Host cell for improving stability of poly(a) plasmid

Abstract

Provided is the use of an E. coli host cell in replicating or expressing a nucleic acid molecule containing a poly(A) sequence, wherein the E. coli host cell comprises down-regulated gyrase activity or down-regulated gyrase expression.

Description

A host cell for improving the stability of poly(A) plasmid

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 202311399354.3 filed on October 25, 2023, the entire contents of which are incorporated herein by reference. Field of the Invention

[0003] This application relates to the use of Escherichia coli host cells for replicating or expressing nucleic acid molecules containing poly(A) sequences, wherein the E. coli host cells have downregulated gyrase activity or downregulated gyrase expression. Using these E. coli host cells to replicate or express nucleic acid molecules containing poly(A) sequences can increase the replication or expression stability of the poly(A) sequences. Background Art

[0004] An mRNA vaccine is a nucleic acid preparation, which is mRNA prepared by transcription, synthesis, and other processes of an exogenous target gene sequence. This mRNA can be introduced into the body's cells through a specific delivery system to express the target protein, thereby stimulating the body to produce a specific immunological response and providing immune protection. Compared with traditional vaccines, mRNA vaccines have several outstanding advantages, such as ease of design, rapid production, low cost, the ability to induce cellular and humoral immunity, and no interaction with genomic DNA. In recent years, various types of mRNA vaccines have made breakthroughs in basic research and clinical research. In particular, their application in viral infectious diseases such as the new coronavirus, influenza virus, HIV, rabies virus, immuno-oncology (IO), personalized cancer (PCV), genetic diseases, and rare diseases has continued to grow.

[0005] The main sequence of a mature mRNA is the coding region, flanked by noncoding regions (UTRs) on both upstream and downstream sides. Eukaryotic mRNA molecules also have a 5' cap and a 3' poly(A) tail at each end. The poly(A) tail plays an important role in maintaining mRNA stability, regulating mRNA translation efficiency, and maintaining mRNA transport.

[0006] Currently, the most efficient method for batch synthesis of mRNA is in vitro transcription (IVT). IVT primarily uses linear DNA as a template to prepare mRNA. The main process steps include transcribing linearized plasmid DNA into mRNA, chemical modification (such as 5'-end capping and 3'-end poly(A) tailing), and separation and purification.

[0007] When synthesizing mRNA via IVT, there are two main ways to add the poly(A) tail.

[0008] The first is enzymatic synthesis, which involves adding poly(A) polymerase from Escherichia coli after mRNA transcription. This method has the advantage of requiring no template and being simple to operate, but it has the limitation of unstable tail length.

[0009] The second method is the co-transcription method, which directly transcribes the poly(A) sequence already present on the template plasmid DNA or PCR product. This method has the advantage of not requiring poly(A) polymerase, reducing process steps and costs. However, it also poses the problem of poly(A) tail deletion. Large-scale plasmid production is typically performed using Escherichia coli fermentation. During amplification, plasmids carrying long poly(A) sequences are inherently unstable, and the poly(A) tail encoding sequence is easily lost during replication, resulting in plasmid impurities. To improve the stability of the poly(A) tail on the plasmid, some scientists have adopted a segmented co-transcription method using poly(A) sequences. Because each poly(A) binding protein, PABP, only binds approximately 30 A residues, a small number of A residues between the two binding proteins acts as a spacer. Replacing the spacer with a non-A base reduces the probability of recombination within the poly(A) tail encoding sequence. Furthermore, the length and base number of the spacer 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 production of poly(A) plasmids can have a certain impact on downstream protein expression. Furthermore, there are reports that low-temperature (30°C) fermentation can improve the integrity of the plasmid poly(A) tail coding sequence. However, low-temperature culture slows bacterial growth, reduces plasmid copy numbers, significantly reduces yields, and prolongs production cycles, making plasmid production difficult.

[0010] Currently, there is no effective method to avoid A base deletion during poly(A) plasmid construction and E. coli amplification. Commercial strains with recombination defects, such as NEB stable and Stabl3, are often used to construct and amplify poly(A)-containing plasmids, but are not always effective. Therefore, finding more stable poly(A) fermentation strains that can reduce the risk of poly(A) tail sequence deletion or impurity without compromising plasmid yield may become a new research direction. However, there are currently few reports on the effects of different strains on poly(A) tail sequence stability.

[0011] Gyrase is a type II DNA topoisomerase crucial for bacterial survival, involved in processes such as DNA replication, repair, recombination, and transcription (Menzel, R., and Gellert, M. (1994) Adv. Pharmacol. 29A:201–225). Gyrase is typically composed of two GyrA subunits and two GyrB subunits. The GyrA subunit is primarily responsible for DNA binding, nicking, and bridging the gaps in the DNA double strand, while the GyrB subunit mediates energy transduction and ATP hydrolysis.

[0012] In particular, gyrase can unwind (+) supercoils caused by replication and transcription, and can introduce (-) supercoils into genomic DNA. In bacteria such as E. coli, gyrase can open a temporary gap in one segment (G segment) of the DNA double strand, allowing the other segment (T segment) to move through the gap to the front of the G segment, thereby converting the DNA (+) supercoil to (-) supercoil. Removing (+) supercoils is a prerequisite for the advancement of the replication fork and is also necessary for the separation of the two double strands produced by replication ( M, et al., (2007) Biochimie. 89(4):490-499).

[0013] At any given moment, at least 300 DNA gyrase molecules are stably bound to the Escherichia coli genome, with an average of about 12 gyrase molecules at each replication fork (Stracy M, et al., (2019) Nucleic Acids Res. 47(1):210-220). When gyrase is inhibited, the transcriptional activity of most genes decreases. For example, overexpression of GyrI, which inhibits gyrase activity, inhibits the growth of the strain (Nakanishi A, et al., (2002) J Biol Chem. 277(11):8949-54). Some antibacterial drugs, such as quinolone antibiotics and coumarins, target gyrase and cause irreversible damage to bacterial DNA (Maxwell, A. (1993) Mol. Microbiol. 9: 681–686; Maxwell, A. (1997) Trends Microbiol. 5: 102–109).

[0014] Summary of the Invention

[0015] The inventors of the present application unexpectedly discovered that when the activity of gyrase is downregulated in host cells, particularly in Escherichia coli host cells, the replication stability and / or expression stability of the poly(A) sequence in the plasmid can be increased while maintaining the plasmid yield and the plasmid supercoiling ratio.

[0016] Thus, in a first aspect, the present application provides a host cell comprising downregulated gyrase activity, or downregulated gyrase expression. In particular, a recombinant host cell is provided, which is modified to comprise downregulated gyrase activity, or downregulated gyrase expression. Wherein, the gyrase comprises GyrA and GyrB. In particular, the gyrase consists of GyrA and GyrB. The host cell, including the recombinant host cell, may comprise downregulated GyrA activity, GyrB activity, or the activity of a gyrase consisting of GyrA and GyrB. The host cell, including the recombinant host cell, may comprise downregulated GyrA expression, GyrB expression, or the expression of a gyrase consisting of GyrA and GyrB.

[0017] Host cells, including recombinant host cells, may comprise down-regulated gyrase activity via mutation of the gyrA gene or the gyrB gene.

[0018] In some embodiments, the host cell can comprise downregulated gyrase activity via a gyrA gene mutation. A host cell comprising a mutated gyrA gene can express a GyrA mutant that can downregulate or reduce gyrase activity compared to the expression product of a gyrA gene lacking the mutation. The host cell can express a GyrA mutant via a gyrA gene mutation. The GyrA mutant can comprise a mutation at position 80 corresponding to SEQ ID NO. 37, or at positions 569 and 586 corresponding to SEQ ID NO. 37. In some embodiments, the host cell can express a GyrA mutant via a gyrA gene mutation. The GyrA mutant can comprise an H80A mutation at position 80 corresponding to SEQ ID NO. 37, or at positions 569 and 586 corresponding to SEQ ID NO. 37, respectively, or at positions A569T and T586A mutations corresponding to SEQ ID NO. 37, respectively. In some embodiments, the E. coli host cell expresses a GyrA mutant by mutating the gyrA gene, wherein the GyrA mutant comprises: i) an H80A mutation at position 80 corresponding to SEQ ID NO. 37, or ii) A569T and T586A mutations at positions 569 and 586 corresponding to SEQ ID NO. 37. In some embodiments, the GyrA mutant may comprise an H80A mutation at position 80 corresponding to SEQ ID NO. 37. In other embodiments, the GyrA mutant may comprise A569T and T586A mutations at positions 569 and 586 corresponding to SEQ ID NO. 37. In some specific embodiments, the GyrA mutant may comprise: i) an H80A mutation at position 80 corresponding to SEQ ID NO. 37; or ii) A569T and T586A mutations at positions 569 and 586 corresponding to SEQ ID NO. 37. In some embodiments, the GyrA mutant may comprise the amino acid sequence of SEQ ID NO. 38, wherein the amino acids at positions 80, 569, and 586 are A, A, and T, respectively. In some embodiments, the GyrA mutant may comprise the amino acid sequence of SEQ ID NO. 38, wherein the amino acids at positions 80, 569, and 586 are A, T, and A, respectively. In some embodiments, the mutated gyrA gene may comprise the nucleotide sequence of SEQ ID NO. 3. In some embodiments, the mutated gyrA gene may comprise the nucleotide sequence of SEQ ID NO. 4.

[0019] In some embodiments, the host cell may comprise downregulated gyrase activity via a gyrB gene mutation. A host cell comprising a mutated gyrB gene may express a GyrB mutant that results in downregulated gyrase activity compared to the expression product of a gyrB gene lacking the mutation. The host cell may express a GyrB mutant via a gyrB gene mutation that comprises an E42D mutation at position 42 corresponding to SEQ ID NO. 39, an R136C mutation at position 136 corresponding to SEQ ID NO. 39, or a D498A mutation at position 498 corresponding to SEQ ID NO. 39. In some embodiments, the E. coli host cell expresses a GyrB mutant by mutating the gyrB gene. The GyrB mutant may comprise: i) an R136C mutation at position 136 corresponding to SEQ ID NO. 39, ii) an E42D mutation at position 42 corresponding to SEQ ID NO. 39, or iii) a D498A mutation at position 498 corresponding to SEQ ID NO. 39. In some embodiments, the GyrB mutant may comprise an R136C mutation at position 136 corresponding to SEQ ID NO. 39. In other embodiments, the GyrB mutant may comprise an E42D mutation at position 42 corresponding to SEQ ID NO. 39. In some embodiments, the GyrB mutant may comprise a D498A mutation at position 498 corresponding to SEQ ID NO. 39. In some embodiments, the GyrB mutant may comprise: i) an R136C mutation corresponding to position 136 of SEQ ID NO. 39, ii) an E42D mutation corresponding to position 42 of SEQ ID NO. 39, or iii) a D498A mutation corresponding to position 498 of SEQ ID NO. 39. In some embodiments, the GyrB mutant may comprise the amino acid sequence of SEQ ID NO. 40, wherein the amino acids at positions 42, 136, and 498 are D, R, and D, respectively. In some embodiments, the GyrB mutant may comprise the amino acid sequence of SEQ ID NO. 40, wherein the amino acids at positions 42, 136, and 498 are E, C, and D, respectively. In some embodiments, the GyrB mutant may comprise the amino acid sequence of SEQ ID NO. 40, wherein the amino acids at positions 42, 136, and 498 are E, C, and D, respectively. In some embodiments, the mutated gyrB gene may comprise the nucleotide sequence shown in SEQ ID NO. 5. In some embodiments, the mutated gyrB gene may comprise the nucleotide sequence shown in SEQ ID NO. 6.In some embodiments, the mutated gyrB gene may comprise the nucleotide sequence shown in SEQ ID NO.7.

[0020] In some embodiments, the host cell may have downregulated gyrase activity due to mutations in the gyrA and gyrB genes. A gyrA gene containing a mutation may express a GyrA mutant, and a gyrB gene containing a mutation may express a GyrB mutant. The gyrase formed by the GyrA and GyrB mutants may have downregulated gyrase activity compared to the gyrase formed by the expression product of a host cell lacking these gyrA and gyrB mutations. In some embodiments, the GyrA mutant may include an H80A mutation at position 80 corresponding to SEQ ID NO. 37, and the GyrB mutant may include an R136C mutation at position 136 corresponding to SEQ ID NO. 39. In some embodiments, the GyrA mutant may comprise the amino acid sequence of SEQ ID NO. 38, wherein the amino acids at positions 80, 569, and 586 are A, A, and T, respectively; and the GyrB mutant may comprise the amino acid sequence of SEQ ID NO. 40, wherein the amino acids at positions 42, 136, and 498 are E, C, and D, respectively. In some embodiments, the mutant gyrA gene may comprise the nucleotide sequence of SEQ ID NO. 3, and the mutant gyrB gene may comprise the nucleotide sequence of SEQ ID NO. 6.

[0021] In some embodiments, the host cell may further comprise one or more of the following mutations: i) a RecA gene mutation, ii) an inserted lacI gene expression cassette, and iii) a RecQ gene knockout. In some embodiments, the host cell may comprise: i) a RecA gene mutation, ii) an inserted lacI gene expression cassette, and iii) a RecQ gene knockout. In other embodiments, the host cell may comprise: i) a mutated RecA gene comprising the nucleotide sequence set forth in SEQ ID NO. 44, ii) an inserted lacI gene expression cassette comprising the nucleotide sequence set forth in SEQ ID NO. 42, or iii) the RecQ gene comprising the nucleotide sequence set forth in SEQ ID NO. 41. In some embodiments, the host cell may comprise a RecA gene mutation, wherein the mutated RecA gene comprises the nucleotide sequence set forth in SEQ ID NO. 44. In other embodiments, the host cell may comprise an inserted lacI gene expression cassette, wherein the inserted lacI gene expression cassette comprises the nucleotide sequence set forth in SEQ ID NO. 42. In some embodiments, the host cell may comprise a RecQ gene knockout, wherein the RecQ gene comprises the nucleotide sequence set forth in SEQ ID NO. 41. In some embodiments, the E. coli host cell may include:

[0022] i) RecA gene mutation, wherein the mutated RecA gene comprises the nucleotide sequence shown in SEQ ID NO. 44,

[0023] ii) inserting a lacI gene expression cassette, wherein the inserted lacI gene expression cassette comprises the nucleotide sequence shown in SEQ ID NO.42, or / and

[0024] iii) RecQ gene knockout, wherein the RecQ gene comprises the nucleotide sequence shown in SEQ ID NO.41.

[0025] In other embodiments, the E. coli host cell may include:

[0026] i) gyrA gene mutation, wherein the mutated gyrA gene comprises the nucleotide sequence shown in SEQ ID NO. 3 or 4,

[0027] ii) RecA gene mutation, wherein the mutated RecA gene comprises the nucleotide sequence shown in SEQ ID NO. 44,

[0028] iii) inserting a lacI gene expression cassette, wherein the inserted lacI gene expression cassette comprises the nucleotide sequence shown in SEQ ID NO. 42, and

[0029] vi) RecQ gene knockout, wherein the RecQ gene comprises the nucleotide sequence shown in SEQ ID NO.41.

[0030] Host cells, including recombinant host cells, can downregulate gyrase expression by knocking down the gyrA gene or the gyrB gene, or knocking out the gyrA gene or the gyrB gene and expressing a GyrA / GyrB vector.

[0031] Host cells can be downregulated by knocking down the gyrA gene, or by knocking out the gyrA gene and introducing a vector expressing GyrA into the host cells. Knockdown of the gyrA gene can be achieved by providing an inhibitory nucleic acid molecule (e.g., shRNA) against GyrA. Recombinant host cells can be downregulated by knocking down the gyrB gene, or by knocking out the gyrB gene and introducing a vector expressing GyrB into the host cells. Knockdown of the gyrB gene can be achieved by providing an inhibitory nucleic acid molecule (e.g., shRNA) against GyrB.

[0032] The host cell can be an E. coli host cell. In particular, the host cell, particularly a recombinant host cell, can be JM108, NEB Stable, Top10, DH5α, DH10B, MG1655, AG1, BL21, DB3.1, DC10B, DH1, E.Cloni 10G, EPI300, EPI400, JM109, JM110, STBL2, STBL3, SURE, TOP10F', XL1-Blue, or XL10-Gold, engineered to contain downregulated gyrase activity or downregulated gyrase expression. In some embodiments, the recombinant host cell can be JM108, NEB Stable, Top10, DH5α, DH10B, or MG1655.

[0033] Host cells, including recombinant host cells, can contain a nucleic acid molecule containing a poly(A) sequence. For example, the host cell can contain a vector, and the vector can contain a nucleic acid molecule containing a poly(A) sequence. Alternatively, the host cell can have a nucleic acid molecule containing a poly(A) sequence integrated into its genome. The poly(A) sequence can contain consecutive A bases, and the number of consecutive A bases can be, for example, 20-250. The 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 the consecutive A base segments can be separated by, for example, 1-20 non-A bases.

[0034] Host cells, including recombinant host cells, can also contain nucleic acid molecules containing poly(T) sequences.

[0035] In a second aspect, the present application provides a method for preparing a recombinant host cell, comprising:

[0036] i) providing a host cell, such as a prokaryotic host cell, comprising a gyrA gene and a gyrB gene, and

[0037] ii) downregulating the activity of gyrase in the host cell, or downregulating the expression level of gyrase in the host cell.

[0038] The host cell in step i) can be an Escherichia coli host cell, in particular an Escherichia coli vector preparation strain, including, but not limited to, JM108, NEB Stable, Top10, DH5α, DH10B, MG1655, AG1, BL21, DB3.1, DC10B, DH1, E.Cloni 10G, EPI300, EPI400, JM109, JM110, STBL2, STBL3, SURE, TOP10F', XL1-Blue and XL10-Gold.

[0039] The gyrase comprises GyrA and GyrB. Specifically, the gyrase is composed of GyrA and GyrB. Step ii) may comprise downregulating GyrA activity, GyrB activity, or the activity of a gyrase composed of GyrA and GyrB. Step ii) may comprise downregulating the expression level of GyrA, GyrB, or the expression level of a gyrase composed of GyrA and GyrB.

[0040] Down-regulating the gyrase activity in step ii) can be achieved by mutation of the gyrA gene or the gyrB gene.

[0041] In some embodiments, downregulating gyrase activity can be achieved by mutating the gyrA gene. An E. coli host cell containing a mutated gyrA gene can express a GyrA mutant. This GyrA mutant can cause downregulation of gyrase activity compared to the expression product of a gyrA gene without the mutation. The GyrA mutant can include an H80A mutation at position 80 corresponding to SEQ ID NO. 37, or an A569T and T586A mutation at positions 569 and 586 corresponding to SEQ ID NO. 37. In some embodiments, the GyrA mutant can include the amino acid sequence of SEQ ID NO. 38, wherein the amino acids at positions 80, 569, and 586 are A, A, and T, respectively. In some embodiments, the GyrA mutant can include the amino acid sequence of SEQ ID NO. 38, wherein the amino acids at positions 80, 569, and 586 are A, T, and A, respectively. In some embodiments, the mutated gyrA gene may comprise the nucleotide sequence shown in SEQ ID NO. 3. In some embodiments, the mutated gyrA gene may comprise the nucleotide sequence shown in SEQ ID NO. 4.

[0042] In some embodiments, downregulating gyrase activity can be achieved by mutating the gyrB gene. An E. coli host cell containing a mutated gyrB gene can express a GyrB mutant. This GyrB mutant can cause downregulation of gyrase activity compared to the expression product of a gyrB gene without the mutation. The GyrB mutant can include an E42D mutation at position 42 corresponding to SEQ ID NO. 39, an R136C mutation at position 136 corresponding to SEQ ID NO. 39, or a D498A mutation at position 498 corresponding to SEQ ID NO. 39. In some embodiments, the GyrB mutant can include the amino acid sequence of SEQ ID NO. 40, wherein the amino acids at positions 42, 136, and 498 are D, R, and D, respectively. In some embodiments, the GyrB mutant can include the amino acid sequence of SEQ ID NO. 40, wherein the amino acids at positions 42, 136, and 498 are E, C, and D, respectively. In some embodiments, the GyrB mutant may comprise the amino acid sequence of SEQ ID NO. 40, wherein the amino acids at positions 42, 136, and 498 are E, R, and A, respectively. In some embodiments, the mutant gyrB gene may comprise the nucleotide sequence of SEQ ID NO. 5. In some embodiments, the mutant gyrB gene may comprise the nucleotide sequence of SEQ ID NO. 6. In some embodiments, the mutant gyrB gene may comprise the nucleotide sequence of SEQ ID NO. 7.

[0043] In some embodiments, downregulating gyrase activity can be achieved by mutating the gyrA and gyrB genes. A gyrA gene containing a mutation can express a GyrA mutant, and a gyrB gene containing a mutation can express a GyrB mutant. The gyrase formed by these GyrA and GyrB mutants can have downregulated gyrase activity compared to the gyrase formed by the expression product of E. coli host cells lacking these gyrA and gyrB mutations. In some embodiments, the GyrA mutant can include an H80A mutation at position 80 corresponding to SEQ ID NO. 37, and the GyrB mutant can include an R136C mutation at position 136 corresponding to SEQ ID NO. 39. In some embodiments, the GyrA mutant may comprise the amino acid sequence of SEQ ID NO. 38, wherein the amino acids at positions 80, 569, and 586 are A, A, and T, respectively; and the GyrB mutant may comprise the amino acid sequence of SEQ ID NO. 40, wherein the amino acids at positions 42, 136, and 498 are E, C, and D, respectively. In some embodiments, the mutant gyrA gene may comprise the nucleotide sequence of SEQ ID NO. 3, and the mutant gyrB gene may comprise the nucleotide sequence of SEQ ID NO. 6.

[0044] In some embodiments, step ii) may include,

[0045] 1) making the gyrA gene contain the nucleotide sequence shown in SEQ ID NO.3,

[0046] 2) making the gyrB gene contain the nucleotide sequence shown in SEQ ID NO.6,

[0047] 3) making the gyrA gene contain the nucleotide sequence shown in SEQ ID NO.4,

[0048] 4) making the gyrB gene contain the nucleotide sequence shown in SEQ ID NO.5, or

[0049] 5) The gyrB gene comprises the nucleotide sequence shown in SEQ ID NO. 7.

[0050] In some embodiments, step ii) may include introducing Cas9 enzyme into the host cell of step i), and

[0051] 1) an sgRNA comprising the nucleotide sequence shown in SEQ ID NO.8, and a donor sequence comprising the nucleotide sequence shown in SEQ ID NO.13,

[0052] 2) an sgRNA comprising the nucleotide sequence shown in SEQ ID NO.9 and a donor sequence comprising the nucleotide sequence shown in SEQ ID NO.14,

[0053] 3) an sgRNA comprising the nucleotide sequence shown in SEQ ID NO.10, and a donor sequence comprising the nucleotide sequence shown in SEQ ID NO.15,

[0054] 4) an sgRNA comprising the nucleotide sequence shown in SEQ ID NO.11 and a donor sequence comprising the nucleotide sequence shown in SEQ ID NO.16, or

[0055] 5) An sgRNA comprising the nucleotide sequence shown in SEQ ID NO. 12, and a donor sequence comprising the nucleotide sequence shown in SEQ ID NO. 17.

[0056] The gene encoding the Cas9 enzyme may be located in a vector. The sgRNA and donor sequence may be located in a vector. Step ii) may include introducing a vector containing the gene encoding the Cas9 enzyme, and a vector containing the sgRNA and donor sequence into the host cell of step i).

[0057] Down-regulating the expression level of gyrase in step ii) can be achieved by knocking down the gyrA gene or the gyrB gene, or by knocking out the gyrA gene or the gyrB gene and introducing a vector expressing GyrA / GyrB into the host cell.

[0058] The downregulation of gyrase expression in step ii) can be achieved by knocking down the gyrA gene, or by knocking out the gyrA gene and introducing a vector expressing GyrA into the host cell. Knocking down the gyrA gene can be achieved by providing an inhibitory nucleic acid molecule (e.g., shRNA) against GyrA. The downregulation of gyrase expression in step ii) can be achieved by knocking down the gyrB gene, or by knocking out the gyrB gene and introducing a vector expressing GyrB into the host cell to downregulate gyrase expression. Knocking down the gyrB gene can be achieved by providing an inhibitory nucleic acid molecule (e.g., shRNA) against GyrB.

[0059] In some embodiments, the E. coli host cell may further comprise any one or more of the following steps: 1) RecA gene mutation, 2) insertion of a lacI gene expression cassette, and 3) RecQ gene knockout. In some embodiments, the E. coli host cell may comprise: 1) RecA gene mutation, 2) insertion of a lacI gene expression cassette, and 3) RecQ gene knockout.

[0060] In some embodiments, the step ii) may include introducing a Cas9 enzyme into the host cell, and

[0061] 1) an sgRNA comprising the nucleotide sequence shown in SEQ ID NO.47 and a donor sequence comprising the nucleotide sequence shown in SEQ ID NO.50,

[0062] 2) an sgRNA comprising the nucleotide sequence shown in SEQ ID NO.46 and a donor sequence comprising the nucleotide sequence shown in SEQ ID NO.49, or

[0063] 3) an sgRNA comprising the nucleotide sequence shown in SEQ ID NO. 45, and a donor sequence comprising the nucleotide sequence shown in SEQ ID NO. 48.

[0064] In a third aspect, the present application provides host cells, particularly prokaryotic host cells, comprising downregulated gyrase activity or downregulated gyrase expression, including the host cells of the present application (including recombinant host cells), and recombinant host cells obtained by the preparation method of the present application, for use in replicating or expressing nucleic acid molecules containing poly(A) sequences or poly(T) sequences.

[0065] The host cell for use in the present application may be an E. coli host cell. In particular, the host cell for use in the present application may be JM108, NEB Stable, Top10, DH5α, DH10B, MG1655, AG1, BL21, DB3.1, DC10B, DH1, E.Cloni 10G, EPI300, EPI400, JM109, JM110, STBL2, STBL3, SURE, TOP10F', XL1-Blue, or XL10-Gold, which have been modified to contain downregulated gyrase activity or downregulated gyrase expression. In some embodiments, the host cell for use in the present application may be JM108, NEB Stable, Top10, DH5α, DH10B, or MG1655, which have been modified to contain downregulated gyrase activity or downregulated gyrase expression.

[0066] In some embodiments, the host cell according to the use of the present application comprises a mutation in the gyrA or gyrB gene that results in decreased gyrase activity. Host cells comprising a mutated gyrA gene can express a GyrA mutant. The GyrA mutant can comprise an H80A mutation at position 80 corresponding to SEQ ID NO. 37, or an A569T and T586A mutation at positions 569 and 586 corresponding to SEQ ID NO. 37. In some embodiments, the GyrA mutant can comprise the amino acid sequence of SEQ ID NO. 38, wherein the amino acids at positions 80, 569, and 586 are A, A, and T, respectively. In some embodiments, the GyrA mutant can comprise the amino acid sequence of SEQ ID NO. 38, wherein the amino acids at positions 80, 569, and 586 are A, T, and A, respectively. In some embodiments, the mutated gyrA gene can comprise the nucleotide sequence of SEQ ID NO. 3. In some embodiments, the mutant gyrA gene may comprise the nucleotide sequence set forth in SEQ ID NO. 4. A host cell comprising a mutant gyrB gene may express a GyrB mutant. The GyrB mutant may comprise an E42D mutation at position 42 corresponding to SEQ ID NO. 39, an R136C mutation at position 136 corresponding to SEQ ID NO. 39, or a D498A mutation at position 498 corresponding to SEQ ID NO. 39. In some embodiments, the GyrB mutant may comprise the amino acid sequence set forth in SEQ ID NO. 40, wherein the amino acids at positions 42, 136, and 498 are D, R, and D, respectively. In some embodiments, the GyrB mutant may comprise the amino acid sequence set forth in SEQ ID NO. 40, wherein the amino acids at positions 42, 136, and 498 are E, C, and D, respectively. In some embodiments, the GyrB mutant may comprise the amino acid sequence of SEQ ID NO.40, wherein the amino acids at positions 42, 136, and 498 are E, R, and A, respectively. In some embodiments, the mutated gyrB gene may comprise the nucleotide sequence of SEQ ID NO.5. In some embodiments, the mutated gyrB gene may comprise the nucleotide sequence of SEQ ID NO.6. In some embodiments, the mutated gyrB gene may comprise the nucleotide sequence of SEQ ID NO.7. In some embodiments, the host cell may comprise mutations in the gyrA and gyrB genes that result in decreased gyrase activity. A gyrA gene containing the mutation may express the GyrA mutant, and a gyrB gene containing the mutation may express the GyrB mutant.The GyrA mutant may comprise an H80A mutation at position 80 corresponding to SEQ ID NO.37, and the GyrB mutant may comprise an R136C mutation at position 136 corresponding to SEQ ID NO.39. In some embodiments, the host cell of the present application may comprise down-regulated gyrase expression by knocking down the gyrA gene or the gyrB gene, or knocking out the gyrA gene or the gyrB gene plus expression of a GyrA / GyrB vector. For example, the host cell may comprise down-regulated gyrase expression by knocking down the gyrA gene, or knocking out the gyrA gene and introducing a vector expressing GyrA into the host cell. Knocking down the gyrA gene may be achieved by providing an inhibitory nucleic acid molecule (e.g., shRNA) directed against GyrA. The host cell may comprise down-regulated gyrase expression by knocking down the gyrB gene, or knocking out the gyrB gene and introducing a vector expressing GyrB into the host cell. Knockdown of the gyrB gene can be achieved by providing an inhibitory nucleic acid molecule (eg, shRNA) against GyrB.

[0067] The host cell according to the use of the present application may further include one or more of the following mutations: i) RecA gene mutation, ii) insertion of a lacI gene expression cassette, and iii) RecQ gene knockout. In some embodiments, the host cell may include: i) RecA gene mutation, ii) insertion of a lacI gene expression cassette, and iii) RecQ gene knockout. In other embodiments, the host cell may include: i) the mutated RecA gene comprises the nucleotide sequence shown in SEQ ID NO.44, ii) the inserted lacI gene expression cassette comprises the nucleotide sequence shown in SEQ ID NO.42, or iii) the RecQ gene comprises the nucleotide sequence shown in SEQ ID NO.41. In some embodiments, the host cell may include a RecA gene mutation, wherein the protein activity expressed by the mutated RecA gene is decreased relative to that of the wild-type RecA gene (such as a recombination-deficient mutation), and the mutated RecA gene may be RecA1. In some specific embodiments, the host cell may include a RecA gene mutation, wherein the mutated RecA gene comprises the nucleotide sequence shown in SEQ ID NO.44. In other embodiments, the host cell can include an inserted lacI gene expression cassette comprising a lacIq promoter, a lacI and / or a terminator. In some specific embodiments, the inserted lacI gene expression cassette comprises the nucleotide sequence set forth in SEQ ID NO. 42. In some embodiments, the host cell can include a knockout of the RecQ gene, wherein the RecQ gene comprises the nucleotide sequence set forth in SEQ ID NO. 41.

[0068] In some embodiments, the E. coli host cell may include:

[0069] i) RecA gene mutation, wherein the mutated RecA gene comprises the nucleotide sequence shown in SEQ ID NO. 44,

[0070] ii) inserting a lacI gene expression cassette, wherein the inserted lacI gene expression cassette comprises the nucleotide sequence shown in SEQ ID NO.42, or / and

[0071] iii) RecQ gene knockout, wherein the RecQ gene comprises the nucleotide sequence shown in SEQ ID NO.41. The nucleic acid molecule containing the poly(A) sequence can be contained in a vector, and the vector is contained in a host cell; or it can be integrated into the genome of the host cell. The poly(A) sequence can comprise continuous A bases, and the number of continuous A bases can be, for example, 20-250. The poly(A) sequence can comprise 2-5 continuous A base segments separated by non-A bases, wherein the number of continuous A bases in each continuous A base segment can be, for example, 10-100, and the continuous A base segments can be separated by, for example, 1-20 non-A bases.

[0072] In a fourth aspect, the present application provides a method for replicating or expressing nucleic acid molecules containing poly(A) sequences or poly(T) sequences using host cells, particularly prokaryotic host cells, comprising downregulated gyrase activity or downregulated gyrase expression, including the host cells of the present application and recombinant host cells prepared by the method of the present application.

[0073] Methods for replicating a nucleic acid molecule containing a poly(A) sequence may comprise:

[0074] i) introducing a vector into a host cell, wherein the vector comprises a nucleic acid molecule containing a poly(A) sequence, and

[0075] ii) culturing the host cells under conditions conducive to vector replication.

[0076] Methods for expressing a nucleic acid molecule comprising a poly(A) sequence may comprise:

[0077] i) introducing a vector into a host cell, wherein the vector comprises a nucleic acid molecule containing a poly(A) sequence,

[0078] ii) optionally, culturing the host cell under conditions favorable for replication of the vector, and

[0079] iii) culturing the host cell under conditions conducive to expression of the vector, or

[0080] The vector is extracted from the host cell in step i) or ii), and the vector is transcribed in vitro.

[0081] Methods for expressing a nucleic acid molecule comprising a poly(A) sequence may comprise:

[0082] i) integrating a nucleic acid molecule containing a poly(A) sequence into the genome of a host cell, and

[0083] ii) culturing the host cell under conditions conducive to the expression of the nucleic acid molecule containing the poly(A) sequence.

[0084] The host cell can be an E. coli host cell. In particular, the host cell can be JM108, NEB Stable, Top10, DH5α, DH10B, MG1655, AG1, BL21, DB3.1, DC10B, DH1, E.Cloni 10G, EPI300, EPI400, JM109, JM110, STBL2, STBL3, SURE, TOP10F', XL1-Blue, or XL10-Gold, engineered to have downregulated gyrase activity or downregulated gyrase expression. In some embodiments, the host cell can be JM108, NEB Stable, Top10, DH5α, DH10B, or MG1655, engineered to have downregulated gyrase activity or downregulated gyrase expression.

[0085] In some embodiments, host cells, including recombinant host cells, may contain a mutation in the gyrA or gyrB gene that results in decreased gyrase activity. Host cells containing a mutated gyrA gene can express a GyrA mutant. The GyrA mutant can contain an H80A mutation at position 80 corresponding to SEQ ID NO. 37, or an A569T and T586A mutation at positions 569 and 586 corresponding to SEQ ID NO. 37. In some embodiments, the GyrA mutant can contain the amino acid sequence of SEQ ID NO. 38, wherein the amino acids at positions 80, 569, and 586 are A, A, and T, respectively. In some embodiments, the GyrA mutant can contain the amino acid sequence of SEQ ID NO. 38, wherein the amino acids at positions 80, 569, and 586 are A, T, and A, respectively. In some embodiments, the mutated gyrA gene can contain the nucleotide sequence of SEQ ID NO. 3. In some embodiments, the mutant gyrA gene may comprise the nucleotide sequence set forth in SEQ ID NO. 4. A host cell comprising a mutant gyrB gene may express a GyrB mutant. The GyrB mutant may comprise an E42D mutation at position 42 corresponding to SEQ ID NO. 39, an R136C mutation at position 136 corresponding to SEQ ID NO. 39, or a D498A mutation at position 498 corresponding to SEQ ID NO. 39. In some embodiments, the GyrB mutant may comprise the amino acid sequence set forth in SEQ ID NO. 40, wherein the amino acids at positions 42, 136, and 498 are D, R, and D, respectively. In some embodiments, the GyrB mutant may comprise the amino acid sequence set forth in SEQ ID NO. 40, wherein the amino acids at positions 42, 136, and 498 are E, C, and D, respectively. In some embodiments, the GyrB mutant may comprise the amino acid sequence set forth in SEQ ID NO.40, wherein the amino acids at positions 42, 136, and 498 are E, R, and A, respectively. In some embodiments, the mutated gyrB gene may comprise the nucleotide sequence set forth in SEQ ID NO.5. In some embodiments, the mutated gyrB gene may comprise the nucleotide sequence set forth in SEQ ID NO.6. In some embodiments, the mutated gyrB gene may comprise the nucleotide sequence set forth in SEQ ID NO.7. In some embodiments, the host cell may comprise mutations in the gyrA and gyrB genes that result in decreased gyrase activity. A gyrA gene comprising the mutation may express the GyrA mutant, and a gyrB gene comprising the mutation may express the GyrB mutant.The GyrA mutant may comprise an H80A mutation at position 80 corresponding to SEQ ID NO. 37, and the GyrB mutant may comprise an R136C mutation at position 136 corresponding to SEQ ID NO. 39.

[0086] Host cells, including recombinant host cells, can, in some embodiments, comprise downregulated gyrase expression by knocking down the gyrA or gyrB gene, or knocking out the gyrA or gyrB gene plus expression of a GyrA / GyrB vector. For example, the host cell can comprise downregulated gyrase expression by knocking down the gyrA gene, or knocking out the gyrA gene and introducing into the host cell a vector expressing GyrA. Knockdown of the gyrA gene can be achieved by providing an inhibitory nucleic acid molecule (e.g., shRNA) directed against GyrA. The host cell can comprise downregulated gyrase expression by knocking down the gyrB gene, or knocking out the gyrB gene and introducing into the host cell a vector expressing GyrB. Knockdown of the gyrB gene can be achieved by providing an inhibitory nucleic acid molecule (e.g., shRNA) directed against GyrB.

[0087] The poly(A) sequence may comprise consecutive A bases, and the number of consecutive A bases may be, for example, 20-250. The poly(A) sequence may comprise 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 the consecutive A base segments may be separated by, for example, 1-20 non-A bases.

[0088] Based on the following specific description and examples, other features and advantages of the current disclosure will become clearer, and specific description and examples should not be interpreted as restrictive. The contents of all documents, Genbank records, patents and published patent applications cited in this application are expressly included in this article by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] The following detailed description is given by way of example but is not intended to limit the present invention to the specific embodiments described, and can be better understood in conjunction with the accompanying drawings.

[0090] Figures 1A and 1B are maps of the target (all-in-one) plasmid (Figure 1A) and the Cas9 protein expression plasmid (Figure 1B) used for Escherichia coli gene mutation in this application.

[0091] FIG2 shows a map of a test plasmid containing a poly(A) sequence.

[0092] 3A-3C show Sanger sequencing peaks of poly(A) sequence purity qualified ( FIG. 3A ), poly(A) sequence purity unqualified ( FIG. 3B ), and poly(A) sequence deletion ( FIG. 3C ).

[0093] FIG4 shows the average supercoiling ratio results of different recombinant genotype strains. DETAILED DESCRIPTION

[0094] Unless otherwise specified, the terms used herein have the ordinary meanings in dictionaries, textbooks, technical reference books, or as generally understood by those skilled in the art. The following descriptions of certain terms are intended only to facilitate understanding of this application and are not intended to be limiting of these terms unless otherwise specified.

[0095] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0096] The term "or" refers to a single element of the listed alternative elements unless the context clearly dictates otherwise.

[0097] The term "comprise" or "include" means that the elements, integers or steps are included, but does not exclude the addition of any other elements, integers or steps. In this article, when the term "comprise" or "include" is used, unless otherwise indicated, combinations of the elements, integers or steps mentioned are also covered.

[0098] "Poly(A) structure" or "poly(A) sequence" refers to a nucleotide sequence comprising consecutive repeats of A bases, including nucleotide sequences in which several consecutive A bases are separated by non-A bases. Similarly, "poly(T) structure" or "poly(T) sequence" refers to a nucleotide sequence comprising consecutive repeats of T bases, including nucleotide sequences in which several consecutive T bases are separated by non-T bases.

[0099] "Host cell" and "strain" herein refer to any cell into which an exogenous nucleic acid molecule (e.g., a vector) can be introduced and which allows the exogenous nucleic acid molecule to be replicated or expressed. The host cell of the present application can be a prokaryotic cell, such as Escherichia coli. "E. coli" and "E. coli" are used interchangeably and include wild-type E. coli and E. coli strains containing artificial or natural mutations, such as JM108, NEB Stable, Top10, DH5α, or DH10B strains.

[0100] "Supercoiling," which includes (+) supercoiling and (-) supercoiling, refers to the helical state of a DNA chain. The main difference between (+) and (-) supercoiling of DNA is that during (+) supercoiling, the DNA chain is excessively coiled compared to its relaxed state, while during (-) supercoiling, the DNA chain is coiled compared to its relaxed state. The DNA of most organisms is (-) supercoiled in its normal state, while (+) supercoiling occurs only during specific cellular functions.

[0101] Gyrase, also known as gyrase or gyrase, is a type of helicase, specifically a type II topoisomerase. This enzyme can introduce (-) supercoils into DNA and convert (+) supercoils into (-) supercoils. It participates in important processes such as replication, transcription, repair, and recombination in prokaryotic cells. Gyrase is composed of GyrA and GyrB subunits. GyrA is primarily responsible for forming and closing gaps in the DNA double helix, while the GyrB subunit primarily mediates energy transduction and ATP hydrolysis. GyrA is encoded by the gyrA gene, and GyrB is encoded by the gyrB gene. It is known that H80A mutation, A569T and T586A double mutation in GyrA, E42D mutation, R136C mutation, and D498A mutation in GyrB can cause a decrease in gyrase activity (Hockings SC, Maxwell A. (2002) J Mol Biol. 318(2):351-9; Oram M, Fisher LM. (1992) J Bacteriol. 174(12):4175-8; Gross CH et al., (2003) Antimicrob Agents Chemother. 47(3):1037-46; Contreras A, Maxwell A. (1992) Mol Microbiol. 6(12):1617-24; Noble CG, Maxwell A. (2002) J Mol Biol. 318(2):361-71).

[0102] "Stability" in this context refers to the structural (base) integrity of poly(A) and poly(T) sequences, as well as the purity of the structural genes. "Integrity" refers to the identity of the poly(A) sequence replicated or expressed in the strain with the poly(A) sequence originally constructed and introduced into the strain.

[0103] Generally speaking, "downregulate" means to reduce. For example, "downregulating" gyrase activity refers to reducing the activity of GyrA, GyrB, or both, by mutating the gyrA and / or gyrB genes, increasing GyrI expression, or the like, including the activity that converts (+) supercoils to (-) supercoils. "Downregulating" gyrase expression refers to reducing the expression of GyrA, GyrB, or both, by knocking down the gyrA and / or gyrB genes, or the like.

[0104] "Knockdown" as used herein refers to the reduction of expression of a target gene or sequence, such as the gyrA and / or gyrB genes herein, by specifically degrading the target mRNA, or interfering with normal RNA translation or splicing, through certain means, such as RNAi, but does not include complete elimination of gyrA and / or gyrB gene expression. "Knockout" refers to the complete elimination of expression of a specific gene or sequence by removing it through certain means, such as homologous recombination.

[0105] "Introduction" refers to the introduction of a vector into a host cell by certain means, such as transfection, transduction, or transformation. "Integration" refers to the insertion of a sequence into the genome by certain means, such as homologous recombination, enabling expression along with the expression of the genomic sequence.

[0106] "Recombinant" cells are cells that have been modified through DNA recombination techniques, such as altering gene sequence, gene expression pattern, or gene expression level. "Recombinant" host cells are cells that have been modified through DNA recombination techniques, such as altering gene sequence, gene expression pattern, or gene expression level, and that can introduce exogenous nucleic acid molecules and allow them to replicate or express.

[0107] "Vector" refers to a naturally occurring or synthetic DNA fragment, including single-stranded and double-stranded DNA fragments, such as chemically synthesized DNA fragments, natural plasmids, or modified viral genomes. Exogenous DNA fragments can be inserted into the vector to clone and / or express the exogenous DNA fragment. The vector may contain, for example, an origin of replication, a selective marker or reporter gene, a multiple cloning site (MCS), etc. The term includes linear DNA fragments (such as PCR products, linearized plasmid fragments, etc.), plasmid vectors, viral vectors, bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), etc. When the vector is double-stranded DNA, the description of the order of the elements and the direction of the element sequence is for one of the DNA chains.

[0108] "In vitro transcription" or "IVT" refers to the process of producing RNA using DNA as a template in a cell-free system in the presence of RNA transcriptase, NTPs, and other conditions, mimicking the in vivo transcription process. When using a plasmid vector as a DNA template, the plasmid is linearized by enzyme digestion using enzyme sites prior to in vitro transcription.

[0109] During mRNA maturation, a poly(A) tail is added to the end. This poly(A) tail significantly impacts the structural stability and translation efficiency of mRNA. Current mRNA production typically includes a poly(A) tail coding sequence in the vector, consisting of a sequence consisting of consecutive repeats of A bases or fragments of consecutive repeats of A bases separated by non-A bases. This poly(A) tail coding sequence often loses a significant number of A-containing nucleotides during host cell propagation and passage. Currently, there is no effective way to prevent this loss. Furthermore, because the poly(A) tail coding sequence does not naturally exist in biological cells, it is difficult to find effective methods to study the reasons for the instability of this sequence structure in vivo.

[0110] The inventors of the present application unexpectedly discovered that when the activity of gyrase in a prokaryotic host cell, such as an Escherichia coli host cell, is downregulated, a vector containing a poly(A) sequence has improved replication stability in the prokaryotic host cell, that is, the loss of A bases in the poly(A) sequence is improved during the replication of the vector.

[0111] It is well known in the art that gyrase is an important DNA helicase in prokaryotic cells, involved in processes such as DNA replication, repair, recombination, and transcription. Drugs that target and inhibit gyrase activity, such as quinolones and coumarins, are used as antibacterial drugs and cause irreversible damage to bacterial DNA.

[0112] Given the important roles of GyrA and GyrB, it is not conceivable to downregulate the activity of GyrA, GyrB, or the gyrase composed of them in a strain for production purposes, except for gene or protein research purposes, because such downregulation is likely to cause the death of the strain.

[0113] Surprisingly, however, downregulating gyrase activity in host cells surprisingly maintained the original plasmid yield. This suggests that by downregulating gyrase activity in host cells, high-stability poly(A) sequence vectors can be produced with higher plasmid yields. This holds significant promise for the production of poly(A) sequence-containing vectors. Furthermore, plasmid quality, such as the supercoiling ratio, is maintained at the original level.

[0114] Gyrase is composed of GyrA and GyrB subunits, encoded by the gyrA gene and the gyrB gene, respectively. Studies have shown that H80A mutations, A569T and T586A double mutations in GyrA, and E42D, R136C, and D498A mutations in GyrB can reduce gyrase activity.

[0115] Using this existing information, the inventors of the present application made corresponding mutations to the gyrA gene or the gyrB gene in the host cell through gene editing, thereby reducing the activity of gyrase.

[0116] Specifically, a vector is used as a medium to introduce a Cas9 enzyme into a host cell, such as an Escherichia coli host cell, in particular JM108, NEB Stable, Top10, DH5α and DH10B as vector preparation strains, and i) an sgRNA comprising the nucleotide sequence shown in SEQ ID NO. 8 and a donor sequence comprising the nucleotide sequence shown in SEQ ID NO. 13, ii) an sgRNA comprising the nucleotide sequence shown in SEQ ID NO. 9 and a donor sequence comprising the nucleotide sequence shown in SEQ ID NO. 14, iii) an sgRNA comprising the nucleotide sequence shown in SEQ ID NO. 10 and a donor sequence comprising the nucleotide sequence shown in SEQ ID NO. 15, iv) an sgRNA comprising the nucleotide sequence shown in SEQ ID NO. 11 and a donor sequence comprising the nucleotide sequence shown in SEQ ID NO. 16, or v) an sgRNA comprising the nucleotide sequence shown in SEQ ID NO. 12 and a donor sequence comprising the nucleotide sequence shown in SEQ ID NO. 17. In the resulting host cell, the gyrA gene comprises the nucleotide sequence shown in SEQ ID NO. 3 or 4, or the gyrB gene comprises the nucleotide sequence shown in SEQ ID NO. 5, 6, or 7. A combination of the above-mentioned sgRNA / donor sequence, such as a combination of i) and iv), can also be introduced into the host cell.

[0117] When any host cell obtained in this way is used to replicate or express a vector containing a poly(A) sequence, the replication stability of the poly(A) sequence is improved compared to a host cell in which the gyrase activity is not downregulated. In particular, this improvement effect becomes more obvious as the number of replications and the number of generations of host cell passages increase.

[0118] The data from mutations in the gyrA gene alone, the gyrB gene alone, and both the gyrA and gyrB genes indicate that gyrase activity is affected by both GyrA and GyrB. In other words, mutations in either GyrA or GyrB can affect gyrase activity.

[0119] By downregulating the expression of gyrase in the host cells, the replication and expression stability of poly(A) in the vector can also be improved.

[0120] Specifically, the expression of gyrase in the host cell can be down-regulated by knocking down the gyrA gene or the gyrB gene, or knocking out the gyrA gene or the gyrB gene plus GyrA / GyrB vector expression, etc. The host cell can include the gyrase expression level that is down-regulated by knocking down the gyrA gene, or knocking out the gyrA gene and introducing a vector expressing GyrA into the host cell, etc. Knocking down the gyrA gene can be achieved by providing an inhibitory nucleic acid molecule (e.g., shRNA) for GyrA. Recombinant host cells can include the gyrase expression level that is down-regulated by knocking down the gyrB gene, or knocking out the gyrB gene and introducing a vector expressing GyrB into the host cell, etc. Knocking down the gyrB gene can be achieved by providing an inhibitory nucleic acid molecule (e.g., shRNA) for GyrB. Knocking out can be performed, for example, by homologous recombination, and the operation of homologous recombination is well known to those skilled in the art. The technology of constructing an expression vector for GyrA or GyrB and introducing it into the host cell is well known to those skilled in the art.

[0121] The recombinant host cells obtained by the above method, as well as the recombinant host cells modified by any other method to contain down-regulated gyrase activity or down-regulated gyrase expression, in particular, the recombinant vector preparation strains modified to contain down-regulated gyrase activity or down-regulated gyrase expression, such as JM108, NEB Stable, Top10, DH5α, DH10B, MG1655, AG1, BL21, DB3.1, DC10B, DH1, E.Cloni 10G, EPI300, EPI400, JM109, JM110, STBL2, STBL3, SURE, TOP10F', XL1-Blue or XL10-Gold, can be used to replicate or express nucleic acid molecules containing poly(A) sequences. In addition, natural mutant strains containing downregulated gyrase activity or downregulated gyrase expression due to various reasons, such as natural mutation of the gyrA gene or gyrB gene, natural knockdown of the gyrA gene or gyrB gene, etc., can also be used to replicate or express nucleic acid molecules containing poly(A) sequences.

[0122] Furthermore, the stability of nucleic acid molecules containing poly (A) sequences can be further improved by combining mutations of other genotypes such as RecA, lacI, and RecQ. Specifically, based on mutations of only the gyrA gene, only the gyrB gene, or the gyrA gene + gyrB gene in the E. coli host cell, the method further comprises mutation of the RecA gene, insertion of a lacI gene expression cassette, or / and knockout of the RecQ gene. After the host cell mutation, the protein activity of the RecA gene expressed relative to the wild-type RecA gene decreases (e.g., a recombination-deficient mutation), and the mutated RecA gene can be RecA1. The site for inserting the lacI gene expression cassette is the yghX gene site of the E. coli host cell, and the lacI gene expression cassette includes a lacIq promoter, lacI, and / or a terminator. The E. coli host cell genotype can be, for example, JM108 [gyrA (H80A), RecA1, lacIq] and JM108 [gyrA (H80A), RecA1, lacIq, RecQ].

[0123] Specifically, one can i) introduce a vector containing a nucleic acid molecule containing a poly(A) sequence into an E. coli host cell, and ii) culture the E. coli host cell under conditions that are conducive to vector replication.

[0124] Alternatively, one can i) introduce a vector into an E. coli host cell, the vector comprising a nucleic acid molecule containing a poly(A) sequence, ii) optionally, culture the E. coli host cell under conditions conducive to vector replication, and iii) culture the E. coli host cell under conditions conducive to vector expression, or extract the vector from the E. coli host cell of step i) or ii) and perform in vitro transcription on the vector.

[0125] The vector can be any suitable vector, such as a plasmid vector, a recombinant adenoviral vector, a recombinant lentiviral vector, etc. Regarding the construction of the vector, the replication conditions and specific operations of various vectors, as well as the expression conditions and specific operations, those skilled in the art can perform them according to actual conditions and needs.

[0126] Alternatively, one may i) integrate a nucleic acid molecule containing a poly(A) sequence into the genome of an E. coli host cell, and ii) culture the E. coli host cell under conditions that favor expression of the nucleic acid molecule containing a poly(A) sequence.

[0127] Those skilled in the art can integrate a nucleic acid molecule containing a poly(A) sequence into the genome of an E. coli host cell, for example, through homologous recombination, and express the nucleic acid molecule containing the poly(A) sequence. Furthermore, those skilled in the art can identify conditions conducive to the expression of nucleic acid molecules containing a poly(A) sequence based on actual conditions.

[0128] Nucleic acid molecules containing poly(A) sequences can encode mRNA with a poly(A) tail.

[0129] The poly(A) sequence can be any poly(A) sequence as long as it comprises continuous repeats of A bases. In some embodiments, the poly(A) sequence can comprise one continuous A base segment. In some embodiments, the continuous A base segment can comprise 20-250 continuous A bases. In some embodiments, the poly(A) sequence can comprise multiple, for example, 2-5, continuous A base segments separated by non-A bases, wherein each continuous A base segment can comprise 10-100 continuous A bases and be separated from each other by 1-20 non-A bases.

[0130] The inventors of the present application tested the replication of various poly(A) sequences, including continuous poly(A) sequences such as 80A, 100A, and 120A, as well as segmented poly(A) sequences such as 30A+70A and 30A+30A+43A, in recombinant host cells engineered to contain downregulated gyrase activity or downregulated gyrase expression. The results showed that the host cells of the present application improved the replication stability of various poly(A) sequences, with a particularly significant effect on the highly unstable 120A sequence.

[0131] The present application also relates to the use of host cells comprising downregulated gyrase activity or downregulated gyrase expression, including natural mutants and recombinant strains, in replicating or expressing nucleic acid molecules containing poly(A) sequences.

[0132] Recombinant host cells obtained by the method of the present application or by any other method and modified to contain downregulated gyrase activity or downregulated gyrase expression, including host cells naturally existing in nature, are all within the scope of protection of the present application.

[0133] The following is a further detailed description of the present application in conjunction with specific embodiments, which are provided by way of example only and do not limit the scope of protection of the present application.

[0134] Example 1. Construction of strain mutants

[0135] The CRISPR-Cas9 technology was used to mutate the gyrA and / or gyrB genes in the genome of the JM108, NEB Stable, Top10, DH5α or DH10B strain, wherein the wild-type gyrA and gyrB gene sequences before mutation are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.

[0136] Specifically, in the JM108 strain, the gyrA gene was mutated so that its expression product contained the H80A, A569T, and T586A mutations. Furthermore, the gyrB gene was mutated so that its expression product contained the E42D, R136C, or D498A mutations.

[0137] 1.1.1 Upload SEQ ID NO. 1 and SEQ ID NO. 2 to the sgRNA design website (www.atum.bio / eCommerce / cas9 / input) and design 20 bp sgRNAs for the fragment containing H80 in gyrA, the fragment containing A569 & T586 in gyrA, the fragment containing E42 in gyrB, the fragment containing R136 in gyrB, and the fragment containing D498 in gyrB, as shown in SEQ ID NOs. 8-12.

[0138] 1.1.2 The above sgRNAs and the corresponding donor sequences SEQ ID NOs. 13-17 were constructed on the target (all-in-one) plasmid. The plasmid map is shown in Figure 1A.

[0139] 1.1.3 Chemically transform the pCas plasmid (see Figure 1B for the plasmid map) into chemically competent JM108, NEB Stable, Top10, DH5α, or DH10B cells. Plate onto solid LB kanamycin-resistant plates and incubate at 30°C for 14 h. Single colonies were selected and verified for positive clones by colony PCR using primers shown in SEQ ID NOs. 18 and 19, followed by Sanger sequencing.

[0140] Table 1. Colony PCR primer sequences

[0141] 1.1.4 The target plasmid was electroporated into the five competent cells containing the pCas plasmid, and the transformation plate was spread onto LB solid kanamycin and spectinomycin double-resistance plates. The plates were incubated at 30°C for 14 h, and single colonies were picked. Positive clones were identified by colony PCR and Sanger sequencing. The primers used were SEQ ID NOs. 20-29 to obtain successfully edited positive clones.

[0142] 1.1.5 Add 0.5 mM IPTG inducer to a 4 mL LB tube containing the positive clone solution and culture at 30°C for 7-8 hours to eliminate the spectinomycin-resistant target plasmid.

[0143] 1.1.6 Incubate 4 mL of the LB tube containing the positive clone at 37°C for 7-8 hours to eliminate the kanamycin-resistant pCas plasmid.

[0144] 1.1.7 Prepare the obtained strain into competent form for later use.

[0145] When preparing a strain containing two of the above gene mutation combinations, first introduce the pCas plasmid as described in 1.1.3. Then, introduce the target plasmid containing the sgRNA and donor sequence corresponding to one mutation as described in 1.1.4. Screen for successfully edited clones. Remove the target plasmid from the strain as described in 1.1.5 and prepare the strain to be competent. Then, return to 1.1.4 and introduce the target plasmid containing the sgRNA and donor sequence corresponding to the other mutation. Screen for successfully edited clones, and then proceed with steps 1.1.5-1.1.7. Specifically, in preparing a strain containing gyrA (H80A) and gyrB (R136C) mutations, first introduce a target plasmid containing the sgRNA and donor sequence corresponding to gyrA (H80A) in 1.1.4, remove the target plasmid from the strain through 1.1.5, prepare the strain to be competent, and then return to 1.1.4 to introduce a target plasmid containing the sgRNA and donor sequence corresponding to gyrB (R136C).

[0146] Similarly, when preparing strains containing three or more combinations of the above gene mutations, follow 1.1.3-1.1.4-1.1.5-(1.1.4-1.1.5) n -1.1.6-1.1.7 in the order of operation, wherein n depends on the type of the above-mentioned gene mutation to be included, and n is greater than or equal to 2.

[0147] The sequences of the gyrA gene after editing are shown in SEQ ID NOs. 3 and 4 (corresponding to H80A and A569T&T586A, respectively), and the sequences of the gyrB gene after editing are shown in SEQ ID NOs. 5-7 (corresponding to E42D, R136C, and D498A, respectively).

[0148] Example 2. Preparation of plasmid containing poly(A) sequence

[0149] Five test plasmids containing continuous poly(A) sequences (SEQ ID NOs. 30-32) or segmented poly(A) sequences (SEQ ID NOs. 33 and 34) were prepared, namely 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).

[0150] The map of the poly(A)-test 1 (80A) plasmid is shown in Figure 2 , wherein the sequence encoding the mRNA portion is shown in SEQ ID NO. 35. The maps of the other test plasmids are identical to Figure 2 , except for the poly(A) sequences, and the sequences encoding the mRNA portions are identical to SEQ ID NO. 35 , except for the poly(A) sequences.

[0151] Specifically, a nucleotide sequence containing a T7 promoter, a sequence encoding a 5'UTR, a sequence encoding EGFP, a sequence encoding a 3'UTR, a poly(A) sequence, and a linearization site BspQI (SEQ ID NO: 35) was synthesized in sequence (GenScript Biotech Co., Ltd.) and assembled into the pVAX1 kanamycin-resistant vector (GenScript Biotech Co., Ltd., SEQ ID NO: 36) using the Gibson method.

[0152] Positive clones were selected for Sanger sequencing, and five test plasmids with correct sequencing were obtained.

[0153] Example 3. Escherichia coli with gyrA gene mutation improves the replication stability of poly(A)

[0154] One plasmid from each of the five test plasmids obtained in Example 2 was randomly selected and transformed into the competent gyrA mutant strain obtained in Example 1, as well as competent commercial strains, including NEB Stable. Eight colonies were selected from each transformed plate and cultured overnight in LB liquid medium at 37°C, 220 rpm. This constituted the first generation. Plasmids were extracted from the first generation culture medium, and the first generation culture medium was used as a seed medium at a 1:1000 dilution and cultured overnight. This constituted the second generation, and continued to be subcultured until the third generation.

[0155] Plasmids extracted from the first and third generation clones were selected and Sanger sequencing was used to verify the effective base number of the poly(A) sequence and the purity qualification rate. The effective base number of poly(A) is defined as the number of bases between the first and last bottom peaks of the poly(A) sequence that do not exceed 50% of the A base. When the error between the effective base number of poly(A) and the initial base number of the transformed plasmid is within 1nt, the bottom peaks of the last three effective A bases are less than 50%, and the bottom peaks of the remaining poly(A) sequence are less than 10%, it is recorded as qualified purity, otherwise it is recorded as unqualified, and the purity qualification rate is calculated based on this. Figures 3A-3C show exemplary Sanger sequencing peak graphs of qualified poly(A) purity (3A), unqualified purity (3B) and poly(A) deletion (3C), respectively.

[0156] The poly(A) base counts and purity pass rates of these subcultured clones were used to determine the stability of poly(A) replication in different mutant host strains. A higher poly(A) base count and a higher poly(A) purity pass rate as determined by Sanger sequencing indicate a more robust poly(A) replication in the mutant host strain.

[0157] Table 2 shows the poly(A) effective bases of poly(A)-test 3 plasmids in the first and third generation strains, and the average purity qualification rates of the five test plasmids in the first and third generation gyrA gene mutant strains.

[0158] Sanger sequencing revealed that in the first-generation NEB Stable strain, the poly(A)-test 3 plasmid had a poly(A) effective base length of 115.6 nt, with an average poly(A) purity pass rate of 62.5% for the five test plasmids. In the third-generation clone, the poly(A)-test 3 plasmid had a poly(A) effective base length of 114.8 nt, with an average poly(A) purity pass rate of 47.5% for the five test plasmids. Furthermore, in the NEB Stable strain with the gyrA gene mutation, both the poly(A) effective base length and the purity pass rate were higher than those of the commercial strain before modification, regardless of whether it was the first or third generation. In the first-generation NEB Stable [gyrA(H80A)] and NEB Stable [gyrA(A569T,T586A)] clones, the poly(A)-test 3 plasmids had effective poly(A) base lengths of 117.6 nt and 116.3 nt, respectively, and the average poly(A) purity pass rates for the five test plasmids were 80.0% and 67.5%, respectively. In the third-generation clones, the poly(A)-test 3 plasmids had effective poly(A) base lengths of 117.6 nt and 116.1 nt, respectively, and the average poly(A) purity pass rates for the five test plasmids were 72.5% and 65.0%, respectively. Similarly, JM108, TOP10, DH5α, and DH10B, with gyrA gene mutations, showed higher poly(A) base lengths and higher purity pass rates compared to their unmutated commercial strains, both in the first and third generations.

[0159] The above data indicate that gyrA gene mutation in E. coli has a positive effect on the replication stability of poly(A) plasmids.

[0160] Table 2. Stability of poly(A) plasmids in commercial strains and gyrA mutant strains

[0161] Example 4. Escherichia coli with gyrB gene mutation improves the replication stability of poly(A)

[0162] Following the procedures of Example 3, one plasmid was randomly selected from each of the five test plasmids obtained in Example 2 and transformed into the competent gyrB gene mutant strain obtained in Example 1, as well as competent commercial strains, including NEB Stable. The number of effective bases in the poly(A) sequences and the purity pass rate of the first- and third-generation clones of these strains were counted.

[0163] Table 3 summarizes the poly(A) effective bases of poly(A)-test 3 plasmids in the first and third generation strains, as well as the average poly(A) purity pass rates of the five test plasmids in the first and third generation gyrB gene mutant strains.

[0164] Sanger sequencing revealed that in the first-generation NEB Stable strain, the poly(A)-test 3 plasmid had a poly(A) effective base length of 115.6 nt, with an average poly(A) purity pass rate of 62.5% for the five test plasmids. The poly(A)-test 3 plasmid in the third-generation clone had a poly(A) effective base length of 114.8 nt, with an average poly(A) purity pass rate of 47.5% for the five test plasmids. In the NEB Stable strain containing the mutant gyrB strain, the poly(A) effective base length and purity pass rate of plasmids in both the first- and third-generation clones were higher than those in the commercial strain before modification. Among them, in NEB Stable[gyrB(E42D)], NEB Stable[gyrB(R136C)], and NEB Stable[gyrB(D498A)], the effective poly(A) base numbers of the poly(A)-test 3 plasmids in the first-generation clones were 116.2 nt, 117.6 nt, and 116.9, respectively, and the average poly(A) purity pass rates of the five test plasmids were 67.5%, 77.5%, and 72.5%, respectively. The effective poly(A) base numbers of the poly(A)-test 3 plasmids in the third-generation clones were 115.2 nt, 117.1 nt, and 116.3 nt, respectively, and the average poly(A) purity pass rates of the five test plasmids were 57.5%, 67.5%, and 60.0%, respectively. Similarly, JM108, TOP10, DH5α, and DH10B with gyrB gene mutations showed higher levels of poly(A) effective base numbers and / or purity qualification rates, especially the purity qualification rates, in both the first and third generations compared to their respective unmutated commercial strains.

[0165] The above data indicate that gyrB gene mutation in Escherichia coli has a positive effect on the replication stability of poly(A) plasmids.

[0166] Table 3. Stability of poly(A) plasmids in commercial strains and gyrB mutant strains

[0167] Example 5. Escherichia coli with gyrA & gyrB gene mutations improves poly(A) replication stability

[0168] Following the procedures of Example 3, one plasmid was randomly selected from each of the five test plasmids obtained in Example 2 and transformed into the competent gyrA and gyrB gene mutant strains obtained in Example 1, as well as competent commercial strains, including NEB Stable. The number of effective bases in the poly(A) sequences and the purity qualification rate of the first- and third-generation clones were counted.

[0169] Table 4 summarizes the poly(A) effective base numbers of the poly(A)-test 3 plasmids in the first and third generation strains, as well as the poly(A) purity qualification rates of the five test plasmids in the first and third generation strains.

[0170] Sanger sequencing revealed that in the first-generation NEB Stable strain, the poly(A)-test 3 plasmid had an effective poly(A) base length of 115.6 nt, with an average poly(A) purity pass rate of 62.5% for the five test plasmids. In the third-generation clone, the poly(A)-test 3 plasmid had an effective poly(A) base length of 114.8 nt, with an average poly(A) purity pass rate of 47.5% for the five test plasmids. In NEB Stable strains with simultaneous mutations in both the gyrA and gyrB genes, both the first and third generations showed higher poly(A) base lengths and higher purity pass rates than the commercial strains before modification. In the NEB Stable strain [gyrA(H80A)gyrB(R136C)], the poly(A)-test 3 plasmid in the first-generation clones had an effective poly(A) base length of 118.1 nt, with an average poly(A) purity pass rate of 87.5% for the five test plasmids. The poly(A)-test 3 plasmid in the third-generation clones had an effective poly(A) base length of 117.9 nt, with an average poly(A) purity pass rate of 75.0% for the five test plasmids. Similarly, JM108, TOP10, DH5α, and DH10B, which have gyrA and gyrB gene mutations, showed higher poly(A) base lengths and higher purity pass rates compared to their unmutated commercial strains, both in the first and third generations.

[0171] The above data indicate that simultaneous mutation of gyrA and gyrB genes in E. coli has a positive effect on the replication stability of poly(A) plasmids.

[0172] Table 4. Stability of poly(A) plasmids in hosts with gyrA and gyrB gene mutations and commercial strains

[0173] Example 6. Plasmid yield in mutant strains

[0174] The poly(A)-test 3 plasmid obtained in Example 2 was selected and transformed into mutant strains JM108[gyrA(H80A)], NEB Stable[gyrA(H80A)], DH10B[gyrA(H80A)], JM108[gyrB(R136C)], and JM108[gyrA(H80A)gyrB(R136C)] with good poly(A) stability, as well as the corresponding commercial strains JM108, NEB Stable, and DH10B. Single clones were randomly selected and transferred to 4 mL of LB liquid medium and cultured at 30°C and 200 rpm for 15 h to obtain seed liquid. The seed liquid was inoculated into 100 mL of LB liquid medium at a dilution of 1:1000, cultured at 37°C and 200 rpm for 13 h, the plasmid was extracted, and the plasmid yield was measured using a nanodrop analyzer.

[0175] Table 5 summarizes the plasmid yields of different strains transformed with the poly(A)-test 3 plasmid. It can be seen that the plasmid yields obtained in the mutant strains are almost the same as those in the non-mutated strains.

[0176] Table 5. Plasmid yields of poly(A) plasmids in gyrA and / or gyrB gene mutant hosts and commercial strains

[0177] Example 7. Supercoiled ratio of plasmids in mutant strains

[0178] The poly(A)-test 2 (100A) plasmid and the poly(A)-test 3 (120A) plasmid obtained in Example 2 were selected and transformed into the commercial strain JM108, as well as the mutant strains JM108 [gyrA (H80A)], JM108 [gyrB (R136C)], and JM108 [gyrA (H80A) gyrB (R136C)], respectively, according to the procedures of Example 3. Single clones were randomly selected for serial passage experiments.

[0179] Plasmids were extracted from the first and third generation strains, 200 ng of the extracted plasmids were subjected to DNA gel electrophoresis, and the supercoiling ratio of the plasmids was analyzed using the fully automatic gel imaging analysis system GIS300.

[0180] The average supercoiling ratio corresponding to each strain is shown in Figure 4. It can be seen that the supercoiling ratio of the plasmid obtained from the mutant strain is almost the same as that of JM108, both greater than 80%.

[0181] In summary, mutation of the gyrA and / or gyrB gene enables the E. coli host to improve the replication stability of the poly(A) plasmid without affecting the quality of the plasmid, such as the supercoiling ratio.

[0182] Example 8. Escherichia coli with gyrA mutations combined with other genotype mutations further improves poly(A) sequence replication stability

[0183] The RecA gene expresses an ATP-dependent DNA recombinase, which helps DNA find and pair with the correct sequence when damaged or in need of replication, thereby facilitating DNA repair or replication. RecA1 is a recombination-deficient mutation in the RecA gene that reduces nonspecific DNA recombination, lowering the probability of recombination with exogenous DNA and making inserted exogenous DNA more stable, facilitating DNA transformation and the extraction of high-purity plasmids.

[0184] The Lac promoter is a common functional element in commercial vectors and is often used as a promoter for blue-white plasmid screening. For example, pUC57 has a multiple cloning site (MCS) located immediately adjacent to the Lac promoter. Inserting mRNA functional sequences into the MCS often results in the appearance of a poly(A) sequence downstream of the Lac promoter. The transcriptional activity of the Lac promoter can lead to decreased stability of the poly(A) sequence. lacIq is a variant of the lacI gene that can express a repressor protein, thereby reducing the transcriptional activity of the Lac promoter under non-induced conditions and enhancing the stability of the downstream poly(A) sequence.

[0185] The RecQ gene belongs to the RecQ helicase family and is involved in DNA unwinding, replication, and DNA damage repair. Knockout of the RecQ gene stabilizes inserted exogenous DNA and facilitates efficient DNA transformation.

[0186] Referring to the method of Example 1, CRISPR-Cas9 technology was used to mutate, knock out, or knock in relevant genes in the genome of Escherichia coli JM108 strain. The gene to be knocked out is as follows: RecQ (SEQ ID NO. 41), the gene to be knocked in is as follows: lacIq promoter + lacI + rrnB T1 terminator + rrnB T2 terminator (SEQ ID NO. 42), and the wild-type gene to be mutated is as follows: RecA (SEQ ID NO. 43). Specifically, the wild-type RecA gene was mutated to RecA1 (SEQ ID NO. 44).

[0187] E. coli mutant strains were constructed and prepared using the method described in Example 1, along with the corresponding competent JM108 [gyrA(H80A), RecA1, lacIq] and JM108 [gyrA(H80A), RecA1, lacIq, RecQ] mutant strains. The sgRNA sequences targeting RecQ, yghX (Gene ID: 2847694), and RecA are shown in SEQ ID NOs. 45-47, the corresponding donor sequences are shown in SEQ ID NOs. 48-50, and the colony PCR primer sequences are shown in Table 6.

[0188] Table 6. Colony PCR primer sequences

[0189] Following the procedures of Example 3, one plasmid was randomly selected from each of the five test plasmids obtained in Example 2 and transformed into competent JM108 [gyrA(H80A), RecA1, lacIq] and JM108 [gyrA(H80A), RecA1, lacIq, RecQ] mutant strains obtained according to the method of Example 1, as well as the corresponding commercial JM108 strain. The number of effective bases in the poly(A) sequence and the purity qualification rate of the first- and third-generation clones were calculated.

[0190] Table 7 summarizes the poly(A) effective base numbers of the poly(A)-test 3 plasmids in the first and third generation strains, as well as the poly(A) purity qualification rates of the five test plasmids in the first and third generation strains.

[0191] Sanger sequencing revealed that the poly(A)-test 3 plasmid in the first-generation JM108 strain had an effective poly(A) base length of 116.9 nt, with an average poly(A) purity pass rate of 55.0% for the five test plasmids. The poly(A)-test 3 plasmid in the third-generation clone had an effective poly(A) base length of 116.5 nt, with an average poly(A) purity pass rate of 47.5% for the five test plasmids. Furthermore, the poly(A) effective base length and purity pass rate of the mutant strains, whether in the first or third generation, were higher than those of the commercial JM108 strain. Among them, in the first-generation JM108[gyrA(H80A), RecA1, lacIq] and JM108[gyrA(H80A), RecA1, lacIq, RecQ] mutant strains, the effective poly(A) base numbers of the poly(A)-test 3 plasmids were 118.6nt and 118.3nt, respectively, and the average poly(A) purity pass rates of the five test plasmids were 83.2% and 85.5%, respectively. In the third-generation clones, the effective poly(A) base numbers of the poly(A)-test 3 plasmids were 117.9nt and 118.1nt, respectively, and the average poly(A) purity pass rates of the five test plasmids were 80.5% and 82.5%, respectively.

[0192] The above data indicate that the combined mutation or knockout of gyrA (H80A) and other genotypes such as RecA1, lacIq, and RecQ can further improve the replication stability of the strain for poly(A) plasmids.

[0193] Table 7. Replication stability of poly(A) plasmids in hosts and commercial strains with combinations of gyrA mutations and other genotype mutations

[0194] Example 9. Escherichia coli with gyrA mutations combined with other genotype mutations further improves the stability of poly(A) plasmid serial replication

[0195] The five test plasmids obtained in Example 2 were transformed into JM108 [gyrA(H80A), RecA1, lacIq, RecQ] and the commercial strain JM108 obtained in Example 1. Sixteen clones were selected from each transformed plate and cultured overnight in LB liquid medium at 37°C, 220 rpm. This culture served as the template for passage (P0 generation). 200 μl of the P0 generation culture solution was mixed with 200 μl of 50% glycerol and stored at -80°C. Plasmids from the P0 generation culture solution were extracted, and the effective base count and peak purity of the poly(A) sequence were verified using Sanger sequencing. Four clones that passed the Sanger sequencing were selected and inoculated with the corresponding P0 generation glycerol stock at a dilution of 1:1000 and cultured overnight. This served as the first generation (P1 generation). The first generation culture solution was also inoculated with the seed solution at a dilution of 1:1000 and cultured overnight. This served as the second generation. Continuous passages were performed until the tenth generation (P10 generation).

[0196] Select the plasmid that extracts from the first generation (P1 generation) to the tenth generation (P10 generation) passage clone, use Sanger order-checking to verify whether the effective base number and the purity of poly (A) sequence are qualified.When effective base number and purity are qualified, be recorded as clone qualified, otherwise be recorded as clone unqualified, calculate clone qualification rate thus.From each generation passage clone qualification rate data, judge that different mutant host bacteria replicate poly (A) structure stability.The passage clone qualification rate data of each generation is higher, illustrates that this mutant host is higher to the stability of replicating poly (A) structure.Table 8 shows the poly (A) clone qualification rate of 5 kinds of test plasmids in the first generation (P1 generation) to the tenth generation (P10 generation) bacterial strain.

[0197] Sanger sequencing revealed that in the first-generation JM108 strain, the average cloning pass rate for the five tested plasmids was 60.7%. This rate decreased rapidly with passage progression, falling below 42.0% by the fifth passage and reaching only 14.6% by the tenth passage. In contrast, the poly(A) cloning pass rate for the JM108 strain harboring the [gyrA(H80A), RecA1, lacIq, RecQ] gene mutations was higher than that of the commercial JM108 strain at both the first and tenth passages. Specifically, the average cloning pass rate for the five tested plasmids in the first-generation JM108 strain harboring the [gyrA(H80A), RecA1, lacIq, RecQ] gene mutations was 87.5% and remained at 42.0% by the tenth passage, exceeding the fifth-generation pass rate of the commercial JM108 strain.

[0198] The above data indicate that the combined mutation or knockout of gyrA(H80A) and other genotypes such as RecA1, lacIq, and RecQ has a positive effect on the stability of poly(A) plasmids.

[0199] Table 8 Cloning qualification rate of poly(A) plasmid in commercial strains and genotype mutant strains

[0200] Especially for plasmids with a more difficult poly(A) structure, such as plasmids with continuous A bases greater than 120 bp, when using the strain of the present application, compared with commercial strains, the advantage of poly(A) replication stability gradually increases with the continuous passage.

[0201] The implementation methods of the present application are not limited to those described in the above embodiments. Without departing from the spirit and scope of the present invention, ordinary technicians in this field can make various changes and improvements to the form and details of the present application, and these all fall within the scope of protection of the present application.

[0202] The nucleotide sequences mentioned herein are as follows.

[0203] SEQ ID NO.1: wild type gyrA

[0204] SEQ ID NO.37: Wild-type GyrA

[0205] SEQ ID NO.38: GyrA mutant

[0206] X1=H or A、X2=A or T、X3=T or A

[0207] SEQ ID NO.2: Wild-type gyrB

[0208] SEQ ID NO.39: Wild-type GyrB

[0209] SEQ ID NO.40: GyrB mutant

[0210] X1=E or D、X2=R or C、X3=D or A

[0211] SEQ ID NO.3: gyrA edited - gyrA (H80A) coding sequence

[0212] SEQ ID NO.4: gyrA edited - gyrA (A569T & T586A) coding sequence

[0213] SEQ ID NO.5: gyrB edited - gyrB (E42D) coding sequence

[0214] SEQ ID NO.6: gyrB edited - gyrB (R136C) coding sequence

[0215] SEQ ID NO.7: gyrB edited - gyrB (D498A) coding sequence

[0216] SEQ ID NO.8: sgRNA targeting gyrA (H80A)

[0217] SEQ ID NO.9: sgRNA targeting gyrA gene (A569T & T586A)

[0218] SEQ ID NO.10: sgRNA targeting gyrB gene (E42D)

[0219] SEQ ID NO.11: sgRNA targeting gyrB gene (R136C)

[0220] SEQ ID NO.12: sgRNA targeting gyrB gene (D498A)

[0221] SEQ ID NO.13: gyrA (H80A) donor sequence

[0222] SEQ ID NO.14: gyrA (A569T & T586A) donor sequence

[0223] SEQ ID NO.15: gyrB (E42D) donor sequence

[0224] SEQ ID NO.16: gyrB (R136C) donor sequence

[0225] SEQ ID NO.17: gyrB (D498A) donor sequence

[0226] SEQ ID NO.30: poly(A) sequence-80A

[0227] SEQ ID NO.31:poly(A) sequence-100A

[0228] SEQ ID NO.32: poly(A) sequence-120A

[0229] SEQ ID NO.33: poly(A) sequence-30&70A

[0230] SEQ ID NO.34: poly(A) sequence-30&30&43A

[0231] SEQ ID NO.35: poly(A)-test 1 (80A) mRNA functional region sequence

[0232] SEQ ID NO.36: pVAX1 vector sequence

[0233] SEQ ID NO.41: RecQ

[0234] SEQ ID NO.42: lacIq promoter+lacI+rrnB T1 terminator+rrnB T2 terminator

[0235] SEQ ID NO.43: RecA

[0236] SEQ ID NO.44: RecA edited-RecA1 coding sequence

[0237] SEQ ID NO.45: sgRNA targeting RecQ

[0238] SEQ ID NO.46: sgRNA targeting yghX

[0239] SEQ ID NO.47: sgRNA targeting RecA

[0240] SEQ ID NO.48: Knockout RecQ donor sequence

[0241] SEQ ID NO.49: Knock-in lacIq promoter + lacI + rrnB T1 terminator + rrnB T2 terminator donor sequence

[0242] SEQ ID NO.50: Mutated RecA to RecA1 donor sequence

[0243] Although the present application has been described in conjunction with one or more embodiments, it should be understood that the present application is not limited to these embodiments. The description in this application is intended to cover all variants and equivalents, all of which are included in the subject matter and scope of the appended claims. All documents cited in this article are incorporated herein by reference in their entirety.

Claims

1. Use of an Escherichia coli host cell in replicating or expressing a nucleic acid molecule containing a poly(A) sequence, wherein the Escherichia coli host cell comprises downregulated gyrase activity or downregulated gyrase expression.

2. The use according to claim 1, wherein the E. coli host cell comprises down-regulated gyrase activity via mutation of the gyrA gene or the gyrB gene.

3. The use according to claim 2, wherein the Escherichia coli host cell expresses a GyrA mutant by mutation of the gyrA gene, and the GyrA mutant comprises: i) contains a H80A mutation at position 80 corresponding to SEQ ID NO. 37, or ii) comprising A569T and T586A mutations at positions 569 and 586 corresponding to SEQ ID NO.

37.

4. The use according to claim 2, wherein the E. coli host cell expresses a GyrB mutant by mutation of the gyrB gene, and the GyrB mutant comprises: i) comprising a R136C mutation at position 136 corresponding to SEQ ID NO. 39, ii) comprising an E42D mutation at position 42 corresponding to SEQ ID NO. 39, or iii) comprising a D498A mutation at position 498 corresponding to SEQ ID NO.

39.

5. The use according to claim 2, wherein The E. coli host cell expresses a GyrA mutant and a GyrB mutant through mutation of the gyrA gene and the gyrB gene, wherein the GyrA mutant comprises an H80A mutation at position 80 corresponding to SEQ ID NO.37, and the GyrB mutant comprises an R136C mutation at position 136 corresponding to SEQ ID NO.

39.

6. The method of any one of claims 1 to 5, wherein the E. coli host cell further comprises one or more of the following mutations: i) RecA gene mutation, ii) inserting the lacI gene expression cassette, and iii) RecQ gene knockout.

7. The use according to claim 6, wherein the Escherichia coli host cell comprises: i) the RecA gene after mutation comprises the nucleotide sequence shown in SEQ ID NO.44, ii) the inserted lacI gene expression cassette comprises the nucleotide sequence shown in SEQ ID NO.42, or iii) The RecQ gene comprises the nucleotide sequence shown in SEQ ID NO.

41.

8. The use according to any one of claims 1 to 7, wherein the E. coli host cell is JM108, NEB Stable, Top10, DH5α, DH10B, MG1655, AG1, BL21, DB3.1, DC10B, DH1, E.Cloni 10G, EPI300, EPI400, JM109, JM110, STBL2, STBL3, SURE, TOP10F', XL1-Blue or XL10-Gold that has been modified to contain down-regulated gyrase activity or down-regulated gyrase expression.

9. The use according to any one of claims 1 to 8, wherein the E. coli host cell comprises a vector, and the vector comprises the nucleic acid molecule containing the poly(A) sequence, or The E. coli host cell comprises the nucleic acid molecule containing the poly(A) sequence in its genome.

10. The use according to any one of claims 1 to 9, wherein The poly(A) sequence in the nucleic acid molecule containing the poly(A) sequence comprises: i) 20-250 consecutive A bases; or ii) 2-5 consecutive A base segments separated by non-A bases, wherein each consecutive A base segment comprises 10-100 consecutive A bases and is separated from each other by 1-20 non-A bases.

11. A recombinant E. coli host cell, which is JM108, NEB Stable, Top10, DH5α, DH10B, MG1655, AG1, BL21, DB3.1, DC10B, DH1, E.Cloni 10G, EPI300, EPI400, JM109, JM110, STBL2, STBL3, SURE, TOP10F', XL1-Blue or XL10-Gold that has been modified to contain down-regulated gyrase activity or down-regulated gyrase expression.

12. A recombinant E. coli host cell comprising down-regulated gyrase activity via mutation of the gyrA gene or the gyrB gene.

13. The recombinant E. coli host cell of claim 11 or 12, which expresses a GyrA mutant, wherein the GyrA mutant comprises: i) contains a H80A mutation at position 80 corresponding to SEQ ID NO. 37, or ii) comprising A569T and T586A mutations at positions 569 and 586 corresponding to SEQ ID NO.

37.

14. The recombinant E. coli host cell of claim 11 or 12, which expresses a GyrB mutant, wherein the GyrB mutant comprises: i) comprising a R136C mutation at position 136 corresponding to SEQ ID NO. 39, ii) comprising an E42D mutation at position 42 corresponding to SEQ ID NO. 39, or iii) comprising a D498A mutation at position 498 corresponding to SEQ ID NO.

39.

15. The recombinant Escherichia coli host cell according to claim 11 or 12, wherein the Escherichia coli host cell expresses a GyrA mutant and a GyrB mutant by mutation of the gyrA gene and the gyrB gene, wherein the GyrA mutant comprises an H80A mutation at position 80 corresponding to SEQ ID NO. 37, and the GyrB mutant comprises an R136C mutation at position 136 corresponding to SEQ ID NO.

39.

16. The recombinant E. coli host cell of any one of claims 11-15, further comprising one or more of the following mutations: i) RecA gene mutation, ii) inserting the lacI gene expression cassette, and iii) RecQ gene knockout.

17. The recombinant E. coli host cell according to any one of claims 11 to 15, further comprising: i) RecA gene mutation, wherein the mutated RecA gene comprises the nucleotide sequence shown in SEQ ID NO.44, ii) inserting a lacI gene expression cassette, wherein the inserted lacIq gene comprises the nucleotide sequence shown in SEQ ID NO.42, or iii) RecQ gene knockout, wherein the RecQ gene comprises the nucleotide sequence shown in SEQ ID NO.

41.

18. The recombinant E. coli host cell according to claim 16 or 17, comprising: i) a gyrA gene mutation, wherein the mutated gyrA gene comprises the nucleotide sequence shown in SEQ ID NO. 3 or 4, ii) RecA gene mutation, wherein the mutated RecA gene comprises the nucleotide sequence shown in SEQ ID NO.44, iii) inserting a lacI gene expression cassette, wherein the inserted lacIq gene comprises the nucleotide sequence shown in SEQ ID NO.42, and vi) RecQ gene knockout, wherein the RecQ gene comprises the nucleotide sequence shown in SEQ ID NO.

41.

19. A method for replicating a nucleic acid molecule containing a poly(A) sequence using an E. coli host cell, The method comprises: i) introducing a vector into the E. coli host cell according to any one of claims 11 to 18, wherein the vector comprises a nucleic acid molecule containing a poly(A) sequence, and ii) culturing the E. coli host cells under conditions conducive to vector replication.

19. A method for expressing a nucleic acid molecule containing a poly(A) sequence using an E. coli host cell, The method comprises: i) introducing a vector into the E. coli host cell according to any one of claims 11 to 18, wherein the vector comprises a nucleic acid molecule containing a poly(A) sequence, ii) optionally, culturing the E. coli host cell under conditions conducive to vector replication, and iii) culturing the E. coli host cell under conditions conducive to vector expression, or Extracting the vector from the E. coli host cell in step i) or ii) and performing in vitro transcription on the vector; or i) integrating into the genome of the E. coli host cell according to any one of claims 11 to 18 an A nucleic acid molecule having a poly(A) sequence, and ii) culturing the E. coli host cell under conditions conducive to the expression of the nucleic acid molecule containing the poly(A) sequence.

20. A method for preparing a recombinant Escherichia coli host cell, comprising: i) providing an E. coli host cell, the E. coli host cell comprising a gyrA gene and a gyrB gene, ii) down-regulating the activity of gyrase in the E. coli host cell, or down-regulating the expression level of gyrase in the E. coli host cell.

21. The method of claim 20, wherein: Step ii) comprises, 1) making the gyrA gene contain the nucleotide sequence shown in SEQ ID NO.3, 2) making the gyrB gene contain the nucleotide sequence shown in SEQ ID NO.6, 3) making the gyrA gene contain the nucleotide sequence shown in SEQ ID NO.4, 4) making the gyrB gene contain the nucleotide sequence shown in SEQ ID NO.5, or 5) The gyrB gene contains the nucleotide sequence shown in SEQ ID NO.

7.

22. The method of claim 21, wherein: Step ii) comprises introducing Cas9 enzyme into the E. coli host cell of step i), and 1) an sgRNA comprising the nucleotide sequence shown in SEQ ID NO.8, and a donor sequence comprising the nucleotide sequence shown in SEQ ID NO.13, 2) an sgRNA comprising the nucleotide sequence shown in SEQ ID NO.11, and a donor sequence comprising the nucleotide sequence shown in SEQ ID NO.16, 3) an sgRNA comprising the nucleotide sequence shown in SEQ ID NO.9, and a donor sequence comprising the nucleotide sequence shown in SEQ ID NO.14, 4) an sgRNA comprising the nucleotide sequence shown in SEQ ID NO.10, and a donor sequence comprising the nucleotide sequence shown in SEQ ID NO.15, or 5) An sgRNA comprising the nucleotide sequence shown in SEQ ID NO.12, and a donor sequence comprising the nucleotide sequence shown in SEQ ID NO.

17.

23. The method of any one of claims 20-22, wherein the E. coli host cell further comprises any one or more of the following steps: 1) RecA gene mutation, 2) inserting the lacI gene expression cassette, and 3) RecQ gene knockout.

24. The method of any one of claims 20 to 23, wherein the E. coli host cell in step i) is selected from JM108, NEB Stable, Top10, DH5α, DH10B, MG1655, AG1, BL21, DB3.1, DC10B, DH1, E.Cloni 10G, EPI300, EPI400, JM109, JM110, STBL2, STBL3, SURE, TOP10F', XL1-Blue or XL10-Gold.

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