Genome editing system for corynebacterium glutamicum

Abstract

The present invention provides a genome editing system for Corynebacterium glutamicum, comprising: a nucleic acid protein complex capable of specifically cleaving DNA in Corynebacterium glutamicum, the nucleic acid protein complex comprising endonuclease and a guide RNA targeting a target sequence; the endonuclease comprises an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence as shown in SEQ ID NO: 1; the guide RNA comprises (1) a nucleic acid sequence as shown in SEQ ID NO: 2 or a nucleic acid sequence having at least 80% sequence identity with the SEQ ID NO: 2, and (2) a recognition sequence complementary or identical to a target sequence, and the length of the recognition sequence is 5-50 nt. The system can realize genome editing in corynebacterium glutamicum, and the editing efficiency is better than that of Cas9 and Cpf1.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and medicine, and more specifically, this invention relates to a genome editing system for Corynebacterium glutamicum. Background Technology

[0002] Currently, the mainstream genome editing technologies for Corynebacterium glutamicum are pK18mobsacB and Cas9 / Cpf1.

[0003] pK18mobsacB is a traditional double-crossover genome editing system that relies on homologous recombination and a negative selection marker (sacB gene). However, it is complex to operate, requires gene integration and removal of the selection marker, has low efficiency, requires a negative selection marker to remove the positive selection marker, and requires strict control of the culture conditions that provide negative selection pressure. Due to the limitations of double-crossover efficiency, it is difficult to insert DNA fragments >3kb.

[0004] The CRISPR-Cas9 / Cpf1 system is an advanced tool in genome editing. The corresponding guide RNA directs Cas9 / Cpf1 to bind to target DNA, causing double-strand breaks. If the sequence of interest is provided, homologous recombination can repair and precisely edit the DNA sequence, offering high efficiency and accuracy. However, the double-strand breaks produced after Cas9 DNA cleavage can cause high cytotoxicity, especially when cellular repair mechanisms are weak, affecting the growth and survival rate of *Corynebacterium glutamicum*. In *Corynebacterium glutamicum*, the conversion efficiency of exogenous DNA is limited, and the relatively long coding sequence of Cas9 / Cpf1 (approximately 4kb), particularly when inserting large genome fragments, may reduce the success rate of the CRISPR system.

[0005] The reRNA-guided endonuclease TnpB has a coding sequence of only about 1.2 kb, which is advantageous for delivery into cells. TnpB has been reported to specifically cleave DNA in E. coli and human cells, but its function in Corynebacterium glutamicum has not yet been demonstrated. If a TnpB strain capable of specifically cleaving DNA in Corynebacterium glutamicum can be screened, it holds promise as a gene-editing tool, offering greater delivery efficiency and improving editing productivity. Summary of the Invention

[0006] The purpose of this invention is to provide a genome editing system for Corynebacterium glutamicum.

[0007] In a first aspect, the present invention provides a genome editing system for Corynebacterium glutamicum, comprising: a nucleic acid protein complex capable of specifically cutting DNA in Corynebacterium glutamicum, said nucleic acid protein complex comprising a nuclease and a guide RNA targeting a target sequence;

[0008] The endonuclease comprises the amino acid sequence described in SEQ ID NO:1, or an amino acid sequence having at least 80% sequence identity with SEQ ID NO:1;

[0009] The guide RNA comprises: (1) the nucleic acid sequence shown in SEQ ID NO:2 or a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO:2, and (2) a recognition sequence that is complementary to or identical to the target sequence, the recognition sequence being 5-50 nt in length.

[0010] In one or more embodiments, typically, the target sequence is preceded by a target-adjacent motif (TAM) sequence that is complementary to or identical to the guide RNA recognition sequence, and in one or more specific embodiments, the TAM sequence is 5'-AGGAG-3'.

[0011] In one or more embodiments, the genome editing system further comprises a recombinant element that facilitates recombination of the sequence of interest into the Corynebacterium glutamicum cleavage site.

[0012] In one or more embodiments, the recombinant element comprises the amino acid sequence described in SEQ ID NO:10 and / or SEQ ID NO:11, or an amino acid sequence having at least 80% sequence identity with SEQ ID NO:10 and / or SEQ ID NO:11.

[0013] In one or more embodiments, the recombinant element comprises the amino acid sequence described in SEQ ID NO:8, or an amino acid sequence having at least 80% sequence identity with SEQ ID NO:8.

[0014] In one or more embodiments, the genome editing system further comprises one or more elements selected from: promoter, replicon, marker gene, terminator, and donor fragment.

[0015] In one or more embodiments, the promoter includes or is selected from: constitutive promoters and inductive promoters.

[0016] In one or more embodiments, the constitutive promoter includes the PJ23119 promoter and the PlacM promoter.

[0017] In one or more embodiments, the inducible promoter includes Ptrc.

[0018] In one or more embodiments, the replicon includes: Corynebacterium glutamicum replicon and Escherichia coli replicon.

[0019] In one or more embodiments, the Corynebacterium glutamicum replicons include pGA1 and pBL1.

[0020] In one or more embodiments, the E. coli replicon includes pMB1 and repA101.

[0021] In one or more embodiments, the marker gene includes or is selected from: resistance genes and fluorescent genes.

[0022] In one or more embodiments, the resistance gene includes a kanamycin resistance gene, a spectinomycin resistance gene, an apramycin resistance gene, and a chloramphenicol resistance gene.

[0023] In one or more embodiments, the terminator includes: rrnB.

[0024] In one or more embodiments, the donor fragment contains a sequence of interest; preferably, the sequence of interest further includes homologous arms at both ends for homologous recombination of the sequence of interest into the target sequence.

[0025] A second aspect of the present invention provides a polynucleotide comprising: a coding sequence for a nuclease and a sequence for a guide RNA;

[0026] The endonuclease comprises the amino acid sequence described in SEQ ID NO:1, or an amino acid sequence having at least 80% sequence identity with SEQ ID NO:1.

[0027] The guide RNA comprises the nucleic acid sequence shown in SEQ ID NO:2 or a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO:2.

[0028] In one or more embodiments, the endonuclease, recognition sequence, or guide RNA is as described in any embodiment of the present invention.

[0029] In one or more embodiments, the polynucleotide further comprises one or more elements selected from: promoter, replicon, marker gene, terminator, donor fragment.

[0030] In one or more embodiments, the promoter, replicon, marker gene, terminator, and donor fragment are as described in any embodiment of the present invention.

[0031] In one or more embodiments, the polynucleotide further comprises: a coding sequence for a recombinant element that facilitates recombination of the sequence of interest into the Corynebacterium glutamicum cleavage site.

[0032] In one or more embodiments, the recombining element is as described in any embodiment of the present invention.

[0033] In one or more embodiments, the coding sequence of the recombinant element comprises the nucleic acid sequence described in SEQ ID NO:12, or a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO:12.

[0034] A third aspect of the present invention provides a nucleic acid construct comprising the polynucleotides described in any embodiment of the present invention.

[0035] In one or more embodiments, the nucleic acid construct contains the polynucleotide in one or more expression frames, and / or the nucleic acid construct contains the plurality of expression frames in one or more vectors.

[0036] In one or more embodiments, the nucleic acid construct further comprises the coding sequence of the recombinant element described in any embodiment of the present invention.

[0037] In one or more embodiments, the nucleic acid construct contains, in two expression frames, the coding sequences of the polynucleotide and the recombinant element described in any embodiment of the present invention, respectively, and / or the nucleic acid construct contains, in two vectors, the coding sequences of the polynucleotide and the recombinant element described in any embodiment of the present invention, respectively.

[0038] In one or more embodiments, the nucleic acid construct comprises a first vector and a second vector, the first vector containing a coding sequence for the endonuclease and a guide RNA sequence, and the second vector containing a coding sequence for a recombinant element that promotes recombination of the sequence of interest into the Corynebacterium glutamicum cleavage site.

[0039] In one or more embodiments, the first vector further includes one or more elements selected from: promoter, replicon, marker gene, donor fragment.

[0040] In one or more embodiments, the first vector comprises or consists of the following elements: the coding sequence of the endonuclease, the sequence of the guide RNA, Corynebacterium glutamicum replicon pBL1, Escherichia coli replicon repA101, a first marker gene, the PlacM promoter, the PJ23119 promoter, and a donor fragment.

[0041] In one or more embodiments, the second vector further includes one or more elements selected from: promoter, replicon, marker gene.

[0042] In one or more embodiments, the second vector includes or is composed of the following elements: a coding sequence for a recombinant element that promotes recombination of the sequence of interest into the Corynebacterium glutamicum cleavage site, Corynebacterium glutamicum replicon pGA1, Escherichia coli replicon pMB1, a second marker gene, a LacI gene, and a Ptrc promoter.

[0043] A fourth aspect of the present invention provides a kit for gene editing of Corynebacterium glutamicum, the kit comprising the genome editing system described in any embodiment of the present invention, or the polynucleotide described in any embodiment of the present invention, or the nucleic acid construct described in any embodiment of the present invention.

[0044] A fifth aspect of the invention provides the application of the genome editing system described in any embodiment of the invention, or the polynucleotide described in any embodiment of the invention, or the nucleic acid construct described in any embodiment of the invention, or the kit described in any embodiment of the invention, in gene editing of target sequences of Corynebacterium glutamicum.

[0045] In one or more embodiments, the gene editing includes: gene knockout and gene insertion.

[0046] A sixth aspect of the present invention provides a method for gene editing a target sequence of Corynebacterium glutamicum, comprising: contacting the genome editing system described in any embodiment of the present invention, or the polynucleotide described in any embodiment of the present invention, or the nucleic acid construct described in any embodiment of the present invention, or the kit described in any embodiment of the present invention with the target sequence of Corynebacterium glutamicum, or delivering it to a cell containing the target sequence of Corynebacterium glutamicum; wherein the target sequence is present in the target sequence of Corynebacterium glutamicum.

[0047] In one or more embodiments, the gene editing includes: gene knockout and gene insertion.

[0048] A seventh aspect of the present invention provides a method for recombining a sequence of interest into a target sequence, comprising the step of contacting a genome editing system, a polynucleotide, or a nucleic acid construct as described in any embodiment of the present invention with the sequence of interest and the target sequence.

[0049] In one or more embodiments, the ends of the sequence of interest further include homologous arms for homologous recombination of the sequence of interest into the target sequence. Attached Figure Description

[0050] Figure 1 Schematic diagram of the structure of plasmid pJYS3-ISClsp3-reRNA-poxB.

[0051] Figure 2 Schematic diagram of pRecE564LT-spc structure.

[0052] Figure 3 PCR verification of Example 2: pJYS3-ISClsp3-reRNA was used to edit the crtYe site of Corynebacterium glutamicum ATCC 13032 to delete DNA fragments.

[0053] Figure 4 Example 3: PCR verification. The avtA gene was knocked out in Corynebacterium glutamicum ATCC 13032 using pJYS3-ISClsp3-reRNA and pRecE564LT-spc helper plasmid.

[0054] Figure 5 Example 4: PCR verification. Genes were inserted into the ATCC 13032poxB site of Corynebacterium glutamicum using pJYS3-ISClsp3-reRNA and pRecE564LT-spc helper plasmids. Detailed Implementation

[0055] To overcome the problems of low gene editing efficiency and difficulty in inserting large fragments in Corynebacterium glutamicum, the inventors have conducted in-depth research and provided a genome editing system for Corynebacterium glutamicum. This genome editing system can effectively achieve genome editing in the Corynebacterium glutamicum ATCC13032 strain, and is more efficient than CRISPR-Cas9 and CRISPR-Cpf1.

[0056] the term

[0057] The term "target gene" or "target sequence" refers to a polynucleotide targeted by a guide RNA, such as a sequence that is complementary to the guide RNA sequence. Perfect complementarity is not required, as long as sufficient complementarity exists to induce hybridization and promote the formation of an endonuclease complex. The target sequence can comprise any polynucleotide, such as DNA or RNA. In some cases, the target sequence is located intracellularly or extracellularly. In this invention, the "target sequence," "target polynucleotide," or "target nucleic acid" can be any endogenous or exogenous polynucleotide for Corynebacterium glutamicum.

[0058] The term "homologous arm" refers to two sequences that are highly homologous to the target DNA sequence on either side of the target gene, which serves as a template during gene editing. Homologous arms are typically located 20–2000 bp (e.g., around 1000 bp) upstream and downstream of the target gene or sequence.

[0059] The term "donor fragment" refers to a sequence that, after a double-strand break is created by a nuclease cleaving a target knockout gene, is simultaneously recombined into the original target knockout gene location by the host's homologous recombination repair system to repair the double-strand break. The donor fragment typically contains upstream and downstream homologous arms of the target site, and optionally also contains the sequence of interest (e.g., the sequence to be knocked in or the insertion sequence). The donor fragment that can be used for recombination into the sequence of interest is also called a "template" or "repair template."

[0060] The terms “guide RNA” and “reRNA” are used interchangeably in this document and refer to RNA molecules that can form complexes with endonucleases and, due to a certain sequence similarity with the target sequence, can target the target sequence with the complexes.

[0061] The terms “polynucleotide,” “nucleic acid sequence,” “nucleotide sequence,” or “nucleic acid fragment” are used interchangeably and refer to single-stranded or double-stranded RNA or DNA polymers, optionally containing synthetic, non-natural, or modified nucleotide bases. Nucleotides are designated by their single-letter names as follows: “A” for adenosine or deoxyadenosine (corresponding to RNA or DNA, respectively), “C” for cytidine or deoxycytidine, “G” for guanosine or deoxyguanosine, “U” for uridine, and “T” for deoxythymidine. Although nucleotide sequences may be represented as DNA sequences (containing T) herein, when referring to RNA, those skilled in the art can readily determine the corresponding RNA sequence (i.e., replacing T with U).

[0062] Sequence “identity” has a generally accepted meaning in the art, and the percentage of sequence similarity between two nucleic acid or polypeptide molecules or regions can be calculated using publicly available techniques (such as BLAST). Sequence identity can be measured along the full length of a polynucleotide or polypeptide or along a region of that molecule. Although many methods exist for measuring the identity between two polynucleotides or polypeptides, the term “identity” is well known to those skilled in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988)).

[0063] The term "construct" or "expression construct" refers to a vector, such as a recombinant vector, suitable for expressing a nucleotide sequence of a gene of interest in an organism. "Expression" refers to the production of a functional product. For example, the expression of a nucleotide sequence can refer to the transcription of the nucleotide sequence (e.g., transcription to generate mRNA or functional RNA) and / or the translation of RNA into a precursor or mature protein.

[0064] The term "expression construct" can be a linear nucleic acid fragment, a circular plasmid, a viral vector, or, in some embodiments, a translatable RNA (such as mRNA).

[0065] The term "expression construct" can include regulatory sequences and nucleotide sequences of the gene of interest from different sources, or regulatory sequences and nucleotide sequences of interest from the same source but arranged in a manner different from those normally found in nature.

[0066] The terms "regulatory sequence" and "regulatory element" are used interchangeably, referring to a nucleotide sequence located upstream (5' non-coding sequence), midway, or downstream (3' non-coding sequence) of a coding sequence that affects the transcription, RNA processing, or stability or translation of the relevant coding sequence. Regulatory sequences may include, but are not limited to, promoters, translational leader sequences, introns, and polyadenylation recognition sequences.

[0067] The term "promoter" refers to a nucleic acid fragment capable of controlling the transcription of another nucleic acid fragment. In some embodiments of the invention, a promoter is a promoter capable of controlling gene transcription in a cell, regardless of whether it originates from the cell. A promoter can be a constitutive promoter, a tissue-specific promoter, a developmentally regulatory promoter, or an inducible promoter.

[0068] The term "operably linked" refers to the linking of a regulatory element (e.g., but not limited to, promoter sequences, transcription termination sequences, etc.) to a nucleic acid sequence (e.g., coding sequences or open reading frames) such that the transcription of the nucleotide sequence is controlled and regulated by the transcriptional regulatory element. Techniques for operably linking regulatory element regions to nucleic acid molecules are known in the art.

[0069] "Introducing" nucleic acid molecules (such as plasmids, linear nucleic acid fragments, RNA, etc.) or proteins into an organism refers to transforming the cells of an organism with the nucleic acid or protein so that the nucleic acid or protein can function in the cell.

[0070] The term "Corynebacterium glutamicum" generally refers to a Gram-positive rod-shaped bacterium used industrially for the large-scale production of amino acids, particularly glutamic acid and lysine. Corynebacterium glutamicum includes, but is not limited to, ATCC 13032, ATCC 14067, and ATCC 13869.

[0071] Genome editing system for Corynebacterium glutamicum

[0072] The present invention provides a genome editing system for Corynebacterium glutamicum, comprising: a nucleic acid protein complex capable of specifically cutting DNA in Corynebacterium glutamicum, wherein the nucleic acid protein complex comprises an endonuclease and guide RNA;

[0073] The endonuclease comprises the amino acid sequence described in SEQ ID NO:1, or an amino acid sequence having at least 80% sequence identity with SEQ ID NO:1 (e.g., sequence identity ≥85%, sequence identity ≥90%, sequence identity ≥92%, sequence identity ≥95%, sequence identity ≥96%, sequence identity ≥97%, sequence identity ≥98%, sequence identity ≥99%, sequence identity ≥99.5% or more).

[0074] The guide RNA comprises: (1) the nucleic acid sequence shown in SEQ ID NO:2 or a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO:2 (e.g., sequence identity ≥85%, sequence identity ≥90%, sequence identity ≥92%, sequence identity ≥95%, sequence identity ≥96%, sequence identity ≥97%, sequence identity ≥98%, sequence identity ≥99%, sequence identity ≥99.5% or more), and (2) a recognition sequence that is complementary to or identical to the target sequence, the recognition sequence having a length of 5-50 nt, for example 10-45 nt, 15-40 nt, 20-35 nt, preferably 20 nt.

[0075] In one or more embodiments, the genome editing system further includes a recombinant element that facilitates recombination of the sequence of interest into the cleavage site of Corynebacterium glutamicum. The recombinant element can reduce the degradation of the donor fragment transferred into the cell to be edited and improve homologous recombination efficiency, thereby increasing editing efficiency.

[0076] In one or more embodiments, the recombinant element comprises the amino acid sequence described in SEQ ID NO:10 and / or 11, or an amino acid sequence having at least 80% sequence identity with SEQ ID NO:10 and / or 11.

[0077] In one or more embodiments, the recombinant element comprises the amino acid sequence described in SEQ ID NO:8, or an amino acid sequence having at least 80% sequence identity with SEQ ID NO:8.

[0078] In the gene editing system of the present invention, the endonuclease (e.g., ISClsp3) plays two roles: first, the reRNA on the helper plasmid guides the endonuclease (e.g., ISClsp3) to cut the target site of Corynebacterium glutamicum genome to achieve genome modification; second, after the genome modification is completed, in order to obtain a host bacterium without recombinant elements for iterative editing, it is necessary to use the reRNA-guided endonuclease (e.g., ISClsp3) to cut the recombinant elements (e.g., helper plasmid) to eliminate the recombinant elements (e.g., helper plasmid).

[0079] In one or more embodiments, the endonuclease (e.g., ISClsp3) cleaves the target knockout gene to create a double-strand break, and assembles upstream and downstream fragments (i.e., homologous arm fragments) of the knockout gene into a donor fragment. The host's homologous recombination repair system repairs the double-strand break while simultaneously recombining the donor fragment into the original gene, thus completing the knockout or knock-in. In one or more embodiments, the donor fragment includes homologous arm fragments of 20–2000 bp (e.g., approximately 1000 bp) upstream and downstream of the insertion site, and optionally also includes the sequence of interest (e.g., the sequence to be knocked in or the insertion sequence).

[0080] In one or more embodiments, the genome editing system further comprises one or more elements selected from: promoter, replicon, marker gene, terminator, and donor fragment.

[0081] In one or more embodiments, the promoter includes or is selected from: constitutive promoters and inductive promoters.

[0082] In one or more embodiments, the constitutive promoter includes the PJ23119 promoter and the PlacM promoter; the inducible promoter includes Ptrc.

[0083] In one or more embodiments, the replicon includes: Corynebacterium glutamicum replicon and Escherichia coli replicon.

[0084] In one or more embodiments, the Corynebacterium glutamicum replicons include pGA1 and pBL1; the Escherichia coli replicons include pMB1 and repA101.

[0085] In one or more embodiments, the marker gene includes or is selected from: resistance genes and fluorescent genes.

[0086] In one or more embodiments, the resistance gene includes a kanamycin resistance gene, a spectinomycin resistance gene, an apramycin resistance gene, and a chloramphenicol resistance gene.

[0087] In one or more embodiments, the terminator includes: rrnB.

[0088] In one or more embodiments, the donor fragment contains a sequence of interest; preferably, the sequence of interest further includes homologous arms at both ends for homologous recombination of the sequence of interest into the target sequence.

[0089] In one or more embodiments, the genome editing system for Corynebacterium glutamicum comprises: a nucleic acid-protein complex capable of specifically cleaving DNA, and a recombinant element that facilitates recombination of the sequence of interest into the cleavage site of Corynebacterium glutamicum.

[0090] The nucleic acid-protein complex contains an endonuclease and guide RNA;

[0091] The endonuclease comprises the amino acid sequence described in SEQ ID NO:1, or an amino acid sequence having at least 80% sequence identity with SEQ ID NO:1 (e.g., sequence identity ≥85%, sequence identity ≥90%, sequence identity ≥92%, sequence identity ≥95%, sequence identity ≥96%, sequence identity ≥97%, sequence identity ≥98%, sequence identity ≥99%, sequence identity ≥99.5% or more).

[0092] The guide RNA comprises: (1) the nucleic acid sequence shown in SEQ ID NO:2 or a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO:2 (e.g., sequence identity ≥85%, sequence identity ≥90%, sequence identity ≥92%, sequence identity ≥95%, sequence identity ≥96%, sequence identity ≥97%, sequence identity ≥98%, sequence identity ≥99%, sequence identity ≥99.5% or more), and (2) a recognition sequence complementary to the target sequence, the recognition sequence having a length of 5-50 nt (e.g., 5-45 nt, 5-40 nt, 8-35 nt, 9-30 nt, 10-25 nt or 15-20 nt, preferably 20 nt).

[0093] Nucleotide endonuclease

[0094] The term "endonuclease" refers to an enzyme that can be used to cleave nucleic acids. Endonucleases described herein can produce DNA or RNA breaks in the target nucleic acid (Cis cleavage) or DNA or RNA breaks in a side-branched nucleic acid substrate (single-stranded nucleic acid substrate) (i.e., nonspecific or non-targeted, Trans cleavage). In some embodiments, the cleavage is a double-stranded DNA break. In some embodiments, the cleavage is a single-stranded DNA break or a single-stranded RNA break.

[0095] In this invention, the nuclease typically refers to a TnpB nuclease, such as the TnpB nuclease (ISClsp3) of Clostridiales bacterium AM23-16LB. It should be understood that although only ISSlsp3 is used as an example in the embodiments, TnpB nucleases derived from other species that have a high sequence identity with ISSlsp3 (e.g., more than 80% sequence identity, preferably more than 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 99.5%) and have similar nucleic acid cleaving functions to ISSlsp3 are all within the scope of protection of this invention.

[0096] ISClsp3 nuclease can be used alone as a restriction enzyme, or it can be combined with other restriction enzymes (such as, but not limited to, Cas enzymes). Combining multiple restriction enzymes can produce additive or synergistic effects, such as increasing digestion efficiency and expanding the range of digestible substrates.

[0097] In some specific implementations, the endonuclease is selected from the group consisting of:

[0098] (i) A protein having the amino acid sequence shown in SEQ ID NO:1;

[0099] (ii) A protein derived from (i) having the nucleic acid cleaving function, formed by substituting, deleting, or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO:1; or

[0100] (iii) A protein having the said nucleic acid cleaving function, whose amino acid sequence is ≥80% identical to the amino acid sequence shown in SEQ ID NO:1 (e.g., sequence identity ≥85%, sequence identity ≥90%, sequence identity ≥92%, sequence identity ≥95%, sequence identity ≥96%, sequence identity ≥97%, sequence identity ≥98%, sequence identity ≥99%, sequence identity ≥99.5% or more).

[0101] Guide RNA

[0102] In this invention, the guide RNA comprises: (1) the nucleic acid sequence shown in SEQ ID NO:2 or a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO:2 (e.g., more than 80% sequence identity, preferably 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, 99.5% or more sequence identity), and (2) a recognition sequence complementary to the target sequence.

[0103] In some embodiments, the identification sequence comprises, or consists of, sequences selected from, the following:

[0104] (i) The sequence shown in any one of SEQ ID NO:3-7, 14;

[0105] (ii) A sequence having one or more substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases) compared to the sequence shown in any of SEQ ID NO:3-7, 14;

[0106] (iii) A sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence identity with any of the sequences shown in SEQ ID NO:3-7, 14;

[0107] (iv) A sequence that hybridizes under stringent conditions with any of the sequences described in (i)-(iii); or

[0108] The complementary sequence of the sequence described in any of (v)(i)-(iii);

[0109] Furthermore, the sequence in any one of (ii)-(v) substantially retains the biological function of the sequence from which it is derived, the biological function of which is the ability to bind complementary to the target sequence and guide the activity of the target sequence.

[0110] Typically, the recognition sequence complementary to the target sequence in the guide RNA is 5-50 nt. The length of the guide RNA containing the recognition sequence is 150-300 nt, preferably 180-250 nt, 190-230 nt, 195-220 nt, 200-210 nt, or, for example, 206 nt.

[0111] In one or more embodiments, typically, the target sequence is preceded by a target-adjacent motif (TAM) sequence that is complementary to or identical to the guide RNA recognition sequence. In one or more specific embodiments, the TAM sequence is 5'-AGGAG-3'.

[0112] In one or more embodiments, when the endonuclease comprises the amino acid sequence described in SEQ ID NO:1, or an amino acid sequence having at least 80% sequence identity with SEQ ID NO:1, the guide RNA comprises or is selected from:

[0113] (i) The sequence shown in any one of SEQ ID NO:15-20;

[0114] (ii) A sequence formed by substitution, deletion or addition of one or more bases compared to the sequence shown in any of SEQ ID NO:15-20;

[0115] (iii) A sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence identity with any of the sequences shown in SEQ ID NO:15-20;

[0116] (iv) A sequence that hybridizes under stringent conditions with any of the sequences described in (i)-(iii); or

[0117] The complementary sequence of the sequence described in any of (v)(i)-(iii);

[0118] Furthermore, the sequence described in any one of (ii)-(v) essentially retains the biological function of the sequence from which it is derived, the biological function of which is the ability to cooperate with endonucleases to cleave nucleic acids and to bind complementaryally to the target sequence to guide the activity of the target sequence.

[0119] Recombination components

[0120] The term "recombinant element" refers to an element that can be used to assist endonucleases in gene editing.

[0121] In some embodiments, the recombinant element comprises: a nucleotide sequence encoding RecE recombinase, a nucleotide sequence encoding RecT recombinase, or a nucleotide sequence encoding both RecE and RecT recombinases; preferably, the recombinant element comprises a nucleotide sequence encoding both RecE and RecT recombinases.

[0122] In some embodiments, the amino acid sequences of the RecE and RecT recombinases are as shown in SEQ ID NO:8 or have at least 80% sequence identity with the sequence shown in SEQ ID NO:8 (e.g., more than 85% sequence identity, preferably more than 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity).

[0123] In some embodiments, the amino acid sequence of the RecE recombinase is as shown in SEQ ID NO:9 or 11 or has at least 80% sequence identity with the sequence shown in SEQ ID NO:9 or 11 (e.g., more than 85% sequence identity, preferably more than 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity).

[0124] In some embodiments, the amino acid sequence of the RecT recombinase is as shown in SEQ ID NO:10 or has at least 80% sequence identity with the sequence shown in SEQ ID NO:10 (e.g., more than 85% sequence identity, preferably more than 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity).

[0125] In some embodiments, the nucleotide sequences encoding RecE and RecT recombinases are as shown in SEQ ID NO:12 or have at least 80% sequence identity with the sequence shown in SEQ ID NO:12 (e.g., more than 85% sequence identity, preferably more than 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity).

[0126] In some specific implementations, the recombinant element is an auxiliary plasmid. The addition of the auxiliary plasmid makes the genome editing system a dual-plasmid system.

[0127] In some embodiments, the helper plasmid contains nucleotide sequences encoding RecE and RecT recombinases. Preferably, the helper plasmid is pRecE564LT-spc plasmid, the nucleotide sequence of which is as shown in SEQ ID NO:13 or has at least 80% sequence identity with the sequence shown in SEQ ID NO:13 (e.g., more than 85% sequence identity, preferably more than 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity).

[0128] Other components of the genome editing system

[0129] In this invention, the genome editing system further includes one or more elements selected from the following: promoter, replicon, marker gene, terminator, target sequence, and donor fragment.

[0130] In some embodiments, the promoter includes or is selected from: a constitutive promoter and an inducible promoter; more preferably, the constitutive promoter includes the PJ23119 promoter and the PlacM promoter, and the inducible promoter includes Ptrc.

[0131] In some embodiments, the replicon includes: Corynebacterium glutamicum replicon and Escherichia coli replicon; more preferably, the Corynebacterium glutamicum replicon includes pGA1 and pBL1; and the Escherichia coli replicon includes pMB1 and repA101.

[0132] In some embodiments, the marker gene includes or is selected from: resistance genes and fluorescent genes; more preferably, the resistance gene includes kanamycin resistance genes, spectinomycin resistance genes, apramycin resistance genes, and chloramphenicol resistance genes. In some embodiments, the target sequence is a gene targeting Corynebacterium glutamicum, such as, but not limited to: the crtYe gene, avtA gene, poxB gene, cg3035 gene, coaE-cg1540 gene, and lldD-cg3230 gene.

[0133] In some implementations, the donor fragment includes a homologous arm.

[0134] In some implementations, the homologous arms are upstream and / or downstream homologous arms of the target sequence. For example, for the crtYe gene, its upstream homologous arms refer to positions 1 to 1000 upstream of the crtYe gene, and its downstream homologous arms refer to positions 367 to 1367 downstream of the crtYe gene. For the avtA gene, its upstream homologous arms refer to positions 11 to 989 upstream of the avtA gene, and its downstream homologous arms refer to positions 1012 to 901 downstream of the avtA gene. For the poxB gene, its upstream homologous arms refer to positions 1 to 1000 upstream of the poxB gene, and its downstream homologous arms refer to positions 1626 to 1024 downstream of the poxB gene.

[0135] In some implementations, the terminator includes: rrnB.

[0136] Polynucleotides

[0137] This invention provides a polynucleotide comprising: a coding sequence for a nuclease, a guide RNA sequence, and a coding sequence for a recombination element that promotes recombination of the sequence of interest to the Corynebacterium glutamicum cleavage site.

[0138] The endonuclease comprises the amino acid sequence described in SEQ ID NO:1, or an amino acid sequence having at least 80% sequence identity with SEQ ID NO:1.

[0139] The guide RNA comprises: (1) the nucleic acid sequence shown in SEQ ID NO:2 or a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO:2, and (2) a recognition sequence complementary to the target sequence.

[0140] In some implementations, the polynucleotide also includes sequences of other elements of the genome editing system.

[0141] It should be understood that, in the polynucleotide, the endonuclease, guide RNA, recombinant element, recognition sequence, and other elements of the genome editing system are as described in any embodiment of the present invention.

[0142] Nucleic acid constructs

[0143] The present invention also provides a nucleic acid construct comprising the polynucleotides described in any embodiment of the present invention.

[0144] In some embodiments, the nucleic acid construct includes the polynucleotide in one or more expression frames. In other embodiments, the nucleic acid construct includes the plurality of expression frames in one or more vectors.

[0145] In some embodiments, the nucleic acid construct comprises a first vector and a second vector, wherein the first vector contains the coding sequence of the endonuclease and the sequence of guide RNA; and the second vector contains the coding sequence of a recombination element that promotes recombination of the sequence of interest to the cleavage site.

[0146] In some preferred embodiments, the first vector further includes one or more elements selected from: promoter, replicon, marker gene, donor fragment (including its homologous arms).

[0147] In some preferred embodiments, the first vector comprises or consists of the following elements: the coding sequence of the endonuclease, the sequence of the guide RNA, Corynebacterium glutamicum replicon pBL1, Escherichia coli replicon repA101, a first marker gene (e.g., a kanamycin resistance gene), a PlacM promoter (for initiating transcription of the endonuclease), a PJ23119 promoter (for initiating transcription of the guide RNA), and a donor fragment (including its homologous arms).

[0148] In some preferred embodiments, the second vector further includes one or more elements selected from: a promoter, a replicon, and a marker gene. In some more preferred embodiments, the second vector includes or consists of: a coding sequence for a recombinant element that promotes recombination of the sequence of interest into the Corynebacterium glutamicum cleavage site, the Corynebacterium glutamicum replicon pGA1, the Escherichia coli replicon pMB1, a second marker gene (e.g., a spectinomycin resistance gene), a LacI gene, and a Ptrc promoter.

[0149] In some preferred embodiments, the first vector (which may be a pJYS3 plasmid, such as pJYS3-ISCba / ISClsp3-reRNA) and the second vector (which may be an auxiliary plasmid pRecE564LT-spc) constitute a dual-vector system for Corynebacterium glutamicum genome editing.

[0150] For example, the elements in the first and / or second vectors can be obtained by PCR amplification and assembled together to obtain the first and / or second vectors. Preferably, the assembly can be performed using the ClonExpressMultiS One Step Cloning Kit (Vazyme) or Gibson.

[0151] In some implementations, the nucleic acid construct may further include a third, fourth, or more vectors, such as, but not limited to, pEcISFba1 plasmid, pEcISEre1 plasmid, etc.

[0152] The term "vector" refers to a nucleic acid molecule capable of delivering another nucleic acid molecule linked to it. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules including one or more free ends, or without free ends (e.g., circular); nucleic acid molecules including DNA, RNA, or both; and a wide variety of other polynucleotides known in the art. Vectors can be introduced into host cells through transformation, transduction, or transfection, thereby enabling the expression of the genetic material elements they carry in the host cells. A vector can be introduced into a host cell to produce transcripts, proteins, or peptides, including proteins, fusion proteins, isolated nucleic acid molecules, etc. (e.g., endonuclease transcripts) as described herein. A vector may contain a variety of elements controlling expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop in which another DNA fragment can be inserted, for example, using standard molecular cloning techniques. Another type of vector is the viral vector, in which a virus-derived DNA or RNA sequence is present in a vector used to package the virus (e.g., retrovirus, replication-defective retrovirus, adenovirus, replication-defective adenovirus, and adeno-associated virus). Viral vectors also contain polynucleotides carried by a virus for transfection into a host cell. Some vectors (e.g., bacterial vectors with bacterial origins of replication and episodic mammalian vectors) are capable of autonomous replication in the host cell into which they are introduced. Other vectors (e.g., non-episodic mammalian vectors) integrate into the host cell's genome after introduction and thereby replicate along with the host genome. Furthermore, some vectors are capable of directing the expression of genes they are operatively linked to. Such vectors are referred to herein as "expression vectors."

[0153] The vector of the present invention can be a cloning vector or an expression vector. In some embodiments, the vector of the present invention is, for example, a plasmid (preferably pJYS3 plasmid), a granule, a bacteriophage, a Cosmid, etc.

[0154] The present invention also provides host cells comprising the isolated nucleic acid molecules or vectors as described above. Such host cells include, but are not limited to, prokaryotic cells such as *Escherichia coli* cells and *Corynebacterium glutamicum* cells. The cells of the present invention can also be cell lines.

[0155] Reagent test kit

[0156] The present invention also provides a kit for gene editing of Corynebacterium glutamicum, the kit comprising the genome editing system, polynucleotide or nucleic acid constructs described in the present invention.

[0157] In some embodiments, the kit further comprises primers, such as primer pairs for amplifying the coding sequences of the endonuclease, guide RNA sequences, recombinant elements, and target sequences. The primers typically comprise an upstream primer and a downstream primer. Exemplary primer sequences are shown in Tables 1 to 6.

[0158] In some embodiments, the kit further comprises one or more buffers. The buffer can be any buffer, including but not limited to sodium carbonate buffer, sodium bicarbonate buffer, borate buffer, Tris buffer, MOPS buffer, HEPES buffer, and combinations thereof. In some embodiments, the buffer is alkaline. In some embodiments, the buffer has a pH from about 7 to about 10.

[0159] In some implementations, the kit also includes instructions for using the genome editing system, polynucleotides, and / or nucleic acid constructs.

[0160] In some embodiments, the components contained in the kit of the present invention can be provided in any suitable container.

[0161] Methods and applications of genome editing

[0162] This invention provides the application of the genome editing system and the polynucleotide and / or nucleic acid constructs described herein in gene editing of target sequences of Corynebacterium glutamicum. Specifically, the gene editing includes gene knockout and gene insertion.

[0163] The present invention also provides a method for gene editing of a target sequence of Corynebacterium glutamicum, comprising: contacting the target sequence of Corynebacterium glutamicum with a genome editing system as described in the present invention, or a polynucleotide and / or nucleic acid construct as described in the present invention, or delivering it to a cell containing the target sequence of Corynebacterium glutamicum; wherein the target sequence is present in the target sequence of Corynebacterium glutamicum.

[0164] In some embodiments, the method further includes contacting the editing template with the target sequence or delivering it to a cell containing the target sequence. In such embodiments, the method repairs the broken target sequence by homologous recombination with a foreign template polynucleotide, wherein the repair results in a mutation, including the insertion, deletion, or substitution of one or more nucleotides of the target sequence. In some embodiments, the mutation results in a change of one or more amino acids in a protein expressed from a gene containing the target sequence.

[0165] The present invention also provides a method for recombining a sequence of interest into a target sequence, which includes the step of contacting the genome editing system or nucleic acid construct described in any embodiment herein with the sequence of interest and the target sequence.

[0166] This invention also provides a method for gene editing a target nucleic acid sequence, comprising the step of contacting a genome editing system or nucleic acid construct as described in any embodiment herein with a donor fragment and a target sequence, the donor fragment comprising two homologous arms complementary to sequences upstream and downstream of the target site. In some embodiments, the two homologous arms in the donor fragment are directly connected, and recombination of the donor fragment results in the deletion of the sequence in the target sequence. In some embodiments, the donor fragment further includes a sequence of interest (a knock-in sequence or an insert sequence) located between the two homologous arms, and recombination of the donor fragment results in the insertion of the sequence of interest into the target site. The length of each homologous arm can be adjusted as needed by those skilled in the art, for example, 20-2000 bp. The homologous arm lengths can be the same or different, for example, 1000 bp.

[0167] The present invention provides a method for altering the expression of a gene product of Corynebacterium glutamicum, comprising: contacting a genome editing system as described in the present invention, a polynucleotide and / or nucleic acid construct as described in the present invention, with a nucleic acid molecule encoding the gene product, or delivering it to a cell containing the nucleic acid molecule, wherein the target sequence is present in the nucleic acid molecule.

[0168] In some embodiments, the method is used to alter the expression of a gene product in vitro or in vitro. In some embodiments, the method is not a method for treating humans or animals as a therapy. In some embodiments, the method does not include the step of modifying human germline genetic characteristics.

[0169] In some embodiments, the nucleic acid molecule is present in an in vitro nucleic acid molecule (e.g., a plasmid). In some embodiments, the nucleic acid molecule is present in a plasmid.

[0170] In some embodiments, the expression of the gene product is altered (e.g., enhanced or reduced). In some embodiments, the expression of the gene product is enhanced. In some embodiments, the expression of the gene product is reduced.

[0171] In some implementations, the gene product is a protein.

[0172] In some specific implementations, the method for gene editing of Corynebacterium glutamicum using the dual-vector system includes:

[0173] 1) The helper plasmid pRecE564LT-spc was transformed into competent Corynebacterium glutamicum cells and screened on BHIS solid plates containing spectinomycin at 30°C.

[0174] 2) Select positive clones, prepare electrocompetent cells, and add 2mM isopropyl-β-D-thiogalactoside to the transfer and incubation medium to induce RecE*T recombinase expression.

[0175] 3) The plasmid pJYS3-ISClsp3-reRNA was transferred into the competent cells obtained in step 2) by electroporation. The bacterial culture was plated on BHIS solid plates containing kanamycin and spectinomycin, and cultured overnight at 30°C. The clones were then verified and screened.

[0176] 4) Select positive clones, inoculate them in BHIS liquid medium and incubate overnight at 30°C. Streak them on BHIS-free solid plates. Select single colonies on BHIS-resistant plates containing kanamycin or spectinomycin for identification. Verify the elimination of plasmid pJYS3-ISCba / ISClsp3-reRNA or helper plasmid pRecE564LT-spc to obtain the edited strain.

[0177] The advantages of this invention include:

[0178] This invention discovers that TnpB (referred to as ISClsp3) derived from Clostridiales bacterium AM23-16LB can achieve specific DNA cleavage in Corynebacterium glutamicum and can be used for genome editing (insertion / knockout). Its editing efficiency is better than that of Cas9 and Cpf1. Using helper plasmids containing recombinant elements can further improve the efficiency of gene editing.

[0179] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0180] Materials and methods

[0181] In the examples, whole-genome synthesis was performed by Nanjing Genscript Biotech Co., Ltd., sequencing was performed by Platinum Biotech (Shanghai) Co., Ltd. or Shanghai Sangon Biotech Co., Ltd., and primer synthesis was performed by Qingke Biotechnology Co., Ltd. or Shanghai Sangon Biotech Co., Ltd.

[0182] The molecular biology experiments described in this article, including plasmid construction, enzyme digestion, ligation, competent cell preparation, transformation, and culture medium preparation, were mainly conducted in accordance with *Molecular Cloning: A Laboratory Manual* (3rd Edition), edited by J. Sambrook and DW. Russell, translated by Huang Peitang et al., Science Press, Beijing, 2002. Specific experimental conditions could be determined through simple experiments if necessary. PCR amplification experiments were performed according to the reaction conditions or instructions provided by the plasmid or DNA template supplier. Adjustments could be made through simple experiments if necessary.

[0183] Example 1: Construction of plasmid pJYS3-ISClsp3-reRNA-poxB targeting the poxB site of Corynebacterium glutamicum ATCC13032

[0184] The primers required for constructing plasmid pJYS3-ISClsp3-reRNA are shown in Table 1. The sequence of plasmid pJYS3 is referenced from addgene#85542.

[0185] Table 1. PCR primer sequences for constructing plasmid pJYS3-ISClsp3-reRNA

[0186]

[0187] 1.1 Amplification of the ISClsp3 fragment

[0188] The ISClsp3 fragment was amplified from the PET-28A-21ISClsp3 plasmid using primers 1249 / 1334.

[0189] 1.2 Amplification of the repA101+pBL1+KanR fragment

[0190] The repA101+pBL1+KanR fragment was amplified from pJYS3_ΔcrtYf (Addgene: 85542) using primers 1224 / 1382. Here, repA101 is an *E. coli* replicon used for amplifying the fragment in *E. coli*; pBL1 is a *Corynebacterium glutamicum* replicon used for gene editing; and the KanR fragment is the kanamycin resistance gene, used to screen for successfully transformed plasmids carrying the gene.

[0191] 1.3 Amplification of reRNA+HDV fragment

[0192] The reRNA fragment targeting poxB (SEQ ID NO: 15) of plasmid ISClsp3 was amplified from plasmid ISClsp3 using primers 1337 / 1294. The HDV fragment was amplified from plasmid ISClsp3 using primers 1378 / 1335. The reRNA fragment and the HDV fragment were ligated into a reRNA+HDV fragment using primers 1378 / 1379. HDV is part of the gene sequence of Hepatitis Delta Virus (HDV) and is capable of self-cleaving; its role is to promote the self-cleavage of the reRNA sequence.

[0193] 1.4 Amplification of donor fragments

[0194] Using primers 1338 / 1339, the downstream homologous arm of the poxB gene (positions 1626 to 1024 downstream of the poxB gene) was amplified from the ATCC 13032 genome (Genebank: GCA_000011325.1). Using primers 1342 / 1343, the upstream homologous arm of the poxB gene (positions 1 to 1000 upstream of the poxB gene) was amplified from the ATCC 13032 genome. Using primers 1340 / 1341, the Phom-hom gene was amplified from the ATCC 13032 genome. Using primers 1380 / 1381, and with the upstream and downstream homologous arms of the poxB gene and the Phom-hom gene as templates, the donor fragment was amplified by PCR.

[0195] 1.5 Amplification of Termination Sequence

[0196] The terminator sequence rrnB was amplified from pJYS3_ΔcrtYf (Addgene: #85542) using primers 1332 / 1377.

[0197] 1.6 Fragment Assembly

[0198] The five fragments obtained from the above steps—ISClsp3, repA101+pBL1+KanR, reRNA+HDV, donor fragment, and terminator fragment—are used. The pJYS3-ISClsp3-reRNA plasmid was assembled using the Basic Seamless Cloning and Assembly Kit (Quanshijin #CU201-03). Sequencing confirmed its correctness.

[0199] The KOD fx used in the above PCR was purchased from Toyobo (Shanghai) Biotechnology Co., Ltd.

[0200] Example 2: Editing the crtYe site of Corynebacterium glutamicum ATCC 13032 with pJYS3-ISClsp3-reRNA to delete DNA fragments.

[0201] The primers used in this embodiment are shown in Table 2.

[0202] Table 2. PCR primer sequences

[0203]

[0204]

[0205] 2.1 Construction of pJYS3-ISClsp3-reRNA-crtYe plasmid

[0206] Using pJYS3-ISClsp3-reRNA-poxB as a template, the backbone-crtYe (containing repA101+pBL1+KanR+PlacM+ISClsp3+rrnB+HDV) and reRNA fragment (SEQ ID NO:17) were amplified using primers 1477 / 1480 and 1476 / 1478, respectively. Using Corynebacterium glutamicum ATCC 13032 as a template, the upstream homologous arm (positions 1 to 1000 upstream of the crtYe gene) and the downstream homologous arm (positions 367 to 1367 downstream of the crtYe gene) were amplified using primers 1483 / 1481 and 1479 / 1482, respectively.

[0207] The amplified fragments were recovered using a gel extraction kit and then analyzed using a homologous recombination kit. The recombinant reaction was performed using the Basic Seamless Cloning and Assembly Kit (Quanshijin #CU201-03). 10 μl of the reaction solution was used to transform DH5α-competent cells. The recovery solution was then plated onto LB agar plates containing kanamycin (final concentration 50 μg / mL) and incubated overnight at 30°C to obtain transformants containing the pJYS3-ISClsp3-reRNA-crtYe plasmid. The plasmid was verified to be correct by sequencing.

[0208] 2.2 Electroporation of pJYS3-ISClsp3-reRNA-crtYe plasmid into Corynebacterium glutamicum ATCC 13032

[0209] Inoculate *Corynebacterium glutamicum* ATCC 13032 into BHIS (37 g / L BHI, 91 g / L D-sorbitol) tubes and incubate overnight at 30°C. Inoculate the overnight culture into 50 mL shake flasks of BHIS medium at a 2% inoculation rate, adding glycine to a final concentration of 4 g / L and 0.1% Tween 80. Incubate at 30°C for 4-6 h until the OD600 reaches approximately 1.0, then incubate on ice for 20 min. Collect the bacterial cells by centrifugation at 4500 rpm for 10 min at 4°C. The bacterial cells were washed twice with 10% glycerol. Five 100 μl competent cell agar plates were prepared from 50 mL of culture medium. 1 μg of pJYS3-ISClsp3-reRNA-crtYe plasmid was added, mixed well, and transferred to a 2 mm electroporation cuvette. Electroporation was performed at 25 μF, 2.5 kV, and 200 Ω. Immediately after electroporation, the cells were transferred to 900 μl of preheated BHIS medium at 46 °C. The medium was then incubated at 46 °C for 6 min, followed by incubation at 30 °C and 250 rpm for 4 h. The bacterial cells were collected by centrifugation and plated onto BHIS plates containing kanamycin (final concentration 25 μg / mL). The plates were incubated at 30 °C for 48 h.

[0210] 2.3 Verification of the deletion fragment at the crtYe site

[0211] Colony PCR verification was performed using primers 1531 / 1532. The positive band was 2.4 kb and the negative band was 3.3 kb.

[0212] Example 3: Knockout of genes avtA, crtYe, and cg3035 in Corynebacterium glutamicum ATCC 13032 using pJYS3-ISClsp3-reRNA and pRecE564LT-spc helper plasmid.

[0213] The primers used in this embodiment are shown in Table 3.

[0214] Table 3. PCR primer sequences

[0215]

[0216]

[0217] 3.1 Construction of plasmids pJYS3-ISClsp3-reRNA-avtA, pJYS3-ISClsp3-reRNA-crtYe, and pJYS3-ISClsp3-reRNA-cg3035

[0218] Using pJYS3-ISClsp3-reRNA-poxB as a template, the backbone-avtA (repA101+pBL1+KanR+PlacM+ISClsp3+rrnB+HDV) and the reRNA fragment targeting avtA (SEQ ID NO:16) were amplified using primers 1507 / 1514 and 1508 / 1509, respectively. Using Corynebacterium glutamicum ATCC 13032 as a template, the upstream and downstream homologous arms of the avtA gene were amplified using primers 1512 / 1513 and 1510 / 1511, respectively. The amplified fragments were recovered using a gel extraction kit and analyzed using a homologous recombination kit. The recombinant reaction was performed using the Basic Seamless Cloning and Assembly Kit (Quanshijin #CU201-03). 10 μl of the reaction solution was used to transform DH5α-competent cells. The recovery solution was then plated onto kanamycin (final concentration 50 μg / mL) LB agar plates and incubated overnight at 30°C. Transformants containing the pJYS3-ISClsp3-reRNA-avtA plasmid were obtained, and the plasmid was verified to be correct by sequencing.

[0219] Using pJYS3-ISClsp3-reRNA-poxB as a template, primers 1477 / 1480 and 1476 / 1478 were used to amplify the backbone-crtYe (repA101+pBL1+KanR+PlacM+ISCLsp3+rrnB+HDV) and reRNA fragments (SEQ ID NO: 17). Using Corynebacterium glutamicum ATCC 13032 as a template, primers 1479 / 1482 and 1483 / 1481 were used to amplify the upstream homologous arms (positions 1 to 1000 upstream of the crtYe gene) and the downstream homologous arms (positions 367 to 1367 downstream of the crtYe gene). As above, gel recovery was used for homologous recombination reaction, transformed into DH5α-competent cells, and the recovery solution was spread on kanamycin LB solid plates and cultured overnight at 30°C to obtain transformants containing the pJYS3-ISClsp3-reRNA-crtYe plasmid. The plasmid was verified to be correct by sequencing. Using pJYS3-ISClsp3-reRNA-poxB as a template, primers 1911 / 1912 and 1913 / 1914 were used to amplify backbone-cg3035 (repA101+pBL1+KanR+PlacM+ISCba+rrnB+HDV) and the reRNA fragment targeting cg3035 (SEQ ID NO:18). Using Corynebacterium glutamicum ATCC 13032 as a template, primers 1915 / 1916 and 1917 / 1918 were used to amplify the upstream homologous arm (position 11 to position 989 upstream of cg3035 gene) and the downstream homologous arm (position 1 to position 1000 downstream of cg3035 gene). As above, gel recovery was used for homologous recombination reaction, transformed into DH5α-competent cells, and the recovery solution was spread on kanamycin LB solid plates and cultured overnight at 30°C to obtain transformants containing the plasmid pJYS3-ISClsp3-reRNA-cg3035. The plasmid was verified to be correct by sequencing.

[0220] Construction of pRecE564LT-spc auxiliary plasmid:

[0221] pGA1+spcR+pMB1 was amplified from pJYS2_crtYf (Addgene: #85544) using primers 1041 / 1038. RecE564-597, RecE597RecT, and LacI fragments were amplified from the E. coli MG 1655 genome using primers 175 / 198, 199 / 174, and 177 / 261, respectively. The ligation constructs Ptrc, gap1, and gap2 fragments were then performed using primers 178-189, 223-232, and 233-244. The amplified fragments were recovered using a gel extraction kit and analyzed using a homologous recombination kit. The recombinant reaction was performed using the Basic Seamless Cloning and Assembly Kit (Fullmetall #CU201-03). 10 μl of the reaction solution was used to transform DH5α-competent cells. The recovery solution was then plated onto LB agar plates containing spectinomycin (final concentration 50 μg / mL) and incubated overnight at 30°C to obtain transformants containing the pRecE564LT-spc plasmid. The plasmid was verified to be correct by sequencing.

[0222] Table 4. Primer sequences used in constructing the pRecE564LT-spc helper plasmid

[0223]

[0224]

[0225] 3.2 Transformation of pRecE564LT-spc helper plasmid

[0226] Using a transformation method similar to that in Example 2, pRecE564LT-spc was transformed into Corynebacterium glutamicum ATCC13032. The bacterial cells were collected by centrifugation and plated onto BHIS plates containing spectinomycin (final concentration 50 μg / mL), and incubated at 30°C for 48 hours. The Corynebacterium glutamicum ATCC 13032 / pRecE564LT-spc strain was obtained.

[0227] 3.3 Transform pJYS3-ISClsp3-reRNA-avtA, pJYS3-ISClsp3-reRNA-crtYe, and pJYS3-ISClsp3-reRNA-cg3035 plasmids

[0228] Corynebacterium glutamicum ATCC 13032 / pRecE564LT-spc strain was inoculated into BHIS tubes containing spectinomycin and cultured overnight at 30°C. The overnight culture was then inoculated at a 2% inoculum into 50 mL shake flasks of BHIS medium, with the addition of glycine (4 g / L) and Tween 80 (0.1%), and isopropyl-β-D-thiogalactoside (2 mM) to induce RecE*T expression. The culture was incubated at 30°C for 4-6 h until the OD600 reached approximately 1.0, followed by a 20 min incubation on ice. The cells were then collected by centrifugation at 4500 rpm for 10 min at 4°C. The bacterial cells were washed twice with 10% glycerol. Five 100 μl competent cell aliquots were prepared from 50 ml of culture medium. 1 μg of plasmids pJYS3-ISClsp3-reRNA-avtA, pJYS3-ISClsp3-reRNA-crtYe, and pJYS3-ISClsp3-reRNA-cg3035 were added to each aliquot, mixed thoroughly, and transferred to a 2 mm electroporation cuvette. Electroporation was performed at 25 μF, 2.5 kV, and 200 Ω. Immediately after electroporation, the cells were transferred to 900 μl of preheated BHIS medium at 46 °C. The medium was then incubated at 46 °C for 6 min, followed by incubation at 30 °C and 250 rpm for 4 h. The bacterial cells were collected by centrifugation and plated onto BHIS plates containing kanamycin and spectinomycin, and incubated at 30 °C for 48 h.

[0229] 3.4 Verify whether the gene knockout at the avtA, crtYe, and cg3035 sites was successful.

[0230] For the avtA site, colony PCR was performed using primers 1533 / 1534, with a positive band of 2.4 kb and a negative band of 3.4 kb.

[0231] For the crtYe site, colony PCR was performed using primers 1531 / 1532, and the positive band was 2.4kb, while the negative band was 3.3kb.

[0232] For the cg3035 site, colony PCR was performed using primers 1919 / 1920. The positive band was 2.2kb and the negative band was 3.2kb.

[0233] Example 4: Genes were inserted into poxB, caoE-cg1540, and lldD-cg3230 of Corynebacterium glutamicum ATCC 13032 using pJYS3-ISClsp3-reRNA and pRecE564LT-spc helper plasmids.

[0234] Table 5 PCR primer sequences

[0235]

[0236]

[0237]

[0238] 4.1 Plasmid Construction

[0239] The construction of pJYS3-ISClsp3-reRNA-poxB is shown in Example 1.

[0240] Using pJYS3-ISClsp3-reRNA-poxB as a template, primers 1921 / 1922 and 1923 / 1924 were used to amplify the backbone-coaE-cg1540 (repA101+pBL1+KanR+PlacM+ISCLsp3+rrnB+HDV) and the reRNA fragment targeting coaE-cg1540 (SEQ ID NO:19). Using Corynebacterium glutamicum ATCC 13032 as a template, primers 1925 / 1926, 1927 / 1928, and 1929 / 1930 were used to amplify the upstream homologous arm (positions 285 upstream to 112 downstream of the coaE gene), the insertion sequence Phom-hom, and the downstream homologous arm (positions 1 to 1000 of the cg1540 gene). As above, gel recovery was used for homologous recombination reaction, transformed into DH5α-competent cells, and the recovery solution was spread on kanamycin LB solid plates and cultured overnight at 30°C to obtain transformants containing the plasmid pJYS3-ISClsp3-reRNA-coaE-cg1540. The plasmid was verified to be correct by sequencing.

[0241] Using pJYS3-ISClsp3-reRNA-poxB as a template, primers 1932 / 1933 and 1934 / 1935 were used to amplify the backbone-lldD-cg3230 (repA101+pBL1+KanR+PlacM+ISCLsp3+rrnB+HDV) and the reRNA fragment targeting lldD-cg3230 (SEQ ID NO:20). Using Corynebacterium glutamicum ATCC 13032 as a template, primers 1936 / 1937, 1938 / 1939 and 1940 / 1941 were used to amplify the upstream homologous arm (positions 264 to 1263 of the lldD gene), the insertion sequence Phom-hom, and the downstream homologous arm (positions 550 upstream to 135 downstream of the cg3230 gene). As above, gel recovery was used for homologous recombination reaction, transformed into DH5α-competent cells, and the recovery solution was spread on kanamycin LB solid plates and cultured overnight at 30°C to obtain transformants containing the plasmid pJYS3-ISClsp3-reRNA-lldD-cg3230. The plasmid was verified to be correct by sequencing.

[0242] 4.2 The plasmids pJYS3-ISClsp3-reRNA-poxB, pJYS3-ISClsp3-reRNA-coaE-cg1540, and pJYS3-ISClsp3-reRNA-lldD-cg3230 were transformed into Corynebacterium glutamicum ATCC 13032 / pRecE564LT-spc strain, respectively, and were validated after culturing for 48 h.

[0243] 4.3 Verify whether the sequence insertion was successful

[0244] Colony PCR was performed using primers 1408 / 1398, 1931 / 1398, and 1942 / 1398 to verify whether a 1.5kb Phom-hom was inserted between poxB, coaE-cg1540, and lldD-cg3230. A positive band was 2.1kb, and a negative band was not observed.

[0245] Example 5: Comparison of ISClsp3 with Cas9 and Cpf1

[0246] In this embodiment, the gene knockout or gene insertion methods using ISClsp3 nuclease in Examples 1-4 are compared with the gene editing methods using Cas9 or Cpf1 nucleases. The results are shown in Table 6.

[0247] The gene editing method using Cas9 nuclease was performed according to the method in Example 2 and the literature Zhang, J., Qian, FH, Dong, F., Wang, QZ, Yang, JJ, Jiang, Y., and Yang, S. (2020). De Novo Engineering of Corynebacterium glutamicum for L-Proline Production. Acs Synthetic Biology 9, 1897-1906.

[0248] The method for gene editing using Cpf1 nuclease is described in Example 2 and the literature Jiang, Y., Qian, FH, Yang, JJ, Liu, YM, Dong, F., Xu, CM, Sun, BB, Chen, B., Xu, XS, Li, Y., et al.

[0249] (2017). CRISPR-Cpf1 assisted genome editing of Corynebacteriumglutamicum. Nature Communications 8,11.

[0250] Table 6. Comparison of Editing Efficiency

[0251]

[0252] sequence

[0253] TnpB (i.e., ISClsp3) nuclease (SEQ ID NO:1) derived from Clostridiales bacterium AM23-16LB:

[0254] MRKLLKSFKTEINPTEEQKTKIRKTIGTCRFIYNFYLAHNKKLHEDGKKFMSSNKFRIWLNNEYLPQHPEYSWIKEAYSKAVTQSVNNGQTAFTRF FNHESAFPNFKKKGKSDVKMYFVKNNPKDCHCERHRIKIPSLGWVRIKEKGYIPTTKDGYVIKSGSVSIKADRYYVSVLVEVSDNKTINHFDEGIGI DLGLKDFAIVSNGRTYQNINKSAKLKKLERQLIREQRCLSRKYENLKKGDATQRANIQKQKLKVQKLHHRIDNIRTDYINKTIAEMVKIKPSYITIE DLNVKGMMKNRHLSKAVASQKFYEFRAKLLAKCNESGIELRVVDRWYPSSKTCHCCGSIKKDLKLSDRIFKCRCGYIEDRDFNAALNLRDAETYSIA

[0255] The sequence of the guide RNA (reRNA) corresponding to ISClsp3 that does not contain the 3' end recognition sequence portion (SEQ ID NO:2):

[0256] TTAAACGCAAATGTAAGTATGTACCGAAGGCTATTTCGGGAATTTACGACTGTGGAGTGTACATGAACTTGTGAGTAGATATGACTACGGTCAATCAAAAACATACACAATGAAGCAGTAAGTAATGTTCGCGAGAACTTACATTATCTCGATGTGGGTATATTTAATCACATTTTGAGTGGCAG

[0257] The recognition sequence for targeting poxB, in the 5' to 3' direction (SEQ ID NO:3)

[0258] tgccagaagaaatagtggaa

[0259] The recognition sequence for targeting avtA, in the 5' to 3' direction (SEQ ID NO:4)

[0260] cacacaaggaaagagaagcc

[0261] The recognition sequence for targeting crtYe, in the 5' to 3' direction (SEQ ID NO:5)

[0262] agtacctagataaataaagg

[0263] The recognition sequence for targeting cg3035, 5' to 3' orientation (SEQ ID NO:6)

[0264] cgcatatttttgggctattc

[0265] The recognition sequence for targeting coaE-cg1540, 5' to 3' orientation (SEQ ID NO:7)

[0266] ttttcctatatcagatttgc

[0267] The recognition sequence for targeting lldD-cg3230, 5' to 3' orientation (SEQ ID NO:14)

[0268] aaaagccacaaacctttccc

[0269] The guide RNA (reRNA) sequence targeting poxB corresponding to ISClsp3, 5' to 3' orientation (SEQ ID NO:15)

[0270] TTAAAACGCAAATGTAAGTTCGTACCGAAGGCTATTTCGGGAATTTACGACTGTGGAGTGTACATGAACTTGTGAGTAGATATGACTACGGTCAATCAAAAACATACACAATGAAGCAGTAAGTAATGTTCGCGAGAACTTACATTATCTCGATGTGGGTATATTTAATCACATTTTGAGTGGCAGtgccagaagaaatagtggaa

[0271] The guide RNA (reRNA) sequence targeting avtA corresponding to ISClsp3, 5' to 3' orientation (SEQ ID NO:16)

[0272] TTAAACGCAAATGTAAGTTCGTACCGAAGGCTATTTCGGGAATTTACGACTGTGGAGTGTACATGAACTTGTGAGTAGATATGACTACGGTCAATCAAAAACATACACAATGAAGCAGTAAGTAATGTTCGCGAGAACTTACATTATCTCGATGTGGGTATATTTAATCACATTTTGAGTGGCAGcacacaaggaaagagaagcc

[0273] The guide RNA (reRNA) sequence targeting crtYe corresponding to ISClsp3, 5' to 3' orientation (SEQ ID NO:17)

[0274] TTAAACGCAAATGTAAGTTCGTACCGAAGGCTATTTCGGGAATTTACGACTGTGGAGTGTACATGAACTTGTGAGTAGATATGACTACGGTCAATCAAAAACATACACAATGAAGCAGTAAGTAATGTTCGCGAGAACTTACATTATCTCGATGTGGGTATATTTAATCACATTTTGAGTGGCAGagtacctagataaataaagg

[0275] The guide RNA (reRNA) sequence targeting cg3035 corresponding to ISClsp3, 5' to 3' orientation (SEQ ID NO:18)

[0276] TTAAACGCAAATGTAAGTTCGTACCGAAGGCTATTTCGGGAATTTACGACTGTGGAGTGTACATGAACTTGTGAGTAGATATGACTACGGTCAATCAAAAACATACACAATGAAGCAGTAAGTAATGTTCGCGAGAACTTACATTATCTCGATGTGGGTATATTTAATCACATTTTGAGTGGCAGcgcatatttttgggctattc

[0277] The guide RNA (reRNA) sequence targeting coaE-cg1540 corresponding to ISClsp3, 5' to 3' orientation (SEQ ID NO: 19)

[0278] TTAAACGCAAATGTAAGTTCGTACCGAAGGCTATTTCGGGAATTTACGACTGTGGAGTGTACATGAACTTGTGAGTAGATATGACTACGGTCAATCAAAAACATACACAATGAAGCAGTAAGTAATGTTCGCGAGAACTTACATTATCTCGATGTGGGTATATTTAATCACATTTTGAGTGGCAGttttcctatatcagatttgc

[0279] The guide RNA (reRNA) sequence targeting lldD-cg3230 corresponding to ISClsp3, 5' to 3' orientation (SEQ ID NO: 20)

[0280] TTAAACGCAAATGTAAGTTCGTACCGAAGGCTATTTCGGGAATTTACGACTGTGGAGTGTACATGAACTTGTGAGTAGATATGACTACGGTCAATCAAAAACATACACAATGAAGCAGTAAGTAATGTTCGCGAGAACTTACATTATCTCGATGTGGGTATATTTAATCACATTTTGAGTGGCAGaaaagccacaaacctttccc

[0281] The amino acid sequence of RecE*T recombinase (SEQ ID NO:8):

[0282] MEHPHNENAGSDPHRDCSDETGEVADPVIVEDIEPGIYYGISNENYHAGPGISKSQLDDIADTPALYLWRKNAPVDTTKTKTLDLGTAFHCRVLEPEEFSNRFIVAPEFNRRTNAGKEEEKAFLMECASTGKTVITAEEGRKIELMYQSVMALPLGQWLVESAGHAESSIYWEDPETGILCRCRPDKIIPEFHWIMDVKTTADIQRFKTAYYDYRYHVQDAFYSDGYEAQFGVQPTFVFLVASTTIECGRYPVEIFMMGEEAKLAGQQEYHRNLRTLSDCLNTDEWPAIKTLSLPRWAKEYAND*MTKQPPI AKADLQKTQGNRAPAAVKNSDVISFINQPSMKEQLAAALPRHMTAERMIRIATTEIRKVPALGNCDTMSFVSAIVQCSQLGLEPGSALGHAYLLPFGNKNEKSGKKNVQLIIGYRGMIDLARRSGQIASLSARVVREGDEFSFEFGLDEKLIHRPGENEDAPVTHVYAVARLKDGGTQFEVMTRKQIELVRSLSKAGNNGPWVTHWEEMAKKTAIRRLFKYLPVSIEIQRAVSMDEKEPLTIDPADSSVLTGEYSVIDNSEE*

[0283] Amino acid sequence of RecE* recombinase (deleted amino acids 2S - 563A and with P597L mutation compared to wild - type RecE) (SEQ ID NO:9);

[0284] MEHPHNENAGSDPHRDCSDETGEVADPVIVEDIELGIYYGISNENYHAGPGISKSQLDDIADTPALYLWRKNAPVDTTKTKTLDLGTAFHCRVLEPEEFSNRFIVAPEFNRRTNAGKEEEKAFLMECASTGKTVITAEEGRKIELMYQSVMALPLGQWLVESAGHAESSIYWEDPETGILCRCRPDKIIPEFHWIMDVKTTADIQRFKTAYYDYRYHVQDAFYSDGYEAQFGVQPTFVFLVASTTIECGRYPVEIFMMGEEAKLAGQQEYHRNLRTLSDCLNTDEWPAIKTLSLPRWAKEYAND*

[0285] Amino acid sequence of RecT recombinase (SEQ ID NO:10):

[0286] MTKQPPIAKADLQKTQGNRAPAAVKNSDVISFINQPSMKEQLAAALPRHMTAERMIRIATTEIRKVPALGNCDTMSFVSAIVQCSQLGLEPGSALGHAYLLPFGNKNEKSGKKNVQLIIGYRGMIDLARRSGQIASLSARVVREGDEFSFEFGLDEKLIHRPGENEDAPVTHVYAVARLKDGGTQFEVMTRKQIELVRSLSKAGNNGPWVTHWEEMAKKTAIRRLFKYLPVSIEIQRAVSMDEKEPLTIDPADSSVLTGEYSVIDNSEE*

[0287] Amino acid sequence of wild-type RecE recombinase (SEQ ID NO:11):

[0288] MSTKPLFLLRKAKKSSGEPDVVLWASNDFESTCATLDYLIVKSGKKLSSYFKAVATNFPVVND

[0289] LPAEGEIDFTWSERYQLSKDSMTWELKPGAAPDNAHYQGNTNVNGEDMTEIEENMLLPISGQ

[0290] ELPIRWLAQHGSEKPVTHVSRDGLQALHIARAEELPAVTALAVSHKTSLLDPLEIRELHKLVRD

[0291] TDKVFPNPGNSNLGLITAFFEAYLNADYTDRGLLTKEWMKGNRVSHITRTASGANAGGGNLT

[0292] DRGEGFVHDLTSLARDVATGVLARSMDLDIYNLHPAHAKRIEEIIAENKPPFSVFRDKFITMPG

[0293] GLDYSRAIVVASVKEAPIGIEVIPAHVTEYLNKVLTETDHANPDPEIVDIACGRSSAPMPQRVTE

[0294] EGKQDDEEKPQPSGTTAVEQGEAETMEPDATEHHQDTQPLDAQSQVNSVDAKYQELRAELH

[0295] EARKNIPSKNPVDDDKLLAASRGEFVDGISDPNDPKWVKGIQTRDCVYQNQPETEKTSPDMN

[0296] QPEPVVQQEPEIACNACGQTGGDNCPDCGAVMGDATYQETFDEESQVEAKENDPEEMEGAE

[0297] HPHNENAGSDPHRDCSDETGEVADPVIVEDIEPGIYYGISNENYHAGPGISKSQLDDIADTPAL

[0298] YLWRKNAPVDTTKTKTLDLGTAFHCRVLEPEEFSNRFIVAPEFNRRTNAGKEEEKAFLMECAS

[0299] TGKTVITAEEGRKIELMYQSVMALPLGQWLVESAGHAESSIYWEDPETGILCRCRPDKIIPEFH

[0300] WIMDVKTTADIQRFKTAYYDYRYHVQDAFYSDGYEAQFGVQPTFVFLVASTTIECGRYPVEIFMMGEEAKLAGQQEYHRNLRTLSDCLNTDEWPAIKTLSLPRWAKEYAND*

[0301] Nucleotide sequence of RecE*T recombinase (SEQ ID NO:12)

[0302] atggaacatccgcacaatgagaatgctggcagcgatccgcatcgcgattgcagtgatgaaactggcgaagtcgcagatcccgtaatcgtagaa

[0303] gacatagagctaggtatttattacggaatttcgaatgagaattaccacgcgggtcccggtatcagtaagtctcagctcgatgacattgctgatactc

[0304] cggcactatatttgtggcgtaaaaatgcccccgtggacaccacaaagacaaaaacgctcgatttaggaactgctttccactgccgggtacttgaac

[0305] cggaagaattcagtaaccgctttatcgtagcacctgaatttaaccgccgtacaaacgccggaaaagaagaagagaaagcgtttctgatggaatgc

[0306] gcaagcacaggaaaaacggttatcactgcggaagaaggccggaaaattgaactcatgtatcaaagcgttatggctttgccgctggggcaatggc

[0307] ttgttgaaagcgccggacacgctgaatcatcaatttactgggaagatcctgaaacaggaattttgtgtcggtgccgtccggacaaaattatccctga

[0308] atttcactggatcatggacgtgaaaactacggcggatattcaacgattcaaaaccgcttattacgactaccgctatcacgttcaggatgcattctaca

[0309] gtgacggttatgaagcacagtttggagtgcagccaactttcgtttttctggttgccagcacaactattgaatgcggacgttatccggttgaaattttca

[0310] tgatgggcgaagaagcaaaactggcaggtcaacaggaatatcaccgcaatctgcgaaccctgtctgactgcctgaataccgatgaatggccag

[0311] ctattaagacattatcactgccccgctgggctaaggaatatgcaaatgactaagcaaccaccaatcgcaaaagccgatctgcaaaaaactcaggg

[0312] aaaccgtgcaccagcagcagttaaaaatagcgacgtgattagttttattaaccagccatcaatgaaagagcaactggcagcagctcttccacgcc

[0313] atatgacggctgaacgtatgatccgtatcgccaccacagaaattcgtaaagttccggcgttaggaaactgtgacactatgagttttgtcagtgcgat

[0314] cgtacagtgttcacagctcggacttgagccaggtagcgccctcggtcatgcatatttactgccttttggtaataaaaacgaaaagagcggtaaaaa

[0315] gaacgttcagctaatcattggctatcgcggcatgattgatctggctcgccgttctggtcaaatcgccagcctgtcagcccgtgttgtccgtgaaggt

[0316] gacgagtttagcttcgaatttggccttgatgaaaagttaatacaccgcccgggagaaaacgaagatgccccggttacccacgtctatgctgtcgca

[0317] agactgaaagacggaggtactcagtttgaagttatgacgcgcaaacagattgagctggtgcgcagcctgagtaaagctggtaataacgggccgt

[0318] gggtaactcactgggaagaaatggcaaagaaaacggctattcgtcgcctgttcaaatatttgcccgtatcaattgagatccagcgtgcagtatcaa

[0319] tggatgaaaaggaaccactgacaatcgatcctgcagattcctctgtattaaccggggaatacagtgtaatcgataattcagaggaataa

[0320] Plasmid sequence containing the RecE*T gene (SEQ ID NO: 13)

[0321]

[0322] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims. Furthermore, all documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference.

Claims

1. A genome editing system for Corynebacterium glutamicum, comprising: a nucleic acid protein complex capable of specifically cleaving DNA in Corynebacterium glutamicum, said nucleic acid protein complex comprising a nuclease and a guide RNA targeting a target sequence; The endonuclease contains the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence that has at least 80% sequence identity with SEQ ID NO:1; The guide RNA comprises: (1) the nucleic acid sequence shown in SEQ ID NO:2 or a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO:2, and (2) a recognition sequence that is complementary to or identical to the target sequence, the recognition sequence being 5-50 nt in length.

2. The genome editing system as described in claim 1, characterized in that, The genome editing system also includes: a recombination element that facilitates recombination of the sequence of interest into the Corynebacterium glutamicum cleavage site; Preferably, the recombinant element comprises the amino acid sequence shown in SEQ ID NO:10 and / or SEQ ID NO:11, or an amino acid sequence having at least 80% sequence identity with SEQ ID NO:10 and / or SEQ ID NO:11; More preferably, the recombinant element comprises the amino acid sequence shown in SEQ ID NO:8, or an amino acid sequence having at least 80% sequence identity with SEQ ID NO:

8.

3. The genome editing system as described in claim 1 or 2, characterized in that, The genome editing system also includes one or more elements selected from the following: promoter, replicon, marker gene, terminator, and donor fragment; Preferably, The promoter includes or is selected from: constitutive promoters and inducible promoters; more preferably, the constitutive promoter includes the PJ23119 promoter and the PlacM promoter; the inducible promoter includes Ptrc; The replicons include: Corynebacterium glutamicum replicons and Escherichia coli replicons; more preferably, the Corynebacterium glutamicum replicons include pGA1 and pBL1; the Escherichia coli replicons include pMB1 and repA101; The marker gene includes or is selected from: resistance gene, fluorescent gene; more preferably, the resistance gene includes kanamycin resistance gene, spectinomycin resistance gene, apramycin resistance gene, chloramphenicol resistance gene; The terminator includes: rrnB; The donor fragment contains the sequence of interest; more preferably, the sequence of interest further includes homologous arms at both ends for homologous recombination of the sequence of interest into the target sequence.

4. A polynucleotide comprising: a coding sequence for a nuclease and a sequence for guide RNA; The endonuclease comprises the amino acid sequence described in SEQ ID NO:1, or an amino acid sequence having at least 80% sequence identity with SEQ ID NO:

1. The guide RNA comprises the nucleic acid sequence shown in SEQ ID NO:2 or a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO:2; Preferably, the endonuclease, recognition sequence, or guide RNA is as described in claim 1; Preferably, the polynucleotide further comprises one or more elements selected from: a promoter, a replicon, a marker gene, a terminator, and a donor fragment; more preferably, the promoter, replicon, marker gene, terminator, and donor fragment are as described in claim 3.

5. The polynucleotide of claim 4, further comprising: a coding sequence for a recombinant element that facilitates recombination of the sequence of interest into the Corynebacterium glutamicum cleavage site; Preferably, the recombination element is as described in claim 2; More preferably, the coding sequence of the recombinant element comprises the nucleic acid sequence described in SEQ ID NO:12, or a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO:

12.

6. A nucleic acid construct comprising the polynucleotide as described in claim 4 or 5; Preferably, the nucleic acid construct contains the polynucleotide in one or more expression frames, and / or the nucleic acid construct contains the plurality of expression frames in one or more vectors.

7. The nucleic acid construct of claim 6, wherein the nucleic acid construct further comprises the coding sequence of the recombinant element of claim 5; Preferably, the nucleic acid construct comprises the coding sequences of the polynucleotide of claim 4 and the recombinant element of claim 5 in two expression frames, respectively, and / or the nucleic acid construct comprises the coding sequences of the polynucleotide of claim 4 and the recombinant element of claim 5 in two vectors, respectively; More preferably, the nucleic acid construct comprises a first vector and a second vector, wherein the first vector contains the coding sequence of the endonuclease and the sequence of the guide RNA, and the second vector contains the coding sequence of a recombination element that promotes recombination of the sequence of interest into the cleavage site of Corynebacterium glutamicum. More preferably, The first vector further includes one or more elements selected from: promoter, replicon, marker gene, donor fragment; more preferably, the first vector includes or is composed of the following elements: The coding sequence of the nuclease, the sequence of the guide RNA, the Corynebacterium glutamicum replicon pBL1, the Escherichia coli replicon repA101, the first marker gene, the PlacM promoter, the PJ23119 promoter, and the donor fragment; The second vector further includes one or more elements selected from the following: promoter, replicon, marker gene; more preferably, the second vector includes or is composed of the following elements: coding sequence of a recombinant element that promotes recombination of the sequence of interest into the Corynebacterium glutamicum cleavage site, Corynebacterium glutamicum replicon pGA1, Escherichia coli replicon pMB1, a second marker gene, LacI gene, and Ptrc promoter.

8. A kit for gene editing of Corynebacterium glutamicum, the kit comprising the genome editing system of any one of claims 1-3, or the polynucleotide of claim 4 or 5, or the nucleic acid construct of claim 6 or 7.

9. The use of the genome editing system of any one of claims 1-3, or the polynucleotide of claim 4 or 5, or the nucleic acid construct of claim 6 or 7, or the kit of claim 8 in gene editing of target sequences of Corynebacterium glutamicum; Preferably, the gene editing includes: Gene knockout, gene insertion.

10. A method for gene editing of a target sequence in Corynebacterium glutamicum, comprising: The genome editing system of any one of claims 1-3, or the polynucleotide of claim 4 or 5, or the nucleic acid construct of claim 6 or 7, or the kit of claim 8, is contacted with the target sequence of Corynebacterium glutamicum or delivered to cells containing the target sequence of Corynebacterium glutamicum. The target sequence is present in the target sequence of Corynebacterium glutamicum; Preferably, the gene editing includes gene knockout and gene insertion.

11. A method for recombining a sequence of interest into a target sequence, comprising the step of contacting the genome editing system of any one of claims 1-3, or the polynucleotide of claim 4 or 5, or the nucleic acid construct of claim 6 or 7 with the sequence of interest and the target sequence; Preferably, the two ends of the sequence of interest further include homologous arms for homologous recombination of the sequence of interest into the target sequence.

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