GyrA(H80A) variants) in SbcC-SbcD knockout host cells, particularly in SbcC-SbcD knockout *E. coli* host cells, can further increase the stability of hairpin sequences such as ITRs or UTRs, or simple repetitive sequences such as poly(A) or poly(T) sequences in plasmids, including replication stability and / or expression stability.

[0014] Building upon the GyrA(H80A) mutation and SbcC-SbcD knockout, the inventors of this application further performed commercially available mcrB, mcrC, hsdM, hsdS, mrr, and hsdr knockouts, as well as endA, recA, and galE mutations on the strain. The results showed that further knockouts and mutations did not adversely affect the effects of GyrA(H80A) mutations and / or SbcC-SbcD knockouts on hairpin sequences such as ITRs or UTRs, or simple repetitive sequences such as poly(A) or poly(T) sequences in stable plasmids. Moreover, they were more beneficial for the introduction and transformation efficiency of exogenous nucleic acid sequences, such as plasmids containing hairpin structures or simple repetitive sequences, thus increasing the yield of exogenous nucleic acid sequences such as plasmids.

[0015] Therefore, in a first aspect, this application provides a host cell that may contain the gyrA and gyrB genes. The gyrA gene expressing GyrA may contain an H80A mutation at position 80 relative to SEQ ID NO:62. The gyrB gene expressing GyrB may contain an R136C mutation at position 136 relative to SEQ ID NO:63. In some embodiments, the host cell may contain both the gyrA and gyrB genes, wherein the gyrA gene expressing GyrA may contain an H80A mutation at position 80 relative to SEQ ID NO:62. In some embodiments, the host cell may contain both the gyrA and gyrB genes, wherein the gyrB gene expressing GyrB may contain an R136C mutation at position 136 relative to SEQ ID NO:63. In some embodiments, the host cell may contain gyrA and gyrB genes, wherein the gyrA gene expressing GyrA may contain an H80A mutation at position 80 relative to SEQ ID NO:62, and the gyrB gene expressing GyrB may contain an R136C mutation at position 136 relative to SEQ ID NO:63.

[0016] The GyrA expressed by the gyrA gene may contain the amino acid sequence shown in SEQ ID NO:62.

[0017] The GyrB gene expressed can contain the amino acid sequence shown in SEQ ID NO:63.

[0018] The host cell may also contain the SbcC or SbcD gene knockout. In some embodiments, the host cell may contain both SbcC and SbcD gene knockouts.

[0019] The host cell may also i) contain gene knockout of mcrB, mcrC, hsdM, hsdS, mrr, and hsdr, and / or ii) contain endA, recA, and galE genes, wherein the EndA, RecA, and GalE expressed by the endA, recA, and galE genes may contain an E208K mutation at position 208 relative to SEQ ID NO:64, a G161D mutation at position 161 relative to SEQ ID NO:65, and an S123F mutation at position 123 relative to SEQ ID NO:66, respectively. The EndA expressed by the endA gene may contain the amino acid sequence shown in SEQ ID NO:64. The RecA expressed by the recA gene may contain the amino acid sequence shown in SEQ ID NO:65. The GalE expressed by the galE gene may contain the amino acid sequence shown in SEQ ID NO:66.

[0020] The host cell may contain nucleic acid molecules with simple repetitive sequences such as poly(A) or poly(T) sequences. For example, the host cell may contain a vector, which may contain nucleic acid molecules with simple repetitive sequences such as poly(A) or poly(T) sequences. The host cell may integrate nucleic acid molecules with simple repetitive sequences such as poly(A) or poly(T) sequences into its genome. The poly(A) sequence may contain consecutive A bases, for example, 20-250 consecutive A bases. The poly(A) sequence may contain 2-5 consecutive A base segments separated by non-A bases, wherein the number of consecutive A bases in each consecutive A base segment may be, for example, 10-100, and each pair of consecutive A base segments may be separated by, for example, 1-20 non-A bases. The poly(T) sequence may contain consecutive T bases, for example, 20-250 consecutive T bases. The poly(T) sequence may contain 2-5 consecutive T base segments separated by non-T bases, wherein the number of consecutive T bases in each consecutive T base segment may be, for example, 10-100, and each pair of consecutive T base segments may be separated by, for example, 1-20 non-T bases.

[0021] The host cell may contain a nucleic acid molecule with a hairpin sequence such as an ITR or UTR. For example, the host cell may contain a vector that contains a nucleic acid molecule with a hairpin sequence such as an ITR or UTR. The host cell may integrate a nucleic acid molecule with a hairpin sequence such as an ITR or UTR into its genome. In some embodiments, the ITR may contain the nucleotide sequence shown in SEQ ID NO:53.

[0022] The host cell can be natural or genetically modified. In particular, the host cell can be genetically modified.

[0023] The host cell can be an *E. coli* host cell, particularly a vector preparation / production strain, including, but not limited to, MG1655, JM108, NEB Stable, Top10, DH5α, and DH10B. Specifically, the host cell can be MG1655, JM108, and NEB Stable. Specifically, the host cell can be MG1655.

[0024] In some embodiments, the host cell may be selected from JM108[gyrA(H80A)], JM108[gyrB(R136C)], MG1655[gyrA(H80A)], MG1655[gyrB(R136C)], NEB stable[gyrA(H80A)], NEB stable[gyrB(R136C)], JM108[ΔsbcC-sbcD-gyrA(H80A)], NEB stable[ΔsbcC-sbcD-gyrA(H80A)], MG1655[ΔsbcC-sbcD-gyrA(H80A)], MG1655[endA-gale15-Δ(mrr-hsdRMS- mcrBC)-recA-ΔsbcC-sbcD-gyrA(H80A)], and MG1655[endA-gale15-Δ(mrr-hsdRMS-mcrBC)-recA-gyrA(H80A)].

[0025] In a second aspect, this application provides a method for preparing recombinant host cells, comprising:

[0026] i) Provide a host cell, such as a prokaryotic host cell, that contains the gyrA and gyrB genes, and

[0027] ii) GyrA that causes gyrA gene expression contains an H80A mutation at position 80 relative to SEQ ID NO:62, or GyrB that causes gyrB gene expression contains an R136C mutation at position 136 relative to SEQ ID NO:63.

[0028] In some embodiments, step ii) may include having GyrA, which causes the gyrA gene to be expressed, contain an H80A mutation at position 80 relative to SEQ ID NO:62. In some embodiments, step ii) may include having GyrB, which causes the gyrB gene to be expressed, contain an R136C mutation at position 136 relative to SEQ ID NO:63. In some embodiments, step ii) may include having GyrA, which causes the gyrA gene to be expressed, contain an H80A mutation at position 80 relative to SEQ ID NO:62, and having GyrB, which causes the gyrB gene to be expressed, contain an R136C mutation at position 136 relative to SEQ ID NO:63.

[0029] The host cell in step i) can be an *E. coli* host cell, particularly an *E. coli* vector preparation / production strain, including, but not limited to, MG1655, JM108, NEB Stable, Top10, DH5α, and DH10B. Specifically, the host cell can be MG1655, JM108, and NEB Stable. Specifically, the host cell can be MG1655.

[0030] Step ii) may include genetically modifying the host cell so that the GyrA gene expressed by the gyrA gene contains an H80A mutation at position 80 relative to SEQ ID NO:62. In some embodiments, step ii) may include introducing a Cas9 enzyme, an sgRNA containing the nucleotide sequence shown in SEQ ID NO:21, and a donor sequence containing the nucleotide sequence shown in SEQ ID NO:30 into the host cell of step i), so that the GyrA gene expressed by the gyrA gene contains an H80A mutation at position 136 relative to SEQ ID NO:63.

[0031] Step ii) may include genetically modifying the host cell so that the GyrB gene expressed contains an R136C mutation at position 136 relative to SEQ ID NO:63. In some embodiments, step ii) may include introducing a Cas9 enzyme, an sgRNA comprising the nucleotide sequence shown in SEQ ID NO:22, and a donor sequence comprising the nucleotide sequence shown in SEQ ID NO:31 into the host cell of step i), so that the GyrB gene expressed contains an R136C mutation at position 136 relative to SEQ ID NO:63.

[0032] Step ii) may include GyrA, which enables the expression of the gyrA gene, to contain the amino acid sequence shown in SEQ ID NO:62.

[0033] Step ii) may include GyrB, which enables the expression of the gyrB gene, to contain the amino acid sequence shown in SEQ ID NO:63.

[0034] The host cell may also contain the sbcC and sbcD genes, and the method may further include knocking out the sbcC and sbcD genes. In some embodiments, the method may include introducing the Cas9 enzyme, sgRNA containing the nucleotide sequence shown in SEQ ID NO:17, and a donor sequence containing the nucleotide sequence shown in SEQ ID NO:26 into the host cell of step i).

[0035] The host cell may also contain the mcrB and mcrC genes, and the method may further include knocking out the mcrB and mcrC genes. In some embodiments, the method may include introducing the Cas9 enzyme into the host cell of step i), as well as an sgRNA containing the nucleotide sequence shown in SEQ ID NO:14 and a donor sequence containing the nucleotide sequence shown in SEQ ID NO:23.

[0036] The host cell may also contain the hsdM and hsdS genes, and the method may further include knocking out the hsdM and hsdS genes. In some embodiments, the method may include introducing the Cas9 enzyme into the host cell of step i), as well as an sgRNA containing the nucleotide sequence shown in SEQ ID NO:15 and a donor sequence containing the nucleotide sequence shown in SEQ ID NO:24.

[0037] The host cell may also contain the mrr and hsdr genes, and the method may further include knocking out the mrr and hsdr genes. In some embodiments, the method may include introducing the Cas9 enzyme into the host cell of step i), as well as an sgRNA containing the nucleotide sequence shown in SEQ ID NO:16 and a donor sequence containing the nucleotide sequence shown in SEQ ID NO:25.

[0038] The host cell may also contain the endA gene, and the method may further include genetically modifying the host cell so that the EndA gene expresses an E208K mutation at position 208 relative to SEQ ID NO:64. In some embodiments, the method may include introducing the Cas9 enzyme, an sgRNA containing the nucleotide sequence shown in SEQ ID NO:18, and a donor sequence containing the nucleotide sequence shown in SEQ ID NO:27 into the host cell of step i).

[0039] The host cell may also contain the recA gene, and the method may further include genetically modifying the host cell so that the RecA expressed by the recA gene contains a G161D mutation at position 161 relative to SEQ ID NO:65. In some embodiments, the method may include introducing the Cas9 enzyme into the host cell of step i), as well as an sgRNA containing the nucleotide sequence shown in SEQ ID NO:19 and a donor sequence containing the nucleotide sequence shown in SEQ ID NO:28.

[0040] The host cell may also contain the galE gene, and the method may further include genetically modifying the host cell so that the GalE expressed by the galE gene contains the S123F mutation at position 123 relative to SEQ ID NO:66. In some embodiments, the method may include introducing the Cas9 enzyme, an sgRNA containing the nucleotide sequence shown in SEQ ID NO:20, and a donor sequence containing the nucleotide sequence shown in SEQ ID NO:29 into the host cell of step i).

[0041] The EndA expressed by the genetically modified endA may contain the amino acid sequence shown in SEQ ID NO:64. The RecA expressed by the genetically modified recA may contain the amino acid sequence shown in SEQ ID NO:65. The GalE expressed by the genetically modified galE may contain the amino acid sequence shown in SEQ ID NO:66.

[0042] In some embodiments, the host cell may further contain the genes mcrB, mcrC, hsdM, hsdS, mrr, hsdr, endA, recA, and galE. The method may further include knocking out the genes mcrB, mcrC, hsdM, hsdS, mrr, and hsdr, and causing the EndA, RecA, and GalE expressed by endA, recA, and galE to contain an E208K mutation at position 208 relative to SEQ ID NO:64, a G161D mutation at position 161 relative to SEQ ID NO:65, and an S123F mutation at position 123 relative to SEQ ID NO:66, respectively.

[0043] This application also provides recombinant host cells prepared by the method, which include the host cells of the first aspect of this application.

[0044] In a third aspect, this application relates to the use of the host cell of the first aspect of this application, or the recombinant host cell prepared by the method of the second aspect of this application, in replicating or expressing nucleic acid molecules containing simple repetitive sequences such as poly(A) or poly(T) sequences, or nucleic acid molecules containing hairpin structure sequences such as ITR or UTR.

[0045] Nucleic acid molecules containing simple repeat sequences such as poly(A) or poly(T) sequences, or nucleic acid molecules containing hairpin structure sequences such as ITR or UTR, can be included in vectors or integrated into the genome of recombinant host cells.

[0046] Specifically, this application provides a method for replicating or expressing nucleic acid molecules containing simple repetitive sequences such as poly(A) or poly(T) sequences, comprising:

[0047] i) Provide the host cell of the first aspect of this application, or the recombinant host cell prepared by the method of the second aspect of this application.

[0048] ii) Introducing nucleic acid molecules containing simple repetitive sequences such as poly(A) or poly(T) sequences into the recombinant host cell, and

[0049] iii) Culture recombinant host cells under conditions that facilitate the replication or expression of nucleic acid molecules containing simple repetitive sequences such as poly(A) or poly(T) sequences.

[0050] In some implementations, the order of steps i) and ii) can be adjusted as needed.

[0051] Alternatively, this application provides a method for replicating or expressing a nucleic acid molecule containing a hairpin structure sequence such as an ITR or UTR, comprising:

[0052] i) Provide the host cell of the first aspect of this application, or the recombinant host cell prepared by the method of the second aspect of this application.

[0053] ii) Introducing a nucleic acid molecule containing a hairpin sequence such as an ITR or UTR into the recombinant host cell, and

[0054] iii) Culture recombinant host cells under conditions that facilitate the replication or expression of nucleic acid molecules containing hairpin structure sequences such as ITR or UTR.

[0055] In some implementations, the order of steps i) and ii) can be adjusted as needed.

[0056] Step ii) in the above method may include introducing a vector into the recombinant host cell, which may contain a nucleic acid molecule containing a simple repeat sequence such as a poly(A) or poly(T) sequence, or a nucleic acid molecule containing a hairpin structure sequence such as an ITR or a UTR. Alternatively, step ii) may include integrating a nucleic acid molecule containing a simple repeat sequence such as a poly(A) or poly(T) sequence, or a nucleic acid molecule containing a hairpin structure sequence such as an ITR or a UTR, into the genome of the recombinant host cell.

[0057] Simple repeating sequences can include, but are not limited to, poly(A) sequences and poly(T) sequences. A poly(A) sequence can contain consecutive A bases, with the number of consecutive A bases being, for example, 20-250. A poly(A) sequence can contain 2-5 consecutive A base segments separated by non-A bases, wherein the number of consecutive A bases in each consecutive A base segment can be, for example, 10-100, and every two consecutive A base segments can be separated by, for example, 1-20 non-A bases. A poly(T) sequence can contain consecutive T bases, with the number of consecutive T bases being, for example, 20-250. A poly(T) sequence can contain 2-5 consecutive T base segments separated by non-T bases, wherein the number of consecutive T bases in each consecutive T base segment can be, for example, 10-100, and every two consecutive T base segments can be separated by, for example, 1-20 non-T bases.

[0058] Sequences containing hairpin structures may include, but are not limited to, inverted terminal repeats (ITRs) and untranslated regions (UTRs), such as UTRs derived from RNA. In some embodiments, the ITR may contain the nucleotide sequence shown in SEQ ID NO:53.

[0059] Specifically, this application provides a method for replicating or expressing nucleic acid molecules containing simple repetitive sequences or nucleic acid molecules containing hairpin structure sequences in Escherichia coli host cells, comprising:

[0060] i) Provide an *E. coli* host cell containing the gyrA and gyrB genes, wherein the gyrA gene expresses GyrA containing an H80A mutation at position 80 relative to SEQ ID NO:62, or the gyrB gene expresses GyrB containing an R136C mutation at position 136 relative to SEQ ID NO:63.

[0061] ii) Introducing nucleic acid molecules containing simple repeat sequences or hairpin structure sequences into host cells, and

[0062] iii) Culture recombinant host cells under conditions that facilitate the replication or expression of the nucleic acid molecules introduced in step ii).

[0063] In some embodiments, this application provides a method for replicating or expressing nucleic acid molecules containing simple repetitive sequences such as poly(A) or poly(T) sequences in a host cell, comprising:

[0064] i) Providing a host cell, such as a prokaryotic host cell, which may contain the gyrA and gyrB genes, wherein the host cell is genetically modified to express GyrA containing the H80A mutation at position 80 relative to SEQ ID NO:62, or genetically modified to express GyrB containing the R136C mutation at position 136 relative to SEQ ID NO:63.

[0065] ii) Introducing nucleic acid molecules containing simple repetitive sequences such as poly(A) or poly(T) sequences into host cells, and

[0066] iii) Culture recombinant host cells under conditions that facilitate the replication or expression of nucleic acid molecules containing simple repetitive sequences such as poly(A) or poly(T) sequences.

[0067] In some implementations, the order of steps i) and ii) can be adjusted as needed.

[0068] In some embodiments, this application also provides a method for replicating or expressing nucleic acid molecules containing hairpin structure sequences such as ITRs or UTRs in host cells, comprising:

[0069] i) Providing a host cell, such as a prokaryotic host cell, which may contain the gyrA and gyrB genes, wherein the host cell is genetically modified to express GyrA containing the H80A mutation at position 80 relative to SEQ ID NO:62, or genetically modified to express GyrB containing the R136C mutation at position 136 relative to SEQ ID NO:63.

[0070] ii) Introducing nucleic acid molecules containing hairpin sequences such as ITRs or UTRs into host cells, and

[0071] iii) Culture recombinant host cells under conditions that facilitate the replication or expression of nucleic acid molecules containing hairpin structure sequences such as ITR or UTR.

[0072] In some implementations, the order of steps i) and ii) can be adjusted as needed.

[0073] In some embodiments of the above method, in step i), the host cell may be genetically modified such that the expressed GyrA contains an H80A mutation at position 80 relative to SEQ ID NO:62, and the expressed GyrB contains an R136C mutation at position 136 relative to SEQ ID NO:63.

[0074] The GyrA expressed by the genetically modified gyrA gene can contain the amino acid sequence shown in SEQ ID NO:62.

[0075] The GyrB gene expressed after genetic modification can contain the amino acid sequence shown in SEQ ID NO:63.

[0076] The host cell in step i) can be an Escherichia coli host cell, particularly an Escherichia coli vector preparation strain, including, but not limited to, MG1655, JM108, NEB Stable, Top10, DH5α, and DH10B. In particular, the host cell can be MG1655, JM108, and NEB Stable.

[0077] In some embodiments, step i) may include introducing the Cas9 enzyme, sgRNA containing the nucleotide sequence shown in SEQ ID NO:21, and a donor sequence containing the nucleotide sequence shown in SEQ ID NO:30 into the host cell of step i), so that the GyrA expressed by the gyrA gene contains an H80A mutation at position 80 relative to SEQ ID NO:62.

[0078] In some embodiments, step i) may include introducing the Cas9 enzyme into the host cell of step i), as well as an sgRNA containing the nucleotide sequence shown in SEQ ID NO:22 and a donor sequence containing the nucleotide sequence shown in SEQ ID NO:31, so that the GyrB gene expressing GyrB contains the R136C mutation at position 136 relative to SEQ ID NO:63.

[0079] Step i) may further include knocking out the sbcC or sbcD gene in the host cell. In some embodiments, step i) may further include knocking out both the sbcC and sbcD genes in the host cell. In some embodiments, step i) may include introducing the Cas9 enzyme, an sgRNA comprising the nucleotide sequence shown in SEQ ID NO:17, and a donor sequence comprising the nucleotide sequence shown in SEQ ID NO:26 into the host cell of step i).

[0080] Step i) may also include knocking out the mcrB and mcrC genes in the host cell. In some embodiments, step i) may include introducing the Cas9 enzyme, an sgRNA comprising the nucleotide sequence shown in SEQ ID NO:14, and a donor sequence comprising the nucleotide sequence shown in SEQ ID NO:23 into the host cell of step i).

[0081] Step i) may also include knocking out the hsdM and hsdS genes in the host cell. In some embodiments, step i) may include introducing the Cas9 enzyme, sgRNA comprising the nucleotide sequence shown in SEQ ID NO:15, and a donor sequence comprising the nucleotide sequence shown in SEQ ID NO:24 into the host cell of step i).

[0082] Step i) may also include knocking out the mrr and hsdr genes in the host cell. In some embodiments, step i) may include introducing the Cas9 enzyme, an sgRNA comprising the nucleotide sequence shown in SEQ ID NO:16, and a donor sequence comprising the nucleotide sequence shown in SEQ ID NO:25 into the host cell of step i).

[0083] Step i) may further include genetically modifying the host cell so that the EndA gene expressed contains an E208K mutation at position 208 relative to SEQ ID NO:64. In some embodiments, step i) may include introducing the Cas9 enzyme, an sgRNA containing the nucleotide sequence shown in SEQ ID NO:18, and a donor sequence containing the nucleotide sequence shown in SEQ ID NO:27 into the host cell of step i).

[0084] Step i) may further include genetically modifying the host cell so that the RecA gene expressed contains a G161D mutation at position 161 relative to SEQ ID NO:65. In some embodiments, step i) may include introducing the Cas9 enzyme, an sgRNA comprising the nucleotide sequence shown in SEQ ID NO:19, and a donor sequence comprising the nucleotide sequence shown in SEQ ID NO:28 into the host cell of step i).

[0085] Step i) may further include genetically modifying the host cell so that the GalE gene expresses an S123F mutation at position 123 relative to SEQ ID NO:66. In some embodiments, step i) may include introducing the Cas9 enzyme, an sgRNA comprising the nucleotide sequence shown in SEQ ID NO:20, and a donor sequence comprising the nucleotide sequence shown in SEQ ID NO:29 into the host cell of step i).

[0086] In some embodiments, step i) may include knocking out the mcrB, mcrC, hsdM, hsdS, mrr, and hsdr genes of the host cell, and causing the EndA, RecA, and GalE genes of the host cell to express an E208K mutation at position 208 relative to SEQ ID NO:64, a G161D mutation at position 161 relative to SEQ ID NO:65, and an S123F mutation at position 123 relative to SEQ ID NO:66.

[0087] The EndA expressed by the genetically modified endA may contain the amino acid sequence shown in SEQ ID NO:64. The RecA expressed by the genetically modified recA may contain the amino acid sequence shown in SEQ ID NO:65. The GalE expressed by the genetically modified galE may contain the amino acid sequence shown in SEQ ID NO:66.

[0088] Step ii) may include introducing a vector into a host cell, which may contain a nucleic acid molecule containing a simple repeat sequence such as a poly(A) or poly(T) sequence, or a nucleic acid molecule containing a hairpin structure sequence such as an ITR or UTR. Alternatively, step ii) may include integrating a nucleic acid molecule containing a simple repeat sequence such as a poly(A) or poly(T) sequence, or a nucleic acid molecule containing a hairpin structure sequence such as an ITR or UTR, into the genome of the host cell.

[0089] Simple repeating sequences can include, but are not limited to, poly(A) sequences and poly(T) sequences. A poly(A) sequence can contain consecutive A bases, with the number of consecutive A bases being, for example, 20-250. A poly(A) sequence can contain 2-5 consecutive A base segments separated by non-A bases, wherein the number of consecutive A bases in each consecutive A base segment can be, for example, 10-100, and every two consecutive A base segments can be separated by, for example, 1-20 non-A bases. A poly(T) sequence can contain consecutive T bases, with the number of consecutive T bases being, for example, 20-250. A poly(T) sequence can contain 2-5 consecutive T base segments separated by non-T bases, wherein the number of consecutive T bases in each consecutive T base segment can be, for example, 10-100, and every two consecutive T base segments can be separated by, for example, 1-20 non-T bases.

[0090] Sequences containing hairpin structures may include, but are not limited to, ITRs and untranslated regions (UTRs), such as UTRs derived from RNA. In some embodiments, the ITR may contain the nucleotide sequence shown in SEQ ID NO:53.

[0091] Accordingly, this application also protects the use of genetically modified GyrA in host cells to express gyrA with an H80A mutation at position 80 relative to SEQ ID NO:62, or GyrB in host cells to express gyrB with an R136C mutation at position 136 relative to SEQ ID NO:63, and the use of knocking out sbcC and / or sbcD genes in host cells in the replication or expression of nucleic acid molecules containing simple repetitive sequences such as poly(A) or poly(T) sequences, or nucleic acid molecules containing hairpin structure sequences such as ITR or UTR.

[0092] Other features and advantages of the present disclosure will become clearer based on the following specific descriptions and embodiments, which should not be construed as limiting. All documents, Genbank records, patents, and published patent applications cited in this application are expressly included herein by reference. Attached Figure Description

[0093] The following detailed description, given by way of example but not intended to limit the invention to the specific embodiments described, can be better understood in conjunction with the accompanying drawings.

[0094] Figure 1 shows a schematic diagram of the AAV2-ITR hairpin structure.

[0095] Figures 2A and 2B show the maps of the multi-target plasmid (A) after inserting sgRNA and donor sequences and the pCas plasmid (B) for expressing the Cas9 protein.

[0096] Figures 3A-3J show the spectra of genITR-test 1F plasmid (A), genITR-test 2F plasmid (B), genITR-test 3F plasmid (C), genITR-test 4F plasmid (D), genITR-test 5F plasmid (E), genITR-test 6F plasmid (F), genITR-test 7F plasmid (G), genITR-test 8F plasmid (H), genITR-test 9 plasmid (I), and genITR-test 10 plasmid (J). In the AH spectra, AAV2-ITR in the circle represents the subsequently added 3'-ITR.

[0097] Figure 4 shows the spectrum of the poly(A)-test1 plasmid.

[0098] Figures 5A-5C show exemplary Sanger sequencing peak diagrams for poly(A) purity met (A), purity not met (B), and poly(A) deletion (C).

[0099] Figure 6 shows the growth curves of JM108[ΔsbcC-sbcD-gyrA(H80A)], NEB stable[ΔsbcC-sbcD-gyrA(H80A)], and MG1655[ΔsbcC-sbcD-gyrA(H80A)] transformed with plasmids.

[0100] Figures 7A and 7B show the plasm growth of JM108[ΔsbcC-sbcD-gyrA(H80A)], NEB stable[ΔsbcC-sbcD-gyrA(H80A)], and MG1655[ΔsbcC-sbcD-gyrA(H80A)] transformed with plasmids (A), MG1655[ΔsbcC-sbcD-gyrA(H80A)] and MG1655[endA-gale15-Δ(mrr-hsdRMS-mcrBC)-recA-ΔsbcC-sbcD-gyrA(H80A)] transformed with plasmids.

[0101] Figure 8 shows the SmaI and AhdI digestion results of plasmids replicated in NEB Stable and MG1655[endA-gale15-Δ(mrr-hsdRMS-mcrBC)-recA-ΔsbcC-sbcD-gyrA(H80A)]. Detailed Implementation

[0102] Unless otherwise specified, the terms used herein have their common meanings as found in dictionaries, textbooks, and technical reference books, or as commonly understood by those skilled in the art. The following descriptions of some terms are for the purpose of understanding this application only and are not intended to impose any particular limitations on these terms, unless otherwise specified.

[0103] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include the plural form of the object referred to, unless the context clearly specifies otherwise.

[0104] The term "or" refers to a single element among the listed selectable elements, unless the context explicitly indicates otherwise.

[0105] The terms “comprising” or “including” mean that the stated elements, integers, or steps are included, but do not exclude the inclusion of any other elements, integers, or steps. In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover combinations of the stated elements, integers, or steps.

[0106] In this article, "simple repeating sequence" refers to a sequence obtained by repeatedly repeating fragments of 1-3 nucleotides as a unit, especially single nucleotide repeating sequences such as poly(A) and poly(T) sequences.

[0107] A "poly(A) structure" or "poly(A) sequence" refers to a nucleotide sequence containing consecutive repeating A bases, as well as a nucleotide sequence in which several consecutive A base segments are separated by non-A bases. Similarly, a "poly(T) structure" or "poly(T) sequence" refers to a nucleotide sequence containing consecutive repeating T bases, as well as a nucleotide sequence in which several consecutive T base segments are separated by non-T bases.

[0108] In this article, "hairpin structure sequence" refers to any sequence capable of forming a hairpin structure, such as the inverted terminal repeat (ITR) sequence derived from adenovirus, and untranslated regions (UTRs) on mRNA structures such as 5'-UTR and 3'-UTR.

[0109] "Host cell" and "strain" both refer to any cell into which a foreign nucleic acid molecule (such as a vector) can be introduced and which can be replicated or expressed. The host cell in this application can be a prokaryotic cell, such as *Escherichia coli*. "*Escherichia coli*" and "E. coli" are used interchangeably, including wild-type *E. coli* and *E. coli* strains containing artificial or natural mutations, such as JM108, NEB Stable, Top10, DH5α, or DH10B strains.

[0110] "Vector preparation / production strains" refer to strains that have good characteristics in vector replication or expression and are usually commercially available.

[0111] "Recombinant" cells refer to cells obtained by altering gene sequences, gene expression patterns, and gene expression levels through DNA recombination technology. "Recombinant" host cells refer to cells obtained by altering gene sequences, gene expression patterns, and gene expression levels through DNA recombination technology, which can introduce foreign nucleic acid molecules and enable the foreign nucleic acid molecules to replicate or be expressed.

[0112] Helicase, also known as gyrase or rotational enzyme, is a type of helicase, specifically belonging to type II topoisomerases. This enzyme can introduce (-) supercoils into DNA and convert (+) supercoils into (-) supercoils, participating in important processes such as replication, transcription, repair, and recombination in prokaryotic cells. Helicase is composed of GyrA and GyrB subunits. GyrA is mainly responsible for forming and closing nicks in the DNA double helix, while the GyrB subunit mainly mediates energy transfer and ATP hydrolysis. GyrA is encoded by the gyrA gene, and GyrB is encoded by the gyrB gene.

[0113] In this paper, "stability" refers to the structural (base) integrity of nucleic acid sequences such as poly(A), poly(T), ITR, and UTR, which remains unchanged, essentially unchanged, or undergoes minimal change during replication or expression in the strain. "Integrity" means that the replicated or expressed nucleic acid sequences such as poly(A), poly(T), ITR, and UTR in the strain are consistent or essentially identical to the sequences initially constructed and transformed into the strain, with no base changes or losses, or minimal base changes or losses.

[0114] "Importation" refers to introducing a vector into a host cell using certain methods, such as transfection, transduction, or transformation. "Integration" refers to embedding a specific sequence into the genome using certain methods, such as homologous recombination, so that it can be expressed along with the genome sequence.

[0115] A "vector" is a naturally occurring or synthetic DNA or RNA fragment used to carry or contain nucleic acid sequences for the introduction of these sequences into a host cell and, optionally, for expression within the host cell. Examples include chemically synthesized DNA fragments, natural plasmids, modified viral genomes, or RNA. A foreign DNA fragment may be inserted into the vector for the cloning and / or expression of that foreign DNA fragment. Vectors may contain, for example, origins of replication, selectivity markers or reporter genes, multiple cloning sites (MCS), etc. The term includes linear DNA fragments (such as PCR products, linearized plasmid fragments, etc.), plasmid vectors, viral vectors, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), etc. When the vector is double-stranded DNA, the description of the element sequence order and orientation refers to one strand of DNA.

[0116] In this article, "replication" refers to the amplification of nucleic acid sequences, nucleic acid molecules, or vectors, i.e., an increase in copy number; while "expression" refers to the transcription and / or translation of nucleic acid sequences or nucleic acid molecules. "Replication stability" refers to the consistency or near-consistency between the original nucleic acid sequence and the newly amplified nucleic acid sequence during the amplification process; "expression stability" refers to the integrity or near-integrity of the nucleic acid sequence during transcription or translation.

[0117] In this article, "gene mutation" refers to a heritable change in the DNA sequence, resulting in changes in the sequence or even structure of the expressed RNA or protein. For example, in this article, the mutation of the gyrA gene causes the H80A mutation in the GyrA protein, leading to a decrease in gyrase activity. "Gene modification" refers to using certain methods, such as gene editing and homologous recombination, to cause a heritable change in the DNA sequence, resulting in changes in the sequence or even structure of the expressed RNA or protein. In this article, the substitution of amino acid residues uses the following naming convention: initial amino acid, position, substituted amino acid. As an example, H80A indicates that at position H80 relative to the original protein sequence, histidine (H) is replaced with alanine (A). When multiple amino acid residues are substituted, they are separated by commas (,"), spaces, or hyphens ("-"), such as A569T and T586A.

[0118] In this article, gene "knockout" refers to the targeted integration of a foreign gene into a specific site on the genome of a target cell through methods such as homologous recombination, so that the gene at that site cannot be expressed, i.e., no RNA or protein is produced, denoted by "Δ". For example, ΔsbcC indicates the knockout of the sbcC gene. When knocking out multiple genes, the knocked-out genes can be connected by hyphens ("-"), such as ΔsbcC-sbcD indicating that both sbcC and sbcD are knocked out, and Δ(mrr-hsdRMS-mcrBC) indicating that mrr, hsdR, hsdM, hsdS, mcrB, and mcrC are all knocked out.

[0119] In host cells used for exogenous gene expression, such as Escherichia coli, some complex exogenous gene structures and sequences, such as hairpin structures (like ITRs) and simple sequence repetitions (like poly(A)), gradually lose their integrity during the replication and expression of the exogenous gene, thus affecting the application of the replicated or expressed exogenous gene.

[0120] For example, the palindromic sequence of AAV-ITR is highly stable and has a high GC content, making it prone to mutation or recombination loss during bacterial replication. However, the integrity of the ITR is crucial for efficient rAAV replication and packaging; ideally, the ITR deletion should be less than 20%. The impact of ITR deletion on rAAV viral packaging and its application in gene therapy is highly complex and can produce unintended biological consequences through vector-host interactions. Using incomplete ITR sequences in the preclinical stage reduces the reproducibility of experiments within and across laboratories and may prevent the achievement of previous effects when using complete ITR sequences in clinical trials.

[0121] For example, RNA drugs, such as RNA vaccines, often contain a non-coding region (UTR) and a 3' poly(A) tail, which are prone to losing their integrity during replication and expression in host cells. The UTR is crucial for RNA translation, while the poly(A) tail plays a vital role in maintaining mRNA stability, regulating translation efficiency, and ensuring mRNA transport.

[0122] Currently, there is no good method to avoid ITR deletion during rAAV-ITR plasmid construction and E. coli amplification. Some recombination-deficient commercial strains, such as JC8111, SURE2, Stabl3, and XL10-Gold, are frequently used for the construction and amplification of plasmids containing ITR structures, but this is not always effective. Compared to wild-type E. coli strains, these strains typically grow slowly and have poorer resistance, resulting in low plasmid yields. Using these strains as host bacteria for plasmid cloning often leads to prolonged production cycles. Knockout of the sbcC and sbcD genes in host cells significantly increases the number of recombinant clones containing complete hairpin structures, but still fails to achieve sufficiently high ITR integrity. Especially when the length of the A-A' region inverted repeat sequence exceeds 30 bp, significant deletion is observed even in ΔsbcC-sbcD mutant strains.

[0123] To improve the stability of poly(A) sequences on plasmids, some scientists have employed a segmented poly(A) sequence co-transcription method. Since each poly(A) binding protein, PABP, binds only about 30 A bases, and a small number of A bases between two binding proteins act as spacers, replacing these spacers with other non-A bases reduces the probability of recombination in the poly(A) tail coding sequence. Furthermore, the length and base composition of the spacer sequence can be optimized to further reduce the risk of poly(A) tail deletion. However, even with these optimizations, the risk of A deletion still exists, and segmented preparation of poly(A) plasmids can affect the expression levels of downstream proteins. Additionally, there are reports that low-temperature (30°C) fermentation can improve the integrity of the poly(A) tail coding sequence in plasmids. However, low-temperature culture slows bacterial growth, reduces plasmid copy number, significantly decreases yield, and prolongs the production cycle, posing challenges to plasmid production.

[0124] The inventors of this application have greatly improved the replication and expression stability of ITR and poly(A) sequences in Escherichia coli host cells by mutating the subunits GyrA or GyrB of the helicase.

[0125] Helicases are important DNA helicases in prokaryotic cells, participating in processes such as DNA replication, repair, recombination, and transcription. Drugs that target helicases and inhibit their activity, such as quinolones and coumarins, are used as antibacterial agents, causing irreversible damage to bacterial DNA. Given the important roles of GyrA and GyrB, downregulating the activity of GyrA, GyrB, or helicases composed of them in bacterial strains for production purposes, except for gene or protein research, is unlikely, as such downregulation would likely lead to strain death. During the evolution of some strains, the GyrA96 mutation has developed to resist the inhibitory effect of the quinolone nalidixic acid on helicases. However, no researchers have found any association between this mutation, or other mutations in helicases, and the replication and expression stability of the ITR and poly(A) sequence.

[0126] The inventors of this application tested reported GyrA and GyrB mutations that may reduce gyrase activity and found that not all mutations significantly improve the replication and expression stability of the ITR and poly(A) sequences. The H80A mutation in GyrA and the R136C mutation in GyrB significantly improve the stability of the ITR and poly(A) sequences in *E. coli* host cells, including replication and expression stability. After multiple passages in host cells, the sequence integrity of the ITR and poly(A) remained at a high level, far exceeding that of commercially prepared vectors without GyrA or GyrB mutations, such as JM108, MG1655, and NEB stable. In the JM108 strain, there was originally a GyrA96 mutation resistant to the quinolone nalidixic acid. Further inclusion of the H80A mutation in GyrA significantly improved the replication stability of the ITR and poly(A) (see, for example, Example 3). Furthermore, the H80A mutation in GyrA is more effective than the R136C mutation in GyrB in stabilizing ITR and poly(A).

[0127] When the gene for the sbcC-sbcD nuclease, which can cleave DNA hairpin structures, is knocked out, the replication / expression stability of the ITR and poly(A) sequences in the host cell is further improved, indicating that the effects of sbcC-sbcD gene knockout and GyrA(H80A) mutation can be superimposed.

[0128] However, genetic modification of host cells may lead to problems such as low transformation efficiency of exogenous nucleic acid sequences (i.e., increased difficulty for exogenous nucleic acid sequences, such as plasmids containing hairpin structures or simple repetitive sequences, to enter cells) and slow amplification rates of plasmid vectors. Alternatively, some strains, such as MG1655, may inherently have these problems.

[0129] Conventionally, knocking out genes in a strain that defend against foreign nucleic acids can improve the strain's nucleic acid conversion efficiency.

[0130] The mrr-hsdRMS-mcrBC system constitutes the restriction system of *E. coli*. The restriction endonucleases it produces can cleave exogenous DNA, promoting further degradation of the exogenous DNA by intracellular nucleases. Knocking out the restriction system in *E. coli* can reduce defense against exogenous DNA, thus promoting the stable existence of circular DNA within the cell.

[0131] endA expresses DNA-specific endonuclease I, which is located in the intermembrane space of the cell wall and can untie all DNA double strands, acting as a "gatekeeper" for cells against foreign DNA. Knocking out / mutating this gene facilitates the passage of circular or linear DNA across the cell membrane into the cell.

[0132] galE expresses uridine diphosphate (UDG) galactose-4-epimerase. Mutations in this gene prevent the production of UDG galactose, resulting in the lipopolysaccharide (LPS) in the cell wall being truncated to the smallest "core," thereby reducing the intervention of foreign DNA uptake.

[0133] The recA gene expresses an ATP-dependent DNA recombinase, which plays a role in the lysogenic recombination of λ-phage with genomic DNA and also has a repair function for DNA radioactive damage. Genotypes resulting from variations in the recA gene can prevent homologous or heterologous DNA recombination, maintain the stability of the inserted DNA, and are beneficial for DNA transformation.

[0134] The mutations in the above genes can improve the transformation efficiency of the strain DNA to a certain extent and reduce the production of multimer structures in plasmids.

[0135] Therefore, based on the sbcC-sbcD gene knockout and GyrA(H80A) mutation, the inventors performed gene knockouts of mcrB, mcrC, hsdM, hsdS, mrr, and hsdr, and gene mutations of endA, recA, and galE in E. coli host cells. This operation successfully improved the transformation efficiency of exogenous nucleic acid sequences and the amplification rate of plasmid vectors, without adversely affecting the replication / expression stability of host cells for ITR or poly(A). For example, compared with strains MG1655 and MG1655[ΔsbcC-sbcD-gyrA(H80A)], strain MG1655[endA-gale15-Δ(mrr-hsdRMS-mcrBC)-recA-ΔsbcC-sbcD-gyrA(H80A)] showed an increased number of long spots and a significantly improved transformation efficiency. In the MG1655[endA-gale15-Δ(mrr-hsdRMS-mcrBC)-recA-ΔsbcC-sbcD-gyrA(H80A)] gene mutant strains, the corresponding poly(A) passaging cloning success rate was higher than that of the commercial strain NEB, regardless of whether it was the first or tenth generation. Stable. Compared with NEB Stable strain, MG1655[endA-gale15-Δ(mrr-hsdRMS-mcrBC)-recA-ΔsbcC-sbcD-gyrA(H80A)] showed a 108% and 107% increase in plasmid yield, respectively. The integrity of the ITR in MG1655[endA-gale15-Δ(mrr-hsdRMS-mcrBC)-recA-ΔsbcC-sbcD-gyrA(H80A)] was significantly higher than that in commercial NEB Stable strain.

[0136] Furthermore, the replication / expression stability of ITR and poly(A) of MG1655[endA-gale15-Δ(mrr-hsdRMS-mcrBC)-recA-ΔsbcC-sbcD-gyrA(H80A)] is higher than that of MG1655[endA-gale15-Δ(mrr-hsdRMS-mcrBC)-recA-ΔsbcC-sbcD] strain, which again shows that the effects of sbcC-sbcD gene knockout and GyrA(H80A) mutation can be additive, even if the host cell has further gene modifications.

[0137] Therefore, this application mainly provides a method for replicating or expressing nucleic acid molecules containing simple repetitive sequences or nucleic acid molecules containing hairpin structure sequences in Escherichia coli host cells, including:

[0138] i) Provide an *E. coli* host cell, which may contain the gyrA gene and the gyrB gene, wherein the gyrA gene expressing GyrA may contain an H80A mutation at position 80 relative to SEQ ID NO:62, or the gyrB gene expressing GyrB may contain an R136C mutation at position 136 relative to SEQ ID NO:63.

[0139] ii) Introducing nucleic acid molecules containing simple repeat sequences (such as poly(A) or poly(T) sequences) or hairpin structure sequences (such as ITR or UTR) into host cells, and

[0140] iii) Culture recombinant host cells under conditions that facilitate the replication or expression of the nucleic acid molecules introduced in step ii) (i.e., nucleic acid molecules containing simple repetitive sequences such as poly(A) or poly(T) sequences, or nucleic acid molecules containing hairpin structure sequences such as ITR or UTR).

[0141] The host cell in step i) can be an Escherichia coli vector strain, including, but not limited to, MG1655, JM108, NEB Stable, Top10, DH5α, and DH10B. In particular, the host cell can be MG1655, JM108, and NEB Stable.

[0142] The GyrA expressed by the gyrA gene may contain the amino acid sequence shown in SEQ ID NO:62.

[0143] The GyrB gene expressed can contain the amino acid sequence shown in SEQ ID NO:63.

[0144] The host cell can also contain gene knockouts of sbcC and sbcD.

[0145] The host cell may also i) contain the endA, recA, and galE genes, wherein the EndA, RecA, and GalE expressed by the endA, recA, and galE genes may contain the E208K mutation at position 208 relative to SEQ ID NO:64, the G161D mutation at position 161 relative to SEQ ID NO:65, and the S123F mutation at position 123 relative to SEQ ID NO:66, and / or ii) contain gene knockout of mcrB, mcrC, hsdM, hsdS, mrr, and hsdr.

[0146] The host cell in step i) can be a natural mutant or obtained through artificial genetic modification.

[0147] In some embodiments, the host cell in step i) may be obtained through artificial genetic modification. This application uses gene editing techniques to mutate and knock out genes in the host cell; those skilled in the art can also use other known methods to achieve the same effect.

[0148] Step ii) may include introducing a vector into the host cell, which may contain a nucleic acid molecule containing a simple repetitive sequence such as a poly(A) or poly(T) sequence, or a nucleic acid molecule containing a hairpin sequence such as an ITR or UTR. Alternatively, step ii) may include integrating a nucleic acid molecule containing a simple repetitive sequence such as a poly(A) or poly(T) sequence, or a nucleic acid molecule containing a hairpin sequence such as an ITR or UTR, into the genome of the host cell. In some embodiments, the host cell in step i) is artificially genetically modified. In this case, step ii) may be performed before genetically modifying the host cell. That is, step ii) may be performed first, such that the cell contains a nucleic acid molecule for expressing a simple repetitive sequence such as a poly(A) or poly(T) sequence, or a nucleic acid molecule containing a hairpin sequence such as an ITR or UTR, before genetic modification of the host cell into gyrA, gyrB, sbcC, sbcD, mcrB, mcrC, hsdM, hsdS, mrr, hsdr, endA, recA, and / or galE.

[0149] Simple repeating sequences can include, but are not limited to, poly(A) sequences and poly(T) sequences.

[0150] Sequences containing hairpin structures may include, but are not limited to, ITR and untranslated region (UTR) sequences, such as UTRs derived from RNA.

[0151] This application also protects host cells, naturally occurring or genetically modified, that may contain the gyrA and gyrB genes. The gyrA gene expressing GyrA may contain an H80A mutation at position 80 relative to SEQ ID NO:62. Alternatively, the gyrB gene expressing GyrB may contain an R136C mutation at position 136 relative to SEQ ID NO:63. In some embodiments, the host cell may contain both the gyrA and gyrB genes, wherein the gyrA gene expressing GyrA may contain an H80A mutation at position 80 relative to SEQ ID NO:62. In some embodiments, the host cell may contain both the gyrA and gyrB genes, wherein the gyrB gene expressing GyrB may contain an R136C mutation at position 136 relative to SEQ ID NO:63. In some embodiments, the host cell may contain gyrA and gyrB genes, wherein the gyrA gene expressing GyrA may contain an H80A mutation at position 80 relative to SEQ ID NO:62, and the gyrB gene expressing GyrB may contain an R136C mutation at position 136 relative to SEQ ID NO:63.

[0152] The host cell may also contain the SbcC or SbcD gene knockout. In some embodiments, the host cell may contain both SbcC and SbcD gene knockouts.

[0153] In some embodiments, this application protects an Escherichia coli host cell comprising the gyrA gene and the gyrB gene, wherein the gyrA gene expresses GyrA containing an H80A mutation at position 80 relative to SEQ ID NO:62, or the gyrB gene expresses GyrB containing an R136C mutation at position 136 relative to SEQ ID NO:63, wherein the Escherichia coli host cell further comprises a gene knockout of SbcC or SbcD.

[0154] In some embodiments, the GyrA expressed by the host cell gyrA gene contains the amino acid sequence shown in SEQ ID NO:62, and the GyrB expressed by the gyrB gene contains the amino acid sequence shown in SEQ ID NO:63.

[0155] In some embodiments, the host cell is genetically modified such that the expressed GyrA contains an H80A mutation at position 80 relative to SEQ ID NO:62, or is genetically modified such that the expressed GyrB contains an R136C mutation at position 136 relative to SEQ ID NO:63.

[0156] The host cell may also i) contain the knockout of genes mcrB, mcrC, hsdM, hsdS, mrr, and hsdr, and / or ii) contain the endA, recA, and galE genes, wherein the EndA, RecA, and GalE expressed by endA, recA, and galE may contain an E208K mutation at position 208 relative to SEQ ID NO:64, a G161D mutation at position 161 relative to SEQ ID NO:65, and an S123F mutation at position 123 relative to SEQ ID NO:66, respectively. The EndA expressed by the endA gene may contain the amino acid sequence shown in SEQ ID NO:64. The RecA expressed by the recA gene may contain the amino acid sequence shown in SEQ ID NO:65. The GalE expressed by the galE gene may contain the amino acid sequence shown in SEQ ID NO:66.

[0157] The host cells used in this application can be naturally occurring or genetically modified, both of which are within the scope of protection of this application. In some embodiments, the host cells are obtained through artificial genetic modification. This modification can be achieved through gene mutation, resulting in mutant proteins. For the gene mutations common in these *E. coli* host cells, those skilled in the art can also perform similar modifications at other sites. For common gene knockouts, those skilled in the art may also achieve similar effects through knockdown.

[0158] This application uses gene editing techniques to mutate and knock out genes in host cells. Those skilled in the art can also use other known methods to achieve the same effect.

[0159] This application also protects the use of these host cells in replicating or expressing nucleic acid molecules containing simple repetitive sequences or nucleic acid molecules containing hairpin structure sequences.

[0160] The following detailed description, in conjunction with specific embodiments, further illustrates the content of this application. These embodiments are given by way of example only and do not limit the scope of protection of this application.

[0161] Example 1. Construction of strain mutants

[0162] Using CRISPR-Cas9 technology, relevant genes were knocked out or mutated in the genomes of *Escherichia coli* strains MG1655, JM108, and NEB stable. Knockout genes included mcrB (SEQ ID NO:1), mcrC (SEQ ID NO:2), hsdM (SEQ ID NO:3), hsdS (SEQ ID NO:4), mrr (SEQ ID NO:5), hsdr (SEQ ID NO:6), sbcC (SEQ ID NO:7), and sbcD (SEQ ID NO:8). Mutated genes included endA, RecA, galE, gyrA, and gyrB. Specifically, endA is mutated to endA1 (C513G, G622A) (SEQ ID NO:9) to encode EndA (E208K) (SEQ ID NO:64); RecA is mutated to RecA1 (G482A, C498T) (SEQ ID NO:10) to encode RecA (G161D) (SEQ ID NO:65); gale is mutated to gale15 (C368T, C483G) (SEQ ID NO:11) to encode GalE (S123F) (SEQ ID NO:66); gyrA is mutated to gyrA (G81T, CAT238-240GCA) (SEQ ID NO:12) to encode GyrA (H80A) (SEQ ID NO:62); and gyrB is mutated to gyrB (G270C, C406T) (SEQ ID NO:13) to encode GyrB (R136C) (SEQ ID NO:64). NO:63).

[0163] The specific steps are as follows.

[0164] 1.1.1 Upload the gene sequence to be knocked out / mutated to the sgRNA design website and design an sgRNA with a length of 20bp, as shown in SEQ ID NO:14-22.

[0165] 1.1.2 The above sgRNA and the corresponding donor sequence SEQ ID NO:23-31 were constructed on an all-in-one target plasmid. The map of the constructed plasmid is shown in Figure 2A.

[0166] 1.1.3 The pCas plasmid expressing the Cas9 protein (plasmid map shown in Figure 2B) was transformed into chemically competent wild-type strains MG1655 (CCTCC NO: M 20241016), NEB Stable, and JM108 using a chemical transformation method. The transformed cells were plated onto LB solid kanamycin-resistant plates and incubated at 30°C for 14 h. Single colonies were picked, and positive clones were obtained by colony PCR using primers SEQ ID NO:32-33 and Sanger sequencing.

[0167] 1.1.4 As needed, the target plasmid constructed in 1.1.2 was electroporated into MG1655, NEB Stable, and JM108 competent cells carrying the pCas plasmid. The cells were transformed into plates, plated onto LB solid kanamycin and spectinomycin double antibody plates, inverted, and incubated at 30°C for 14 h. Single colonies were picked, and positive clones were identified by colony PCR and Sanger sequencing. The primers used were selected from SEQ ID NO:34-51, and successfully edited positive clones were obtained.

[0168] 1.1.5 Add 0.5 mM IPTG inducer to 4 mL of LB tube containing the positive clone culture to the successfully edited positive clone and incubate at 30 °C for 7-8 h to eliminate the target plasmid for spectinomycin resistance.

[0169] 1.1.6 Place 4 mL of LB tube containing positive clone bacterial solution in 37℃ for 7-8 h to eliminate kanamycin-resistant pCas plasmids.

[0170] 1.1.7 Prepare competent cells from the obtained strain for later use.

[0171] When preparing strains with multiple knockouts / mutations of the above genes, follow the procedures outlined in 1.1.3-1.1.4-1.1.5-(1.1.4-1.1.5). nThe operation is performed in the order of 1.1.6-1.1.7, where n depends on the types of gene knockout / mutation to be included, and n is greater than or equal to 1. That is, first, the pCas plasmid is introduced in 1.1.3, then the target plasmid containing one sgRNA and donor sequence is introduced in 1.1.4, and a positive clone strain with successful editing is obtained by screening. The target plasmid is removed from the strain in 1.1.5, and the strain is prepared as a competent state. Then, 1.1.4 is repeated, and a target plasmid containing another sgRNA and donor sequence is introduced. A positive clone strain with successful editing is obtained by screening. At this point, depending on whether a third target plasmid needs to be introduced, the target plasmid is removed from the strain in 1.1.5, and the strain is prepared as a competent state, and 1.1.4 is repeated, or the order of 1.1.5-1.1.7 is followed. For example, when preparing a strain containing the knockout of mcrB-mcrC and the mutant gyrA (H80A), first introduce a target plasmid containing the sgRNA corresponding to the knockout of mcrB-mcrC and the donor sequence in 1.1.4, then remove the target plasmid from the strain in 1.1.5, and prepare the strain as competent cells. Then, return to 1.1.4, introduce a target plasmid containing the sgRNA corresponding to the mutant gyrA (H80A) and the donor sequence, and then proceed according to 1.1.5-1.1.7.

[0172] Table 1. Primer sequences for colony PCR

[0173] The strain variants constructed in this application are shown in Table 2.

[0174] Table 2. Strain variants constructed in this application

[0175] Example 2. Preparation of plasmids containing ITR or poly(A) sequences

[0176] Ten test plasmids containing wild-type AAV2-ITR sequences derived from the adeno-associated virus (AAV) genome were constructed.

[0177] Among them, plasmids genITR-test1, genITR-test2, genITR-test3, genITR-test4, genITR-test5, genITR-test6, genITR-test7, and genITR-test8 contain only a single ITR, namely the 5'-ITR (SEQ ID NO:52). The plasmid sequences of genITR-test1 and genITR-test2 are shown in SEQ ID NO:54 and 55, respectively. Approximately 50 ng of each of these eight plasmids was synthesized and transformed into 100 μL of JM108[ΔsbcC-sbcD] competent cells. The cells were then placed on ice for 30 min, followed by heat shock at 42°C for 90 s, and then on ice for 3 min. 800 μL of LB medium was added, and the cells were incubated at 37°C with shaking for 45 min. 100 μL of the recovered medium was then spread onto LB solid antibiotic plates, which were then inverted and incubated overnight at 37°C. Four clones were selected from each of the transformed plates, cultured in a shake culture, and plasmids were extracted and sequenced using Sanger sequencing to obtain the correctly sequenced ITR test plasmids. Before further testing, the genITR-test2 plasmid was digested with BsmBI, and the remaining seven plasmids were digested with AarI. The wild-type AAV2 3'-ITR sequence (SEQ ID NO:53) synthesized by annealing was ligated to the above eight AarI / BsmBI linearized vectors using T4 enzyme to obtain 20 μl of reaction product. The correctly ligated clone contains two complete AAV2-ITR sequences. The plasmid maps are shown in Figures 3A-3H, and are respectively named genITR-test1F, genITR-test2F, genITR-test3F, genITR-test4F, genITR-test5F, genITR-test6F, genITR-test7F, and genITR-test8F. The AAV2-ITR highlighted in the box is the subsequently ligated 3'-ITR (SEQ ID NO:53).

[0178] genITR-test 9 and genITR-test 10 contain 5'-ITR (SEQ ID NO: 52) and 3'-ITR sequences (SEQ ID NO: 53). The plasmid maps are shown in Figures 3I-3J, and the plasmid sequences are shown in SEQ ID NO: 56 and 57. Approximately 50 ng of each plasmid was synthesized and transformed into 100 μL of JM108[ΔsbcC-sbcD] competent cells. The cells were then placed on ice for 30 min, followed by heat shock at 42°C for 90 s, and then on ice for 3 min. 800 μL of LB medium was added, and the cells were incubated at 37°C with shaking for 45 min. 100 μL of the recovered medium was spread onto LB solid antibiotic plates, and the plates were inverted and incubated overnight at 37°C. Four clones from each transformed plate were selected for shaking culture, plasmid extraction was performed, and Sanger sequencing was conducted to obtain the correctly sequenced test plasmids.

[0179] In addition, five test plasmids containing consecutive 80 A's, 100 A's, 120 A's, or segmented poly(A) sequences (SEQ ID NO: 58 and 59) were prepared: poly(A)-test 1 (80A), poly(A)-test 2 (100A), poly(A)-test 3 (120A), poly(A)-test 4 (30 & 70A), and poly(A)-test 5 (30 & 30 & 43A). The map of poly(A)-test 1 (80A) plasmid is shown in Figure 4, where the sequence encoding the mRNA portion is as shown in SEQ ID NO: 60. Except for the different poly(A) sequences, the maps of the other test plasmids are the same as in Figure 4, and except for the different poly(A) sequences, the sequences encoding the mRNA portion are the same as in SEQ ID NO: 60. Specifically, nucleotide sequences containing the T7 promoter, 5'UTR, EGFP-encoding sequence, 3'UTR, poly(A) sequence, and linearization site BspQI, such as SEQ ID NO:60 (GenScript Biotechnology Co., Ltd.), were synthesized and assembled into the pVAX1 kanamycin resistance vector (GenScript Biotechnology Co., Ltd., SEQ ID NO:61) using the Gibson method. Positive clones were selected for Sanger sequencing, yielding five correctly sequenced poly(A) test plasmids.

[0180] Example 3. gyrA gene mutation improves the stability of ITR replication in Escherichia coli.

[0181] Take 8 μL of the ligation products of the eight 5'-ITR-containing restriction plasmids obtained in Example 2 and the wild-type AAV2 3'-ITR, and transform them into 100 μL of the gyrA gene mutant strains (MG1655[gyrA(H80A)], JM108[gyrA(H80A)] and NEB stable[gyrA(H80A)]), gyrB gene mutant strains (MG1655[gyrB(R136C)], JM108[gyrB(R136C)] and NEB stable[gyrB(R136C)]) and the corresponding unmutated commercial strains (MG1655, NEB Stable and JM108) obtained in Example 1, which were prepared as competent cells. Eight clones were picked from each transformed plate, for a total of 576 clones. They were cultured overnight in LB liquid medium at 37°C and 220 rpm. Plasmids were extracted from the overnight culture and the integrity of the ITR was analyzed by SmaI and AhdI restriction enzyme digestion.

[0182] Both the 5'-ITR and 3'-ITR sequences of the plasmid contain SmaI and AhdI restriction sites. Therefore, the integrity of the ITR structure can be determined by whether SmaI or AhdI digestion cleaves the plasmid. Add 300 ng of plasmid and 1 μL of SmaI or AhdI enzyme to a 20 μL reaction volume and incubate at 37°C for 60 min. The presence of undigested bands on the electrophoresis gel after digestion indicates incomplete digestion. The proportion of the brightness of this incompletely digested band relative to the total brightness of the bands in that lane represents the proportion of the ITR-deficient plasmid in the total plasmid. When the proportion of the incompletely digested band is <15%, it indicates that the proportion of the intact ITR plasmid is >85%, defined as ITR integrity >85%.

[0183] Specifically, plasmids were digested with SmaI and AhdI, respectively. The size of the digested fragments was verified by DNA gel electrophoresis. The proportions of fragments incompletely digested by SmaI and AhdI were analyzed using Gel Image System (GIS) software. A digestion rate of less than 15% incomplete digestion was considered successful; otherwise, it was considered unsuccessful. The SmaI and AhdI digestion success rates were statistically analyzed for all clones of each cell type. The SmaI and AhdI digestion success rates can be used to assess the stability of ITR replication in different mutant host cells. That is, a higher SmaI and AhdI digestion success rate indicates higher stability of the mutant host in replicating the ITR structure.

[0184] Table 3 summarizes the ITR integrity data of all clones of the 8 test plasmids in 9 strains. Enzyme digestion results showed that the commercial strain JM108 had a SmaI digestion pass rate of 45.1% and an AhdI digestion pass rate of 34.6%. In contrast, the JM108 strain with the mutated gyrA gene had a SmaI digestion pass rate of 67.7% and an AhdI digestion pass rate of 48.6%. This indicates that compared to JM108, the JM108 [gyrA(H80A)] strain showed a 22.6% increase in SmaI digestion pass rate and a 14% increase in AhdI digestion pass rate. Similarly, the JM108 [gyrB(R136C)] strain with the mutated gyrB gene showed a 17.4% increase in SmaI digestion pass rate and a 9.2% increase in AhdI digestion pass rate compared to JM108. Similarly, MG1655 and NEB stable strains with mutated gyrA or gyrB genes showed improved SmaI and AhdI digestion rates compared to their unmutated commercial strains. Furthermore, the SmaI and AhdI digestion rates were higher in strains with mutated gyrA genes than in strains with mutated gyrB genes. Overall, the ITR integrity of strains with mutated gyrA or gyrB genes was higher than that of unmutated strains, with the ITR integrity of strains with mutated gyrA genes being higher than that of strains with mutated gyrB genes. This indicates that mutated E. coli with either gyrA or gyrB genes have a positive effect on the replication stability of ITR plasmids, and that gyrA genes play a greater role in maintaining stable ITR replication compared to gyrB genes.

[0185] Table 3. Stability of ITR plasmids in commercial and mutant strains

[0186] Example 4. The combination of gyrA gene mutation and sbcC and sbcD gene knockout improves the stability of ITR or poly(A) replication in E. coli.

[0187] Take 8 μL of the ligation products of the eight plasmids containing only 5'-ITR obtained in Example 2 and wild-type AAV2 3'-ITR, and transform them into 100 μL of the competent strains obtained in Example 1 that have knocked out the sbcC and sbcD genes (JM108[ΔsbcC-sbcD], NEB stable[ΔsbcC-sbcD], MG1655[ΔsbcC-sbcD]) and the strains that have knocked out the sbcC and sbcD genes and have mutated the gyrA gene (JM108[ΔsbcC-sbcD-gyrA(H80A)], NEB stable[ΔsbcC-sbcD-gyrA(H80A)], MG1655[ΔsbcC-sbcD-gyrA(H80A)]). Following the procedures in Example 3, spot picking and bacterial culture were performed, plasmid extraction was carried out, and the 384 clones obtained were digested with SmaI and AhdI enzymes, respectively.

[0188] For GMP or clinical testing-grade plasmids, there are higher standards for plasmid integrity. Therefore, based on the statistical analysis of the proportion of clones with incomplete SmaI and AhdI digestion rates <15% for each host cell type, we further statistically analyzed the proportion of clones with incomplete SmaI digestion rates <10% and the proportion of clones with incomplete AhdI digestion rates <10%.

[0189] Table 4 summarizes the ITR integrity data of all clones of the eight test plasmids in six strains. It can be seen that for strain JM108[ΔsbcC-sbcD], which only knocked out the sbcC and sbcD genes, the pass rates for SmaI and AhdI incomplete digestion ratios <15% were 71.4% and 67.3%, respectively. For strain JM108[ΔsbcC-sbcD-gyrA(H80A)], the pass rates for SmaI and AhdI incomplete digestion ratios <15% were further improved to 87.7% and 89.9%, respectively. It is particularly noteworthy that when the incomplete digestion ratio standard was increased from <15% to <10%, strain JM108[ΔsbcC-sbcD-gyrA(H80A)] showed a greater advantage in ITR plasmid replication stability compared to JM108[ΔsbcC-sbcD]. Similarly, similar results were observed in NEB stable and MG1655 strains, where the SmaI / AhdI digestion pass rate of strains with additional mutations in the gyrA gene, especially those with a SmaI incomplete digestion band ratio of <10%, was better than that of strains that only knocked out sbcC and sbcD.

[0190] The data above indicate that mutating the gyrA gene based on the knockout of the sbcC and sbcD genes can further improve the stability of ITR plasmid replication. Especially when high plasmid integrity is required, this genotype can significantly improve the plasmid production success rate.

[0191] Table 4. Stability of ITR plasmids in commercial and mutant strains

[0192] In addition, 1 μl of the five poly(A) test plasmids obtained in Example 2 were transformed into 100 μl of competent gyrA gene mutant combinations sbcC and sbcD gene knockout strains (NEB stable [ΔsbcC-sbcD-gyrA(H80A)], MG1655 [ΔsbcC-sbcD-gyrA(H80A)], MG1655 [ΔsbcC-sbcD-gyrA(H80A)]) and their corresponding commercial strains (JM108, NEB Stable, MG1655). Cells were incubated on ice for 15 min, heat-shocked at 42°C for 45 s, and then incubated on ice for 3 min. Afterwards, 800 μl of LB liquid medium was added, and the cells were revived at 37°C and 220 rpm for 45 min. 10 μl of the revival solution was spread onto LB solid antibiotic plates, and the plates were incubated at 37°C for 15 h. Eight clones were selected from each transformed plate and cultured overnight in LB liquid medium at 37°C and 220 rpm. This was the first generation. Plasmids were extracted from the first generation bacterial culture, and the first generation bacterial culture was used as a seed culture to inoculate LB liquid medium at a ratio of 1:1000 and cultured overnight at 37°C and 220 rpm. This was the second generation, and the culture was continuously passaged to the third generation.

[0193] Plasmids extracted from first- and third-generation passaged clones were selected, and Sanger sequencing was used to verify the effective base number and purity of the poly(A) sequence. The effective base number of poly(A) was defined as the number of bases between the first base and the last A base whose basal peak does not exceed 50%. When the error between the effective base number of poly(A) and the initial base number of the transformed plasmid was within 1 nt, the basal peak of the last 3 effective A bases was below 50%, and the basal peak of the remaining poly(A) sequence was below 10%, the purity was considered acceptable; otherwise, it was considered unacceptable. The purity acceptance rate was calculated accordingly. Figures 5A-5C show exemplary Sanger sequencing peak diagrams of poly(A) purity acceptable (A), purity unacceptable (B), and poly(A) deletion (C), respectively. From the effective base number and purity acceptance rate data of these passaged clones, the stability of poly(A) structure replication by different mutant host bacteria was determined. The more effective bases in the poly(A) sequence obtained by Sanger sequencing, the higher the poly(A) purity qualification rate, indicating that the mutant host has a higher stability in replicating the poly(A) structure.

[0194] Table 5 shows the effective poly(A) bases of poly(A)-test 3 plasmid in the first and third generation strains, as well as the average purity pass rate of the five test plasmids in the first and third generation mutant strains.

[0195] Sanger sequencing revealed that in the first-generation NEB Stable strain, the effective poly(A) base count of the poly(A)-test 3 plasmid was 115.6 nt, and the average poly(A) purity qualification rate of the five test plasmids was 62.5%. In the third-generation clone, the effective poly(A) base count of the poly(A)-test 3 plasmid was 114.8 nt, and the average poly(A) purity qualification rate of the five test plasmids was 47.5%. However, in the NEB Stable strains with the gyrA gene mutation combination of sbcC and sbcD gene knockout, both in the first and third generations, the corresponding effective poly(A) base count and purity qualification rate were higher than those of the unmodified commercial strains. In the first-generation NEB stable [ΔsbcC-sbcD-gyrA(H80A)], the effective poly(A) base count of the poly(A)-test 3 plasmid was 117.8 nt, and the average poly(A) purity pass rate of the five test plasmids was 81.0%. In the third-generation clone, the effective poly(A) base count of the poly(A)-test 3 plasmid was 117.1 nt, and the average poly(A) purity pass rate of the five test plasmids was 73.0%. Similarly, the gyrA gene mutation combinations sbcC and sbcD gene knockout JM108 and MG1655, compared with their respective unmutated commercial strains, showed higher levels of effective poly(A) base count and purity pass rate in both the first and third generations.

[0196] The data above indicate that the gyrA gene mutation combination of sbcC and sbcD gene knockout is beneficial not only to the replication stability of ITR plasmids, but also to the replication stability of poly(A) plasmids.

[0197] Table 5. Stability of poly(A) plasmids in commercial and mutant strains

[0198] Example 5. Gene editing of RecA and endA improves the performance of MG1655 strain

[0199] The growth performance of JM108[ΔsbcC-sbcD-gyrA(H80A)], NEB stable[ΔsbcC-sbcD-gyrA(H80A)] and MG1655[ΔsbcC-sbcD-gyrA(H80A)] was further evaluated.

[0200] Take 50 ng of pUC57 (GenBank: LT671993.1) and pAAV-tTA (GenBank: MZ708023.1) plasmids, and transform them into 100 μL of test strain cells according to the method and steps in Example 2. Add 800 μL of LB medium to each cell for recovery and incubate at 37°C for 45 min. Then, spread 50 μL of the culture medium onto plates, place the transformed plates in a 37°C incubator, and incubate for 16 h. Observe and count the plaque growth of each strain.

[0201] Next, three clones were picked from each plate and inoculated into test tubes containing 4 mL of LB medium. The culture was incubated at 37°C and 220 rpm for 15 hours; this culture served as the seed culture. To maintain the initial OD of the culture... 600 Similarly, first measure the OD of all cloned seed solutions. 600 Calculate the required volume of bacterial culture to be transferred based on the measured OD value, and transfer it to a 100 mL LB medium shake flask, controlling the initial OD of all clones. 600 The OD value was 0.02. After transfer, the bacterial culture was incubated at 37℃ and 220 rpm. Samples were taken every hour for the first 7 hours, and then every 2 hours thereafter. 600 Then, plot the growth curves for each strain.

[0202] As shown in Figures 6 and 7A, compared with NEB stable[ΔsbcC-sbcD-gyrA(H80A)] and JM108[ΔsbcC-sbcD-gyrA(H80A)], MG1655[ΔsbcC-sbcD-gyrA(H80A)] grows faster and reaches a plateau with OD 600 The highest; although MG1655[ΔsbcC-sbcD-gyrA(H80A)] grows quickly, it has the fewest spots when transforming the same amount of plasmid, that is, the transformation efficiency is relatively low, but it is comparable to the transformation efficiency of wild-type MG1655.

[0203] Furthermore, following the steps described above, the pUC57 (GenBank: LT671993.1) and pAAV-tTA (GenBank: MZ708023.1) plasmids were transformed into strains MG1655[ΔsbcC-sbcD-gyrA(H80A)] and MG1655[endA-gale15-Δ(mrr-hsdRMS-mcrBC)-recA-ΔsbcC-sbcD-gyrA(H80A)], respectively. As shown in Figure 7B, compared to strain MG1655[ΔsbcC-sbcD-gyrA(H80A)], strain MG1655[endA-gale15-Δ(mrr-hsdRMS-mcrBC)-recA-ΔsbcC-sbcD-gyrA(H80A)] exhibited an increased number of plaques, indicating a significantly improved transformation efficiency.

[0204] Example 6. The combination of gyrA gene mutation and sbcC and sbcD gene knockout improves the stability of ITR plasmid replication through continuous passage.

[0205] Following the transformation steps in Example 4, 8 μl of the correctly sequenced plasmids genITR-test9 and genITR-test10 obtained in Example 2 were used to transform 100 μl of MG1655[endA-gale15-Δ(mrr-hsdRMS-mcrBC)-recA-ΔsbcC-sbcD], MG1655[endA-gale15-Δ(mrr-hsdRMS-mcrBC)-recA-ΔsbcC-sbcD-gyrA(H80A)], and the commercial strain NEB Stable, respectively. Six clones were picked from each transformed plate and cultured in LB liquid medium at 37°C and 220 rpm for 12 h. This was the first generation of bacteria. Plasmids were extracted from the first-generation bacterial culture. Simultaneously, the first-generation bacterial culture was used as a seed culture and inoculated at a ratio of 1:1000 for 12 hours. This constituted the second generation. This method was used to continuously passage the bacteria up to the tenth generation. Even-numbered generation plasmids were extracted for SmaI and AhdI restriction enzyme digestion verification. The proportion of incomplete SmaI and AhdI digestion in each clone was analyzed. Clones with <15% incomplete SmaI and AhdI digestion were considered to have passed both SmaI and AhdI digestion. The SmaI and AhdI digestion pass rates for each cell were statistically analyzed.

[0206] Table 6 summarizes the average SmaI and AhdI digestion rates of clones from the second, fourth, sixth, eighth, and tenth generations during the passage of the two test plasmids. Taking the genITR-test 9 plasmid passage as an example, the proportion of incomplete SmaI digestion increases with the number of passages, indicating a decreasing trend in ITR integrity. Among them, the pass rate of SmaI and AhdI digestion in the second generation of NEB stable strain was 66.7%, which dropped to 33.3% in the second generation and 0% in the tenth generation, showing the most significant decrease in ITR integrity. In contrast, the pass rates of the second and tenth generations of the sbcC and sbcD knockout strains were 83.3% and 50%, respectively, showing higher ITR integrity compared to NEB stable. Furthermore, the pass rates of the gyrA gene further mutated based on the sbcC and sbcD knockout strains were 100% and 83.3%, respectively, meaning that the digestion pass rate in the tenth generation was still higher than that of the second generation plasmid of NEB stable and not lower than that of the second generation plasmid of the strain that only knocked out sbcC and sbcD. Similarly, during the genITR test with 10 plasmid passages, the pass rates of SmaI and AhdI digestion in each generation of strain MG1655[endA-gale15-Δ(mrr-hsdRMS-mcrBC)-recA-ΔsbcC-sbcD-gyrA(H80A)] were higher than those of strain MG1655[endA-gale15-Δ(mrr-hsdRMS-mcrBC)-recA-ΔsbcC-sbcD], indicating higher ITR stability and integrity during passage.

[0207] In conclusion, mutating the gyrA gene in strain MG1655[endA-gale15-Δ(mrr-hsdRMS-mcrBC)-recA-ΔsbcC-sbcD] can further enhance the stability of ITR during passage.

[0208] Table 6. Passage stability of ITR plasmids in commercial and mutant strains

[0209] Example 7. The combination of gyrA gene mutation and sbcC and sbcD gene knockout improves the stability of continuous passage replication of poly(A) plasmid.

[0210] Following the transformation steps in Example 2, the five poly(A) test plasmids obtained in Example 2 were transformed into MG1655 [endA-gale15-Δ(mrr-hsdRMS-mcrBC)-recA-ΔsbcC-sbcD-gyrA(H80A)] and the commercial strain NEB Stable, respectively. Sixteen clones were picked from each transformed plate and cultured overnight in LB liquid medium at 37°C and 220 rpm. This strain served as the template for passage (P0 generation). 200 μl of P0 generation bacterial culture was mixed with 200 μl of 50% glycerol and stored at -80°C. The plasmids from the P0 generation bacterial culture were extracted, and Sanger sequencing was used to verify the effective base count and peak purity of the poly(A) sequence. Four clones that passed Sanger sequencing were selected from each of the following groups. They were inoculated with the corresponding P0 generation glycerol bacteria at a ratio of 1:1000 and cultured overnight. This bacteria was the first generation (P1 generation). The culture was continuously passaged to the tenth generation (P10 generation).

[0211] Plasmids extracted from passaged clones from generation 1 (P1) to generation 10 (P10) were selected, and Sanger sequencing was used to verify the effective base count and purity of the poly(A) sequence. Clones were considered successful if the effective base count and purity were within acceptable limits, and unsuccessful if they did not meet these limits. The clone success rate was calculated accordingly. The success rate data for each generation of passaged clones were used to assess the stability of poly(A) replication in different mutant host strains. A higher success rate for each generation indicates greater stability of the mutant host in replicating the poly(A) structure. Table 7 shows the poly(A) clone success rates of the five test plasmids in strains from generation 1 (P1) to generation 10 (P10).

[0212] Sanger sequencing showed that in the first generation of NEB Stable strains, the average passage cloning success rate of the five tested plasmids was 64.7%. With each generation, the passage cloning success rate of the NEB Stable strain decreased rapidly, falling below 42% by the fifth generation and only 17.6% by the tenth generation. However, in the MG1655[endA-gale15-Δ(mrr-hsdRMS-mcrBC)-recA-ΔsbcC-sbcD-gyrA(H80A)] gene mutant strain, the corresponding poly(A) passage cloning success rate was higher than that of the commercially available NEB Stable strain in both the first and tenth generations. Among them, in the first-generation MG1655[endA-gale15-Δ(mrr-hsdRMS-mcrBC)-recA-ΔsbcC-sbcD-gyrA(H80A)] gene mutant strain, the average passage cloning success rate of the five test plasmids was 85.7%, and the average passage cloning success rate was still 42.9% in the tenth generation, which is higher than the passage cloning success rate of the fifth generation of the commercial strain NEB Stable.

[0213] The above data indicate that the *E. coli* with the *gyrA(H80A)&SbcC-SbcD&RecA1&galE15&EndA1&mrr-hsdRMS-mcrB,C* gene mutation has a positive effect on the passage stability of the poly(A) plasmid.

[0214] Table 7. Cloning success rate of poly(A) plasmids in commercial strains and genotype mutant strains

[0215] Example 8. Fermentation of strain MG1655[endA-gale15-Δ(mrr-hsdRMS-mcrBC)-recA-ΔsbcC-sbcD-gyrA(H80A)]

[0216] Following the transformation steps in Example 4, 8 μl of the correctly sequenced plasmids genITR-test9 and genITR-test10 obtained in Example 2 were taken and transformed into 100 μl of MG1655[endA-gale15-Δ(mrr-hsdRMS-mcrBC)-recA-ΔsbcC-sbcD-gyrA(H80A)] and the commercial strain NEB Stable, respectively. One single clone was picked from the transformation plate and placed into 4 mL of LB resistant liquid medium and cultured overnight at 30°C and 180 rpm (as primary seed culture). The next morning, the matured single-tube seed culture was collected and inoculated into 100 mL of LB medium at a 1:1000 inoculation rate. It was incubated at 37°C and 180 rpm for 8 hours (shaking flasks were used as secondary seed culture). In the afternoon, after feeding and setting the parameters for the fermenter, it was inoculated into 4 L of high-density medium at a 1:1000 inoculation rate (Carnes AE, Williams JA. Plasmid fermentation process for DNA immunization applications. Methods Mol). Biol. 2014; 1143:197-217. doi:10.1007 / 978-1-4939-0410-5_13.PMID:24715290. Before inoculation, the pH of the culture medium in the tank was between 6.5 and 7.0, the rotation speed was 300 rpm, and the tank pressure was 0.05 MPa. After the pressure in the tank stabilized, the dissolved oxygen was calibrated to 100%. After calibration, the pH was controlled between 6.5 and 7.0, and the rotation speed and dissolved oxygen were set in series to control the dissolved oxygen at about 30%. After 8 hours of fermentation, a fed-batch method was followed according to the reference (Carnes AE, Williams JA. Plasmid fermentation process for DNA immunization applications. Methods Mol Biol. 2014; 1143:197-217. doi:10.1007 / 978-1-4939-0410-5_13.PMID:24715290), with an average feed rate of 70 mL / h and a fermentation time of 19 h. Samples were taken at 19 h of fermentation, and the fermentation broth was diluted and OD was measured. 600 To ensure an OD value <1, a certain volume of fermentation broth was used to extract plasmids using a kit. The plasmid concentration was measured using a Nanodrop analyzer, and the plasmid yield in the fermenter was calculated.

[0217] Table 8 summarizes the OD values ​​of the fermentation products of each strain after 19 hours of fermentation. 600Plasmid yield and SmaI / AhdI digestion results were analyzed. After 19 hours of fermentation with the commercial NEB Stable strain, the yields of the two tested plasmids were 253 mg / L and 286 mg / L, respectively; while the yields of MG1655 [endA-gale15-Δ(mrr-hsdRMS-mcrBC)-recA-ΔsbcC-sbcD-gyrA(H80A)] were 396 mg / L and 430 mg / L, representing increases of 108% and 107% respectively compared to the NEB Stable strain. Although the OD of NEB stable after fermentation... 600 It was significantly higher than MG1655[endA-gale15-Δ(mrr-hsdRMS-mcrBC)-recA-ΔsbcC-sbcD-gyrA(H80A)], but the latter's unit yield was about 3.5 times that of NEB stable, meaning that compared to NEB stable, it had less cell volume but higher plasmid volume.

[0218] The extracted plasmids were digested with SmaI and AhdI, and the proportion of incomplete SmaI / AhdI digestion bands was analyzed. Table 8 and Figure 8 show the digestion results. It can be seen that after 19 h of fermentation, the incomplete SmaI digestion bands of the two test plasmids in the commercial NEB Stable strain were 19.2% and 27.3%, respectively, while the incomplete SmaI digestion bands of MG1655[endA-gale15-Δ(mrr-hsdRMS-mcrBC)-recA-ΔsbcC-sbcD-gyrA(H80A)] were 8.0% and 10.1%, respectively. Similarly, the incomplete AhdI digestion bands of the two plasmids in the NEB stable strain were about 20%, while the incomplete AhdI digestion of the mutant strain plasmid was no higher than 3.4%, and the ITR integrity was significantly higher than that of the commercial NEB Stable strain. This indicates that, compared with existing technologies, mutant strains have the advantages of higher plasmid yield and more complete ITR regions.

[0219] Table 8. Evaluation of plasmid production for strain MG1655 [endA-gale15-Δ(mrr-hsdRMS-mcrBC)-recA-ΔsbcC-sbcD-gyrA(H80A)]

[0220] In summary, mutations in the gyrA gene promote ITR replication in *E. coli*. Further mutation of the gyrA gene, in addition to knocking out the sbcC and sbcD genes, significantly enhances ITR stability during replication and passage. Combined with other gene mutations, the strain MG1655 [endA-gale15-Δ(mrr-hsdRMS-mcrBC)-recA-ΔsbcC-sbcD-gyrA(H80A)] exhibits plasmid yields more than twice that of the commercial strain NEB stable during fermentation. Particularly for the wild-type AAV2-ITR, which has a more complex ITR structure, the plasmids containing this type of ITR from the strain described in this application demonstrate significantly better replication stability than commercial strains. This application also used different ITR sequences and different plasmids to transform the host cells, and the plasmid passage stability consistently showed advantages over commercial strains (data not shown).

[0221] Furthermore, following the steps in Example 2, the poly(A)-test4 plasmid obtained in Example 2 was transformed into MG1655[endA-gale15-Δ(mrr-hsdRMS-mcrBC)-recA-ΔsbcC-sbcD-gyrA(H80A)] and the commercial strain NEB Stable. The seed culture and fermentation process were the same as the fermentation process of the ITR plasmid described above. Table 9 shows the fermentation plasmid yield of poly(A)-test4 plasmid in the commercial strain NEB Stable and the mutant host strain. It can be seen that the fermentation plasmid yield of the mutant strain is significantly higher than that of the commercial strain NEB Stable, with an increase of approximately 1.7 times.

[0222] The above data indicate that the *E. coli* with the gyrA(H80A)&SbcC-SbcD&RecA1&galE15&EndA1&mrr-hsdRMS-mcrB,C gene mutation also has a positive effect on the fermentation yield of the poly(A) plasmid.

[0223] Table 9. Fermentation yield of poly(A) plasmid in commercial strains and genotype mutant strains

[0224] The embodiments of the present invention are not limited to those described above. Without departing from the spirit and scope of the present invention, those skilled in the art can make various changes and improvements to the present invention in form and detail, and these are all considered to fall within the protection scope of the present invention.

[0225] Some of the nucleotide and amino acid sequences mentioned in this article are shown below.

[0226] SEQ ID NO:1:mcrB

[0227] SEQ ID NO:2:mcrC

[0228] SEQ ID NO:3:hsdM

[0229] SEQ ID NO:4:hsdS

[0230] SEQ ID NO:5:mrr

[0231] SEQ ID NO:6:hsdr

[0232] SEQ ID NO:7:sbcC

[0233] SEQ ID NO:8:sbcD

[0234] SEQ ID NO:9:endA - The bold underline indicates mutation points, including C513G and G622A.

[0235] SEQ ID NO:64:EndA(E208K)

[0236] SEQ ID NO:10:RecA - The bold underline indicates mutation points, including G482A and C498T.

[0237] SEQ ID NO:65:RecA(G161D)

[0238] SEQ ID NO:11:galE - The bold underline indicates the mutation points, including C368T and C483G.

[0239] SEQ ID NO:66:GalE(S123F)

[0240] SEQ ID NO:12:gyrA - The bold underline indicates mutation points, including G81T and CAT238-240GCA.

[0241] SEQ ID NO:62:GyrA(H80A)

[0242] SEQ ID NO:13:gyrB - The bold underline indicates mutation points, including G270C and C406T.

[0243] SEQ ID NO:63:GyrB(R136C)

[0244] SEQ ID NO:14: sgRNA targeting mcrB-mcrC

[0245] SEQ ID NO:15: sgRNA targeting hsdM-hsdS

[0246] SEQ ID NO:16: sgRNA targeting mrr-hsdr

[0247] SEQ ID NO:17: sgRNA targeting sbcC-sbcD

[0248] SEQ ID NO:18: sgRNA targeting endA

[0249] SEQ ID NO:19: sgRNA targeting RecA

[0250] SEQ ID NO:20: sgRNA targeting gale

[0251] SEQ ID NO:21: sgRNA targeting gyrA

[0252] SEQ ID NO:22: sgRNA targeting gyrB

[0253] SEQ ID NO:23: Knockout mcrB-mcrC donor sequence

[0254] SEQ ID NO:24: Knockout hsdM-hsdS donor sequence

[0255] SEQ ID NO:25: Knockout MRRR-HSDR donor sequence

[0256] SEQ ID NO:26: Knockout sbcC-sbcD donor sequence

[0257] SEQ ID NO:27: Mutant endA is the donor sequence for endA1

[0258] SEQ ID NO:28: Mutant RecA is a RecA1 donor sequence

[0259] SEQ ID NO:29: Mutant gale is a gale15 donor sequence

[0260] SEQ ID NO:30: Mutant gyrA as gyrA (H80A) donor sequence

[0261] SEQ ID NO:31: Mutant gyrB is the gyrB(R136C) donor sequence

[0262] SEQ ID NO:52: Wild-type AAV2-ITR 5'-ITR sequence (Bold underline indicates AhdI restriction site, bold italic underline indicates SmaI restriction site)

[0263] SEQ ID NO:53: Wild-type AAV2-ITR 3'-ITR sequence (Bold underline indicates AhdI restriction site, bold italics indicates SmaI restriction site)

[0264] SEQ ID NO:54: genITR-test1 sequence of 5' end ITR test plasmid (underlined ITR sequence, bold wavy line indicates AhdI restriction site, bold italic wavy line indicates SmaI restriction site)

[0265] SEQ ID NO:55: genITR-test2 sequence of 5' end ITR test plasmid (underlined ITR sequence, bold wavy line indicates AhdI restriction site, bold italic wavy line indicates SmaI restriction site)

[0266] SEQ ID NO:56: Test plasmid genITR-test9 sequence (underline indicates ITR sequence, bold wavy line indicates AhdI restriction site, bold italic wavy line indicates SmaI restriction site)

[0267] SEQ ID NO:57: Test plasmid genITR-test10 sequence (underline indicates ITR sequence, bold wavy line indicates AhdI restriction site, bold italic wavy line indicates SmaI restriction site)

[0268] SEQ ID NO:58:poly(A) sequence-30&70A

[0269] SEQ ID NO:59:poly(A) sequence-30&30&43A

[0270] SEQ ID NO:60:mRNA functional region sequence of poly(A)-test1(80A)

[0271] SEQ ID NO:61: pVAX1 vector sequence

[0272] Although this application has been described in conjunction with one or more embodiments, it should be understood that this application is not limited to these embodiments. The description in this application is intended to cover all variations and equivalents, all of which are included within the spirit and scope of the appended claims. All references cited herein are incorporated herein by reference in their entirety.

Claims

1. An Escherichia coli host cell comprising a gyrA gene and a gyrB gene, wherein the gyrA gene expresses GyrA containing an H80A mutation at position 80 relative to SEQ ID NO:62, or the gyrB gene expresses GyrB containing an R136C mutation at position 136 relative to SEQ ID NO:63, wherein the Escherichia coli host cell further comprises a gene knockout of SbcC or SbcD.

2. The *E. coli* host cell of claim 1, wherein the *GyrA* expressed by the *gyrA* gene comprises the amino acid sequence shown in SEQ ID NO:

62. The GyrB gene expresses GyrB, which contains the amino acid sequence shown in SEQ ID NO:

63.

3. The *E. coli* host cell as described in claim 1 or 2, wherein the expressed GyrA contains an H80A mutation at position 80 relative to SEQ ID NO:62, or the expressed GyrB contains an R136C mutation at position 136 relative to SEQ ID NO:63, is genetically modified.

4. The *E. coli* host cell according to any one of claims 1-3, further comprising i) gene knockout of mcrB, mcrC, hsdM, hsdS, mrr, and hsdr, and / or ii) genes containing endA, recA, and galE, wherein the EndA, RecA, and GalE expressed by the endA, recA, and galE genes contain an E208K mutation at position 208 relative to SEQ ID NO:64, a G161D mutation at position 161 relative to SEQ ID NO:65, and an S123F mutation at position 123 relative to SEQ ID NO:

66.

5. The *Escherichia coli* host cell according to any one of claims 1-4, wherein it is MG1655, JM108, or NEB Stable.

6. A method for replicating or expressing a nucleic acid molecule containing a simple repeat sequence or a nucleic acid molecule containing a hairpin structure sequence in an *Escherichia coli* host cell, comprising: i) Provide an *E. coli* host cell containing the gyrA and gyrB genes, wherein the gyrA gene expresses GyrA containing an H80A mutation at position 80 relative to SEQ ID NO:62, or the gyrB gene expresses GyrB containing an R136C mutation at position 136 relative to SEQ ID NO:

63. ii) Introducing nucleic acid molecules containing simple repeat sequences or hairpin structure sequences into host cells, and iii) Culture recombinant host cells under conditions that facilitate the replication or expression of the nucleic acid molecules introduced in step ii).

7. The method of claim 6, wherein the GyrA expressed by the gyrA gene comprises the amino acid sequence shown in SEQ ID NO:

62. The GyrB expressed by the gyrB gene contains the amino acid sequence shown in SEQ ID NO:

63.

8. The method of claim 6 or 7, wherein the host cell in step i) is MG1655, JM108, or NEB Stable.

9. The method of any one of claims 6-8, wherein the host cell comprises the knockout of the sbcC and sbcD genes.

10. The method of claim 6 or 9, wherein the host cell further i) contains endA, recA, and galE genes, wherein the EndA, RecA, and GalE expressed by the endA, recA, and galE genes contain an E208K mutation at position 208 relative to SEQ ID NO:64, a G161D mutation at position 161 relative to SEQ ID NO:65, and an S123F mutation at position 123 relative to SEQ ID NO:66, and / or ii) contains gene knockout of mcrB, mcrC, hsdM, hsdS, mrr, and hsdr.

11. The method of any one of claims 6-10, wherein the host cell in step i) is genetically modified such that the expressed GyrA contains an H80A mutation at position 80 relative to SEQ ID NO:62, or is genetically modified such that the expressed GyrB contains an R136C mutation at position 136 relative to SEQ ID NO:

63.

12. The method of claim 11, further comprising, prior to step i), introducing a Cas9 enzyme, an sgRNA comprising the nucleotide sequence shown in SEQ ID NO:21, and a donor sequence comprising the nucleotide sequence shown in SEQ ID NO:30 into the host cell, such that the GyrA expressed by the host cell contains an H80A mutation at position 80 relative to SEQ ID NO:62, or The host cell of step i) is introduced with the Cas9 enzyme, an sgRNA containing the nucleotide sequence shown in SEQ ID NO:22, and a donor sequence containing the nucleotide sequence shown in SEQ ID NO:31, so that the GyrB expressed by the host cell contains the R136C mutation at position 136 relative to SEQ ID NO:

63.

13. The method of any one of claims 6-12, wherein the simple repeating sequence comprises a poly(A) sequence or a poly(T) sequence.

14. The method of claim 13, wherein the poly(A) sequence comprises consecutive A bases, wherein the number of consecutive A bases is 20-250; or the poly(A) sequence comprises 2-5 consecutive A base segments separated by non-A bases, wherein the number of consecutive A bases in each consecutive A base segment is 10-100, and every two consecutive A base segments are separated by 1-20 non-A bases.

15. The method of any one of claims 6-14, wherein the sequence containing the hairpin structure comprises an inverted terminal repeat (ITR) or an untranslated region (UTR).

16. The method of any one of claims 6-15, wherein step ii) comprises introducing a vector into a host cell, the vector comprising the nucleic acid molecule containing a simple repeat sequence or the nucleic acid molecule containing a hairpin structure sequence; or integrating the nucleic acid molecule containing a simple repeat sequence or the nucleic acid molecule containing a hairpin structure sequence into the genome of the host cell.

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