A vector combination for single base editing in halomonas and application thereof

Abstract

This invention provides a single-base editing system based on the dSpRY-PmCDA1-UGI ​​fusion protein and its application in *Halomonas*. By constructing a combination containing the dSpRY-PmCDA1-UGI ​​deaminase and an sgRNA expression vector, efficient and precise single-base editing of the *Halomonas* genome is achieved. This system does not rely on homologous arm or double-strand break repair mechanisms, significantly improving editing efficiency; it has almost no requirements on the PAM sequence of the editing site and supports simultaneous editing of multiple gene sites. Specific implementations show that this invention can be applied to gene function research, metabolic pathway regulation, and other fields.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, specifically to a single-base editing tool based on the CRISPR / dSpRY-PmCDA1-UGI ​​system, suitable for precise genome editing of Halomonas sp. and its derivative strains. Background Technology

[0002] Halomonas (such as Halomonas bluephagenesis TD) are important substrate strains for biomanufacturing due to their tolerance to high salt and alkali conditions. Traditional gene editing relies on homologous recombination, which is inefficient and time-consuming; while CRISPR / Cas9 improves efficiency, it still requires double-strand break repair, which can easily lead to genome damage. Existing single-base editing technologies have strict PAM limitations (e.g., only supporting NGG). Summary of the Invention

[0003] Technical problems to be solved

[0004] This invention aims to provide a highly efficient, flexible, and PAM-restricted single-base editing system to accelerate the metabolic engineering of Halomonas bacteria.

[0005] Technical solution

[0006] 1. Carrier combination design

[0007] • dSpRY-PmCDA1-UGI ​​expression vector: The dSpRY-PmCDA1-UGI ​​fusion protein is used and driven by the PCas promoter. SpRY is a variant of SpCas9, which has almost no requirements for the PAM region sequence; dSpRY is a mutated SpRY with no DNA cleavage activity.

[0008] • sgRNA expression vector: a single sgRNA or multiple copies of sgRNA in tandem, regulated by the J23119 series promoters, targeting specific sites in the genome.

[0009] 2. Editing Strategy

[0010] • Utilizing the deaminase activity of PmCDA1-UGI, the target C is converted to T (or A to G) under the guidance of the dSpRY-sgRNA complex, without the need for double-strand breaks.

[0011] • Supports multiple editing: Enables simultaneous inactivation or point mutation of multiple genes through tandem sgRNA.

[0012] Therefore, the present invention provides a method for single-base editing of *Halomonas*, comprising the following steps: introducing dSpRY, cytidine deaminase, uracil glycosylation inhibitor (UGI), and sgRNA targeting a gene in *Halomonas* into *Halomonas* to achieve single-base editing in the genome of *Halomonas*.

[0013] In one specific embodiment, the coding gene for dSpRY, the coding gene for cytidine deaminase, the coding gene for UGI, and the DNA transcribed from the sgRNA are introduced into the *Halomonas* to achieve single-base editing in the genome of the *Halomonas*.

[0014] In a preferred embodiment, the gene encoding dSpRY is shown in SEQ ID NO:1, or the amino acid sequence of dSpRY is shown in SEQ ID NO:2.

[0015] In one specific embodiment, the cytidine deaminase is cytidine deaminase 1 (PmCDA1) of the marine lamprey (Petromyzon marinus) or a variant thereof (e.g., evoPmCDA1), and / or the UGI is a glycosylation inhibitor derived from Bacillus subtilis phage PBS1.

[0016] In a preferred embodiment, the encoding gene of PmCDA1 is shown in SEQ ID NO:3, or the amino acid sequence of PmCDA1 is shown in SEQ ID NO:4.

[0017] In a preferred embodiment, the gene encoding the UGI is shown in SEQ ID NO:5, or the amino acid sequence of the UGI is shown in SEQ ID NO:6.

[0018] In one specific embodiment, the dSpRY, cytidine deaminase, and uracil glycosylation inhibitor (UGI) are expressed as a fusion protein (from N-terminus to C-terminus): dSpRY-PmCDA1-UGI.

[0019] In a preferred embodiment, the fusion protein dSpRY-PmCDA1-UGI ​​optionally includes a linker between dSpRY and PmCDA1 or between PmCDA1 and UGI.

[0020] In a preferred embodiment, the fusion protein comprises (from N-terminus to C-terminus): dSpRY-connector-PmCDA1-UGI.

[0021] In a preferred embodiment, the gene encoding the adapter is shown in SEQ ID NO:7, or the amino acid sequence of the adapter is shown in SEQ ID NO:8.

[0022] In one embodiment, the method is performed using a carrier combination or kit, wherein the carrier combination or kit comprises:

[0023] • The dSpRY-PmCDA1-UGI ​​expression vector expresses the fusion protein dSpRY-PmCDA1-UGI; and

[0024] • An sgRNA expression vector containing the sgRNA sequence targeting a gene in the genome of Halomonas.

[0025] In one specific embodiment, the dSpRY-PmCDA1-UGI ​​expression vector and / or the sgRNA expression vector are driven by a constitutive promoter or an inducible promoter; preferably (1) the fusion protein dSpRY-PmCDA1-UGI ​​is expressed by a constitutive promoter Pcas promoter, and / or the sgRNA sequence is expressed by a J23119 series promoter; or (2) the inducible promoter is selected from the following inducible expression systems: P Tac and its mutant (isopropyl galactothioglycoside IPTG induction system), P BAD and its mutant (arabinose-induced system), P Lux and its mutant (acylhomoserine lactone AHL-induced system), P Tet and its mutant (ahydrotetracycline aTc-induced system), P Van and its mutants (vanillic acid-induced system).

[0026] In a preferred embodiment, the Pcas promoter is shown as SEQ ID NO:9.

[0027] In a preferred embodiment, the J23119 series promoter is shown in SEQ ID NO:10.

[0028] In one specific embodiment, the sgRNA expression vector expresses a single copy of sgRNA or tandem multiple copies of sgRNA, wherein the multiple copies of sgRNA can be the same or different, i.e., they can target the same target gene or different target genes in the genome of *Halomonas*. In the case of multiple copies of sgRNA, the promoters of each sgRNA can be the same or different, and can be constitutive or inducible promoters independently. The promoter can be the J23119 series promoters described above or promoters in the inducible expression system.

[0029] In one specific embodiment, the Halomonas includes Halomonas bluephagenesis or its derivatives, Halomonas campaniensis or its derivatives, Halomonas aydingkolgenesis or its derivatives, preferably Halomonas bluephagenesis TD1.0 (see Zhao, H. et al. (2017) Novel T7-like expression systems used for Halomonas. Metab Eng 39, 128-140), Halomonas bluephagenesis TD01 (CGMCC No. 4353), Halomonas aydingkolgenesis M1 (CGMCC No. 19880), and Halomonas campaniensis LS21 (CGMCC No. 6593). All of the above CGMCC strains have been deposited and disclosed in previous patent applications. For example, CGMCC No. 4353, CGMCC No. 19880, and CGMCC No. 6593 have been published in CN119464346A.

[0030] Another aspect of the present invention provides a vector combination or kit for single-base editing of Halomonas bacteria, comprising:

[0031] • The dSpRY-PmCDA1-UGI ​​expression vector expresses the fusion protein dSpRY-PmCDA1-UGI; and

[0032] • An sgRNA expression vector containing the sgRNA sequence of a target gene in Halomonas.

[0033] In one specific embodiment, the dSpRY-PmCDA1-UGI ​​expression vector and / or the sgRNA expression vector are driven by a constitutive promoter or an inducible promoter; preferably (1) the fusion protein dSpRY-PmCDA1-UGI ​​is expressed by a constitutive promoter Pcas promoter, and / or the sgRNA sequence is expressed by a J23119 series promoter; or (2) the inducible promoter is selected from the following inducible expression systems: P Tac and its mutant (isopropyl galactothioglycoside IPTG induction system), P BAD and its mutant (arabinose-induced system), P Lux and its mutant (acylhomoserine lactone AHL-induced system), P Tetand its mutant (ahydrotetracycline aTc-induced system), P Van and its mutants (vanillic acid-induced system).

[0034] In one specific embodiment, the fusion protein dSpRY-PmCDA1-UGI ​​includes a linker between dSpRY and PmCDA1 or between PmCDA1 and UGI.

[0035] In a preferred embodiment, the elements in the carrier are as defined above in relation to the method of the present invention.

[0036] In a preferred embodiment, the Halomonas single-base editing method of the present invention is implemented by the above-described combination of vectors or kits for Halomonas single-base editing.

[0037] Another aspect of the present invention provides the use of the method, carrier combination, or kit according to the present invention in any of the following:

[0038] 1) Single-base editing of the genome of Halomonas (C→T or A→G);

[0039] 2) Preparation of Halomonas mutants;

[0040] 3) Modification of Halomonas strains;

[0041] 4) Research on gene function in Halomonas halosa;

[0042] 5) Regulation of metabolic pathways in Halomonas;

[0043] 6) Increase the yield of polyhydroxy fatty acid esters (PHA) from Halomonas bacteria;

[0044] 7) Enhance the salt tolerance or stress resistance of Halomonas; and

[0045] 8) Optimize the allocation of carbon metabolic flux in Halomonas.

[0046] Technical effect

[0047] • PAM has broad compatibility, expanding the range of target selection;

[0048] • High editing efficiency (≥50% for single genes, ≥10% for three genes);

[0049] • Avoid double-strand break (DSB) toxicity and reduce strain fitness loss.

[0050] By constructing a combination containing dSpRY-PmCDA1-UGI ​​deaminase and an sgRNA expression vector, efficient and precise single-base editing of the *Halomonas* genome was achieved. This system does not rely on homologous arm or double-strand break repair mechanisms, significantly improving editing efficiency; it has almost no requirements on the PAM sequence of the editing site and supports simultaneous editing of multiple gene sites. Experimental data indicate that this invention can be applied to gene function research, metabolic pathway regulation, and other fields. Attached Figure Description

[0051] The above features and advantages of the present invention will become more apparent from the following detailed description taken in conjunction with the accompanying drawings.

[0052] in:

[0053] Figure 1 This is a schematic diagram of the dSpRY-PmCDA1-UGI ​​expression vector pL9;

[0054] Figure 2 This is a schematic diagram of the sgRNA expression vector psgRNA;

[0055] Figure 3 This demonstrates the efficiency of editing the phaC gene; and

[0056] Figure 4 This is a schematic diagram of the triple sgRNA vector psgRNAX3. Detailed Implementation

[0057] Unless otherwise stated, the terms used herein have their general technical meanings as understood by those skilled in the art. For definitions and terms in this field, those skilled in the art are particularly recommended to refer to Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, Plainsview, New York (1989); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999).

[0058] As used herein, the term "Halomonas" refers to a class of halophilic microorganisms capable of growing under extreme conditions such as high pH, ​​high salinity, and high temperature. Because the growth of non-halophilic microorganisms is inhibited under high salinity and alkaline conditions, bacteria of this genus are resistant to contamination by other microorganisms and are often used to establish open fermentation systems that are low in energy, freshwater, and fixed capital, and can operate continuously.

[0059] As used herein, the term "promoter" generally refers to a deoxyribonucleic acid (DNA) sequence that enables the transcription of a specific gene. Promoters are recognized by RNA polymerase, which initiates transcription to synthesize RNA. In RNA synthesis, promoters interact with transcription factors that regulate gene transcription, controlling the initiation time and extent of gene expression (transcription). A promoter comprises a core promoter region and a regulatory region, located in the regulatory sequence controlling gene expression, upstream of the transcription start site (5' direction of the DNA antisense strand), and does not have a coding function itself. Based on their mode of action and function, promoters are classified into three categories: constitutive promoters (which maintain continuous activity in most or all tissues), specific promoters (tissue-specific or developmentally specific), and inducible promoters (regulated by external chemical or physical signals).

[0060] In this application, the term "Cas9 protein" or "Cas9 nuclease," also known as Csn1 or Csx12, generally refers to a class of proteins in the type II CRISPR / Cas system that are involved in both crRNA biosynthesis and the destruction of invading DNA. Cas9 proteins typically comprise a RuvC nuclease domain and an HNH nuclease domain, which cleave the two distinct strands of a double-stranded DNA molecule, respectively. Cas9 proteins have been described in various bacterial species such as *Streptococcus thermophilus*, *Listeria innocua*, and *Streptococcus pyogenes*. For example, the amino acid sequence of the Cas9 protein of *Streptococcus pyogenes* can be found in the SwissProt database accession number Q99ZW2; the amino acid sequence of the Cas9 protein of *Neisseria meningitides* can be found in the UniProt database accession number A1IQ68; the amino acid sequence of the Cas9 protein of *Streptococcus thermophilus* can be found in the UniProt database accession number Q03LF7; and the amino acid sequence of the Cas9 protein of *Staphylococcus aureus* can be found in the UniProt database accession number J7RUA5.

[0061] In this application, the term "SpCas9" refers to Cas9 derived from Streptococcus pyogenes. SpCas9 is one of the earliest Cas9 proteins to be widely studied and applied in CRISPR gene editing. It requires a 20-nucleotide guide sequence and an NGG PAM (prototype adjacent sequence) site to recognize target DNA.

[0062] In this application, the term "SpRY" refers to an engineered SpCas9 mutant, which is a SpG (L1111R / A1322R) derivative containing A61R, N1317R, and R1333P substitutions. The engineered SpCas9 mutant SpRY is almost entirely free from PAM contamination, recognizing PAM sequences encompassing NRN and NYN (Y is C / T) (NRN>NYN). See Russell T. Walton et al., "Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants," Science 10.1126 / science.aba8853 (2020).

[0063] In this application, the term "dSpRY" refers to SpRY that has no DNA cleavage activity after mutation.

[0064] The gRNA described in this application can bind to sequences in target nucleic acids. Nucleic acids (or portions thereof) targeting the genome can interact with the target nucleic acid in a sequence-specific manner through hybridization (i.e., base pairing). The gRNA described in this application can be a single-stranded guide RNA (sgRNA), and the nucleotide sequence of the sgRNA can vary depending on the sequence of the target nucleic acid.

[0065] In the CRISPR / Cas system of this application, the gRNA sequence can be programmed to hybridize with a target nucleic acid adjacent to a PAM sequence recognizable by the Cas protein used in the system. The gRNA may or may not perfectly match the target sequence. Cas proteins typically have a specific PAM sequence that can be recognized in the target DNA.

[0066] The gRNAs used in the CRISPR system described in this application can be synthesized by chemical methods, such as high-performance liquid chromatography (HPLC). For example, two or more RNA molecules can be linked together. Longer RNAs (e.g., RNA encoding Cas9) can be obtained through enzymatic reactions. In the art, various types of RNA modifications can be introduced during or after the chemical and / or enzymatic synthesis of RNA, for example, modifications to enhance stability, reduce innate immune responses, and / or enhance other properties.

[0067] CRISPR / Cas9-based gene editing systems include at least one gRNA scaffold. The gRNA scaffold facilitates the binding of Cas9 to gRNA and endonuclease activity. The gRNA scaffold is a polynucleotide sequence immediately following the gRNA targeting sequence. The gRNA targeting sequence and the gRNA scaffold together form a polynucleotide.

[0068] As used herein, the term "linker" refers to a covalent linker, non-covalent linker, chemical group, or molecule that can be used to connect two proteins or domains of a fusion protein (e.g., any two of dSpRY, PmCDA1, and UGI). In some embodiments, the linker can be an amino acid or multiple amino acids, for example, a linker can be about 5 to 100 amino acids in length. For example, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, or 90 to 100 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGSETPGTSESATPES, which may also be referred to as an XTEN linker. In some embodiments, the linker can be GGGGSGGGGSAEYVRALFDFNGNDEEDLPFKKGDILRIRDKPEEQWW

[0069] NAEDSEGKRGMIPVPYVEKYSGDYKDHDGDYKDHDIDYKDDDDKSRLESGDYKDHDGDYKDHDIDYKDDDDKSR(SEQ ID NO:8), SGGS(SEQ ID NO:11), SGGSSGSETPGTSESATPESSGGS(SEQ ID NO:12), SGGSSGGSSGSETPGTSESATPESSGGSSGGS(SEQ ID NO: 13), GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS (SEQ ID NO: 14), SGGSGGSGGS (SEQ ID NO: 15), (GGGS)n, (GGGGS)n, (G)n, (EAAAK)n, (GGS)n, etc., where n=1, 2, 3, 4, 5 or 6.

[0070] The term "comprising" or "including" as used in this invention is an open-ended description, encompassing all specified components or steps described, as well as other specified components or steps that do not substantially affect the meaning; when used to describe the sequence of a protein or nucleic acid, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still possess the activity described in this invention.

[0071] The term "and / or" as used in this invention encompasses all combinations of items connected by the term, and should be considered as if each combination had been individually listed herein. For example, "A and / or B" includes "A", "A and B", and "B". As another example, "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C", and "A and B and C".

[0072] In one specific embodiment, the present invention provides a *Haloxylon ammodendron* single-base editing vector assembly, comprising:

[0073] • dSpRY-PmCDA1-UGI ​​expression vector: encodes the fusion protein dSpRY-PmCDA1-UGI, driven by a constitutive promoter (such as Pcas);

[0074] • sgRNA expression vector: contains the sgRNA sequence targeting the gene and is driven by a constitutive promoter (such as the J23119 series promoters).

[0075] The dSpRY-PmCDA1-UGI ​​variant is PAM compatible.

[0076] In one specific embodiment, the present invention provides a method for single-base editing of the genome of Halomonas, comprising the following steps:

[0077] a) The dSpRY-PmCDA1-UGI ​​expression vector was introduced into a Halomonas host to obtain recombinant bacteria;

[0078] b) Construct an editing vector containing the target gene sgRNA sequence and transform it into recombinant bacteria;

[0079] c) Target single-base editing is achieved through deaminase-mediated base switching (C→T or A→G).

[0080] The method supports simultaneous editing of single or multiple gene loci, with an editing efficiency of ≥10%.

[0081] In one specific embodiment, the present invention provides the application of the vector combination of the present invention in the study of gene function in Halomonas.

[0082] Especially suitable for:

[0083] • Precise gene knockout (by introducing a stop codon or removing a start codon);

[0084] • Regulating gene expression (e.g., point mutations regulating enzyme activity);

[0085] • Construct a metabolic pathway mutation library.

[0086] In one specific embodiment, the present invention provides the application of the vector combination of the present invention in the modification of industrial strains, including:

[0087] • Increase the production of polyhydroxyalkanoates (PHA);

[0088] • Enhance salt tolerance or stress resistance; and / or

[0089] • Optimize carbon metabolic flux allocation.

[0090] The above only summarizes some aspects of the present invention and is not, and should not be considered as limiting the present invention in any way.

[0091] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0092] Unless otherwise specified, the experimental methods used in the examples are conventional methods.

[0093] Unless otherwise specified, all reagents and materials used in the examples are commercially available.

[0094] Example 1

[0095] Expression vector construction and single gene editing

[0096] Carrier construction

[0097] The fusion gene dSpRY-PmCDA1-UGI ​​(with an adapter between dSpRY and PmCDA1) was cloned into pSEVA321 (GenBank accession No. JX560332.1) and controlled by the Pcas promoter to obtain pL9. Figure 1 The gene sequences of the above-mentioned elements dSpRY, PmCDA1, UGI, linker, and Pcas promoter are shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, and SEQ ID NO:9, respectively.

[0098] • Design and synthesis of three sgRNAs targeting phaC (PHB synthase) (SEQ ID NO:16), sgRNA-1 (SEQ ID NO:17):

[0099] ctcaaggtgtttctcaagaagGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT,

[0100] sgRNA-2 (SEQ ID NO:18):

[0101] aacagtataaaggcctgcttgaGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT,

[0102] sgRNA-3 (SEQ ID NO:19):

[0103] ggcaaactcagtcgcgattgctaGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT,

[0104] The psgRNAs were assembled into pSEVA341 (GenBank accession No. JX560334.1) to obtain three different psgRNAs (see schematic diagram). Figure 2 The sgRNA is controlled by the J23119 series of promoters. The sequences of the J23119 series of promoters are shown in SEQ ID NO:10.

[0105] Plasmid conjugation and verification

[0106] • pL9 and individual psgRNAs were introduced into Halomonas bluephagenesis TD1.0 via conjugation (see Zhao, H. et al. (2017) Novel T7-like expression systems used for Halomonas. Metab Eng 39, 128-140), and cultured for 48 h.

[0107] • Scrape the obtained colonies from the plate and extract genomic DNA. Perform PCR amplification on the phaC gene (primer 1 (5'→3', the same below): gggagagtgagtatgctgtcag (SEQ ID NO:20), primer 2: gccaaagcagccactgatc (SEQ ID NO:21)), construct a library from the obtained product and perform high-throughput sequencing to detect the editing efficiency.

[0108] Editing efficiency calculation method:

[0109] The PCR products were subjected to next-generation high-throughput sequencing using the BGI T7 sequencing platform. After removing adapters and low-quality sequences, the sequencing results were assembled. The editing efficiency was determined by detecting the proportion of ordinary codons converted to stop codons (CAA->TAA, CAG->TAG) in the assembled sequences.

[0110] Results: All sgRNAs mediated phaC editing, with an editing efficiency greater than 50%. Figure 3 ).

[0111] Example 2

[0112] Simultaneous editing of multiple genes

[0113] • Design sgRNAs targeting the prpC (encoding 2-methylcitrate synthase) gene (SEQ ID NO:22),

[0114] sgRNA-prpC:

[0115] cgcaaaacagcgcaggactccgGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT (SEQ ID NO: 23),

[0116] sgRNA targeting lpxL (encoding lauroyl acyltransferase) (SEQ ID NO:24),

[0117] sgRNA-lpxL:

[0118] agccaggcaacaacatgcattgGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT (SEQ ID NO: 25),

[0119] Together with sgRNA-2 (SEQ ID NO:18) targeting phaC, a triple sgRNA vector psgRNAX3 was constructed. Figure 4 ).

[0120] • pL9 and psgRNAX3 were introduced into H. bluephagenesis TD1.0 via conjugation and cultured for 48 h.

[0121] • Scrape the obtained colonies from the plate, place them in LB60 solution and mix well. Serially dilute the obtained LB60 bacterial suspension, plate it on antibiotic-free LB60 plates, and incubate overnight to obtain colonies. Select 96 colonies and perform PCR amplification on phaC, prpC, and lpxL (phaC amplification primers are the same as above; prpC amplification primer 1: gctgaatagccgttaacaccatc (SEQ ID NO:26), amplification primer 2: tagccatcaagctcggcttg (SEQ ID NO:27); lpxL amplification primer 1: ccttcgcttaaacggttatcagtatg (SEQ ID NO:28), amplification primer 2: taccagccccatcccattg (SEQ ID NO:29)). Perform Sanger sequencing on the obtained products to detect editing efficiency. The editing efficiency calculation method is the same as in Example 1.

[0122] LB medium contains: 5 g / L yeast extract (OXID, catalog number LP0021), 10 g / L peptone (OXID, catalog number LP0042), 10 g / L NaCl, and the remainder is water. Adjust the pH to 7.0-7.2 and autoclave.

[0123] LB60 medium is LB medium containing 60 g / L NaCl, and the other components and preparation conditions are the same as LB medium.

[0124] Results: 10.4% of the strains simultaneously inactivated phaC, prpC and lpxL.

[0125] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0126] It should also be noted that the various specific technical features described in the above embodiments can be combined in any suitable manner without contradiction. To avoid unnecessary repetition, the present invention will not describe the various possible combinations separately. Furthermore, various different embodiments of the present invention can also be arbitrarily combined, as long as they do not violate the spirit of the present invention, and should also be regarded as the content disclosed by the present invention.

Claims

1. A method for single-base editing of Halomonas bacteria, comprising the following steps: By introducing dSpRY, cytidine deaminase, uracil glycosylation inhibitor (UGI), and sgRNA targeting the gene in *Halomycosis* into *Halomycosis* for expression, single-base editing of the *Halomycosis* genome was achieved.

2. The method according to claim 1, wherein the coding gene for dSpRY, the coding gene for cytidine deaminase, the coding gene for UGI, and the DNA transcribed from the sgRNA are introduced into the *Halomonas* to achieve single-base editing in the genome of the *Halomonas*.

3. The method according to claim 1 or 2, wherein the cytidine deaminase is cytidine deaminase 1 (PmCDA1) of petrified lamprey or a variant thereof (e.g., evoPmCDA1), and / or the UGI is a glycosylation inhibitor UGI derived from Bacillus subtilis phage PBS1.

4. The method according to claim 1 or 2, wherein the method is performed using a carrier combination or kit, wherein the carrier combination or kit comprises: • The dSpRY-PmCDA1-UGI ​​expression vector expresses the fusion protein dSpRY-PmCDA1-UGI; and • An sgRNA expression vector containing the sgRNA sequence of a target gene in the genome of Halomonas.

5. The method according to claim 4, wherein the dSpRY-PmCDA1-UGI ​​expression vector and / or the sgRNA expression vector are driven by a constitutive promoter or an inducible promoter; preferably (1) the fusion protein dSpRY-PmCDA1-UGI ​​is expressed by a constitutive promoter Pcas promoter, and / or the sgRNA sequence is expressed by a J23119 series promoter; or (2) the inducible promoter is selected from the following inducible expression systems: P Tac and its mutant (isopropyl galactothioglycoside IPTG induction system), P BAD and its mutant (arabinose-induced system), P Lux and its mutant (acylhomoserine lactone AHL-induced system), P Tet and its mutant (ahydrotetracycline aTc-induced system), P Van and its mutants (vanillic acid-induced system).

6. The method of claim 5, wherein, optionally, a linker is included between dSpRY and PmCDA1 or between PmCDA1 and UGI in the fusion protein dSpRY-PmCDA1-UGI.

7. A vector combination or kit for single-base editing of Halomonas, comprising: • The dSpRY-PmCDA1-UGI ​​expression vector expresses the fusion protein dSpRY-PmCDA1-UGI; and • An sgRNA expression vector containing the sgRNA sequence of a target gene in Halomonas.

8. The vector combination or kit according to claim 7, wherein the dSpRY-PmCDA1-UGI ​​expression vector and / or the sgRNA expression vector are driven by a constitutive promoter or an inducible promoter; preferably (1) the fusion protein dSpRY-PmCDA1-UGI ​​is expressed by a constitutive promoter Pcas promoter, and / or the sgRNA sequence is expressed by a J23119 series promoter; or (2) the inducible promoter is selected from the following inducible expression systems: P Tac and its mutant (isopropyl galactothioglycoside IPTG induction system), P BAD and its mutant (arabinose-induced system), P Lux and its mutant (acylhomoserine lactone AHL-induced system), P Tet and its mutant (ahydrotetracycline aTc-induced system), P Van and its mutants (vanillic acid-induced system).

9. The vector combination or kit according to claim 8, wherein in the fusion protein dSpRY-PmCDA1-UGI, a linker is included between dSpRY and PmCDA1 or between PmCDA1 and UGI.

10. The use of the method according to any one of claims 1 to 6 or the carrier combination or kit according to any one of claims 7 to 9 in any of the following: 1) Single-base editing of the genome of Halomonas (C→T or A→G); 2) Preparation of Halomonas mutants; 3) Modification of Halomonas strains; 4) Research on gene function in Halomonas halosa; 5) Regulation of metabolic pathways in Halomonas; 6) Increase the yield of polyhydroxyalkanoates (PHA) from Halomonas bacteria; 7) Enhance the salt tolerance or stress resistance of halomonas; and 8) Optimize the distribution of carbon metabolic flux in halomonas.

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