New retron editing system and its application in gene editing of corynebacterium glutamicum

Abstract

The present disclosure discloses a new Retron editing system and its application in Corynebacterium glutamicum gene editing, specifically discloses a new Retron editing system and demonstrates its application in Corynebacterium glutamicum gene editing, belonging to the technical field of biotechnology and genetic engineering. The new gene editing system provided by the present disclosure is obtained by modifying the existing Retron editing system, which improves the efficiency and capacity of gene editing. The gene editing system of the present disclosure is applied to the construction of a DNA fragment genomic in situ saturation mutation library, which has high construction efficiency, high mutation site coverage and comprehensive mutation types. This method has broad application prospects in the fields of multi-gene editing regulation, DNA regulatory sequence in situ library construction, protein coding gene in situ library construction, enzyme high-throughput screening and the like.

Description

Technical Field

[0001] This disclosure belongs to the fields of biotechnology and genetic engineering technology, and relates to a novel Retron editing system and its application in gene editing of Corynebacterium glutamicum, specifically to the novel Retron editing system and its application in editing DNA fragments at arbitrary locations in the entire Corynebacterium glutamicum genome. Background Technology

[0002] Corynebacterium glutamicum, a soil-derived Gram-positive bacterium, is considered a biosafety microorganism and a key microorganism in the industrial production of amino acids, used to produce over 6 million tons of amino acids annually. In recent years, it has developed into a crucial industrial production platform, widely used in the production of various products such as amino acids, bio-based material monomers, and recombinant proteins. These target compounds have broad applications in pharmaceuticals, food, animal feed, and cosmetics, possessing immense economic value. With the increasing market demand for amino acids, how to modify the core genomic elements of Corynebacterium glutamicum to enable its efficient synthesis of target compounds is a pressing issue that my country's biofermentation industry needs to address.

[0003] Currently, the modification of key enzymes and regulatory elements in the metabolic pathway of Corynebacterium glutamicum is mostly accomplished through traditional mutagenesis screening, random mutation, or saturation mutation at a small number of sites. These mutation methods are inefficient, and the target sites and mutation types are very limited, making it difficult to obtain highly effective and diverse mutants at the whole genome scale.

[0004] Therefore, developing efficient and precise genome editing technologies for Corynebacterium glutamicum with high mutation site coverage and diverse mutation types is an important technical means to achieve the modification of core components related to production and high-throughput screening. It is also a strong support for increasing the yield of target compounds and enhancing the competitiveness of the bio-fermentation industry. Summary of the Invention

[0005] The problem the invention aims to solve

[0006] In view of the problems existing in the prior art, such as in order to further improve the gene editing efficiency and capability in Corynebacterium glutamicum, this disclosure provides a gene editing method based on the Retron system, thereby improving the efficiency and capability of gene editing.

[0007] Solution for solving the problem

[0008] [1]. A novel Corynebacterium glutamicum gene editing system, the gene editing system comprising a plasmid expressing a Retron and a recombinase RecT, wherein the Retron is Retron-73, which comprises non-coding RNA (ncRNA) and reverse transcriptase (RT).

[0009] [2]. The gene editing system described in [1] uses a bicistronic structure to express ncRNA and RT.

[0010] [3]. Gene editing systems as described in [1] or [2], wherein,

[0011] The nucleotide sequence of the ncRNA includes the nucleotide sequence shown in SEQ ID NO:6, or a sequence that has more than 90% homology with the sequence shown in SEQ ID NO:6;

[0012] The amino acid sequence of the RT includes the amino acid sequence shown in SEQ ID NO:7, or a sequence that has more than 90% homology with the sequence shown in SEQ ID NO:7.

[0013] The amino acid sequence of the recombinase RecT includes that shown in SEQ ID NO:8, or a sequence that has more than 90% homology with the sequence shown in SEQ ID NO:8.

[0014] [4]. The gene editing system as described in [3], wherein the ncRNA sequence contains an editing template sequence; the editing template sequence includes a mutation sequence of the site to be edited, and upstream and downstream homologous arm sequences of the site to be edited; preferably, the total length of the upstream and downstream homologous arm sequences is 40bp to 150bp, preferably 50bp to 140bp, and more preferably 70bp to 130bp;

[0015] Optionally, the editing template sequence can be inserted at any position in the ncRNA sequence. Optionally, it can be inserted between 87bp and 88bp, 86bp and 87bp, 87bp and 88bp, 86bp and 87bp, 88bp and 89bp, 89bp and 90bp, or 90bp and 91bp in the ncRNA sequence.

[0016] [5]. The gene editing system as described in [4] is characterized in that the bicistronic structure comprises:

[0017] a) First cistron: Contains a promoter operatively linked to the polynucleotide encoding the RT;

[0018] b) Second cistron: Contains a promoter and a terminator operatively linked to the polynucleotide encoding the ncRNA;

[0019] Optionally, use P tac The promoter expression RT uses the P11F promoter and the terminator T. rrnB Express ncRNA;

[0020] Preferably, the promoter P tac The sequence is shown in SEQ ID NO:1;

[0021] Preferably, the sequence of the P11F promoter is shown in SEQ ID NO:9;

[0022] Preferably, the terminator T rrnB As shown in SEQ ID NO:10.

[0023] [6]. The gene editing system as described in [5], wherein the plasmid expressing Retron further contains a resistance gene that can increase the stability of the plasmid;

[0024] Preferably, the resistance gene is a zithromycin resistance gene;

[0025] Preferably, the nucleotide sequence of the zebumycin resistance gene is shown in SEQ ID NO:14.

[0026] [7]. The gene editing system as described in any one of [1] to [6], wherein the plasmid expressing recombinase RecT contains a paraphrased per gene, the per gene being shown in SEQ ID NO:12;

[0027] Optionally, the sequence of the RecT plasmid expressing the recombinase is shown in SEQ ID NO:13.

[0028] [8]. The gene editing system as described in any one of [1] to [7], wherein the plasmid sequence expressing Retron is shown in SEQ ID NO:11 or 15.

[0029] [9]. Any one of (A) to (C):

[0030] (A) A polynucleotide encoding a gene editing system as described in any of [1] to [8];

[0031] (B) A carrier containing the polynucleotide described in (A);

[0032] (C) Recombinant cells containing (A) or (B);

[0033] Optionally, the recombinant cells include prokaryotic cells; alternatively, the recombinant cells include Escherichia coli and Corynebacterium glutamicum, preferably Corynebacterium glutamicum.

[0034]

[10] . A method for editing a target nucleic acid, wherein the method comprises: causing the gene editing system described in any one of [1] to [8] to bind to a target nucleotide and edit the target nucleic acid; optionally, the target nucleic acid is a single target nucleic acid or multiple target nucleic acids.

[0035]

[11] . The method according to

[10] , wherein the method includes the steps of introducing the gene editing system of any one of [1] to [8] into a host cell, constructing a recombinant cell, and then culturing the recombinant cell in a culture system;

[0036] Optionally, the culture system includes TSB, LBG, BHI, or CGIII medium;

[0037] Preferably, the culture system contains TSB, LBG, or BHI medium;

[0038] More preferably, the LBG culture medium consists of: glucose, 5 g / L; yeast extract, 5 g / L; tryptone, 10 g / L; and NaCl, 10 g / L.

[0039] More preferably, the BHI culture medium consists of: brain and heart extract, 37.1 g / L;

[0040] More preferably, the CGIII culture medium consists of: glucose, 5 g / L; yeast extract, 10 g / L; tryptone, 10 g / L; and NaCl, 25 g / L.

[0041]

[12] . Application of the gene editing system as described in any of [1] to [8], the polynucleotide as described in [9], the vector or the recombinant cell as described in the gene editing system at any position on the genome;

[0042] Optionally, the nucleotide sequence includes any gene coding and non-coding sequence;

[0043] Optionally, the non-coding sequence includes promoters, DNA elements such as RBS, and ncRNA;

[0044] Optionally, the gene coding sequence includes a gene encoding green fluorescent protein.

[0045]

[13] . A method for constructing a genome mutant library, the method comprising the steps of converting the gene editing system described in any one of [1] to [8] into a host cell and editing nucleotides at any position on the genome of the host cell;

[0046] Optionally, the nucleotide sequence includes any gene coding and non-coding sequence;

[0047] Optionally, the non-coding sequence includes DNA elements and ncRNA, and may further be a promoter and / or RBS;

[0048] Optionally, the gene coding sequence includes a gene encoding green fluorescent protein.

[0049] The effects of the invention

[0050] The novel gene editing system disclosed herein improves gene editing efficiency and enables highly efficient editing of single and multiple genes by modifying existing editing systems. This gene editing system allows for the construction of in-situ saturated mutant libraries of the genome with high efficiency, high coverage, and diverse mutation types. Attached Figure Description

[0051] Figure 1 The process of constructing library editing plasmids is shown;

[0052] Figure 2 The diversity distribution of the GFP gene editing plasmid mutant library is shown;

[0053] Figure 3 The diversity distribution of the *Corynebacterium glutamicum* genome mutation library for the *gfp* gene is shown;

[0054] Figure 4 The fluorescence intensity distribution of cells before and after gfp gene editing is shown. Detailed Implementation

[0055] Various exemplary embodiments, features, and aspects of this disclosure will be described in detail below. The term "exemplary" as used herein means "serving as an example, embodiment, or illustration." Any embodiment illustrated herein as "exemplary" is not necessarily to be construed as superior to or better than other embodiments.

[0056] Furthermore, to better illustrate this disclosure, numerous specific details are set forth in the following detailed description. Those skilled in the art will understand that this disclosure can be practiced without certain specific details. In other instances, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the main points of this disclosure.

[0057] Unless otherwise stated, all units used in this specification are international standard units, and all numerical values ​​and ranges appearing in this disclosure should be understood to include systematic errors that are unavoidable in industrial production.

[0058] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0059] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0060] In this specification, "optional" and "optionally" mean that the events or circumstances described below may or may not occur, and the description includes both cases where the events or circumstances occur and cases where the events or circumstances do not occur.

[0061] While the disclosure supports the definition of the term "or" as merely a substitute and "and / or", the term "or" in the claims means "and / or" unless expressly stated as merely a substitute or as mutually exclusive. In this specification, the term "and / or", when used to connect two or more options, should be understood to mean any one or any two or more of the options.

[0062] When used in claims or description, the optional / preferred "numerical range" includes both the numerical endpoints at both ends of the range and all natural numbers covered in the middle of the numerical endpoints relative to the aforementioned numerical endpoints.

[0063] The terms “preferred” and “ideal” as used herein are not intended to limit the scope of the claimed disclosure or to imply that certain features are critical, necessary, or even important to the structure or function of the claimed disclosure. Rather, these terms are merely used to emphasize alternative or additional features that may or may not be used in particular embodiments of the disclosure.

[0064] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0065] As used herein, the term "bicistronic expression system" comprises a polynucleotide sequence encoding a polypeptide to be expressed and sequences controlling its expression, such as a promoter and optional enhancer sequences. The promoters of this disclosure are operatively linked to the gene to be expressed (i.e., a transcription unit) or separated from it by insertion into DNA (e.g., through the 5' untranslated region of a heterologous gene). Preferably, the flanking parts of the expression system are one or more suitable restriction sites to enable the insertion of the expression cassette into a vector and / or its removal from the vector. Therefore, the expression systems according to this disclosure can be used to construct expression vectors, particularly bacterial expression vectors.

[0066] As used in this article, the term "promoter" refers to a DNA regulatory region typically located upstream of a gene that provides a control point for regulating gene transcription.

[0067] As used in this article, the term "terminator" is a DNA sequence whose main function is to provide a transcription termination signal to RNA polymerase, thereby ending the transcription process of RNA.

[0068] As used in this article, the term “operably linked” refers to a functional relationship between two or more DNA segments, particularly a gene sequence to be expressed and those sequences that control its expression.

[0069] As used herein, the term "host cell" means any cell type readily adaptable to a gene editing system comprising the gene editing system of this disclosure, or a polynucleotide, nucleic acid construct, or recombinant expression vector encoding the gene editing system. The term "recombinant cell" encompasses a host cell that differs from the parent cell after the introduction of a polynucleotide, nucleic acid construct, or recombinant expression vector, specifically achieved through transformation.

[0070] As used herein, the term "transformation" has the meaning commonly understood by those skilled in the art as the process of introducing exogenous DNA into a host. Methods of transformation include any method of introducing nucleic acids into cells, including but not limited to electroporation, calcium phosphate precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method.

[0071] As used herein, the term "library" is used in its known meaning in the fields of cell biology and molecular biology, referring to a collection of different nucleic acid fragments / molecules. One particular type of library is a library containing random mutants generated through random mutagenesis. Another example is a designed (or synthetic) library (e.g., through the introduction of the gene editing system disclosed herein) containing specially engineered different nucleic acid fragments / molecules.

[0072] The technical solution of this disclosure is described in detail below:

[0073] <Gene Editing System>

[0074] The Retron system is a bacterial genetic element composed of a polynucleotide that simultaneously encodes non-coding RNA (ncRNA) and reverse transcriptase (RT). The RT uses the ncRNA as a template to reverse transcribe and produce multiple copies of single-stranded DNA (msDNA) covalently linked to the ncRNA. These msDNAs contain ssDNA templates for single-stranded recombination, i.e., sequences targeting the target nucleic acid, which can serve as templates for single-stranded recombination. Gene editing can then be achieved using the phage recombinase RecT. This disclosure screened four Retron systems from *E. coli*—Retron-Ec86, Retron-Ec73, Retron-Ec107, and Retron-Ec484—and selected Retron-Ec73, which exhibited high editing efficiency. Further modifications were then made to further improve the editing efficiency.

[0075] Therefore, in this disclosure, the gene editing system includes a plasmid expressing Retron and recombinase RecT, wherein the Retron includes an ncRNA sequence and an RT sequence, the ncRNA sequence having an editing template sequence inserted therein, and the ncRNA and RT are expressed using a bicistronic structure.

[0076] In some alternative implementations, the edit template sequence can be inserted at any position in the ncRNA sequence. For example, it can be inserted between 87bp and 88bp, 86bp and 87bp, 87bp and 88bp, 86bp and 87bp, 88bp and 89bp, 89bp and 90bp, and 90bp and 91bp of the ncRNA sequence shown in SEQ ID NO:6.

[0077] In some exemplary embodiments, the coding sequence of the reverse transcriptase is preceded by a ribosome binding site (RBS) sequence that initiates translation.

[0078] In some exemplary embodiments, the recombinase RecT is derived from *Escherichia coli*, with the protein sequence having the NCBI accession number NP_415865 and the specific gene sequence having the NCBI GeneID 945917. Further, the recombinase RecT is expressed using the pRecT plasmid; the sequence of the pRecT plasmid is disclosed in patent CN112111469B, which is incorporated herein by reference.

[0079] In some implementations, the promoter P is utilized. tac Initiate the expression of the non-coding RNA and the reverse transcriptase; optionally, the promoter P tac Contains the sequence shown in SEQ ID NO:1:

[0080] TTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGCTCACAATTTCACACAGGAAACAGAATT.

[0081] In some implementations, the bicistronic structure is used to express non-coding RNA and reverse transcriptase.

[0082] The bicistronic expression structure includes:

[0083] a) First cistron: Contains a promoter P operatively linked to the polynucleotide encoding the reverse transcriptase. tac ;

[0084] b) Second cistron: Contains a promoter P11F operatively linked to a polynucleotide encoding the non-coding RNA.

[0085] In some exemplary embodiments, the first cistron and the second cistron each contain polynucleotides encoding different target fragments, enabling the bicistron expression system to express the reverse transcriptase and non-coding RNA.

[0086] In some alternative embodiments, the second cis-transformer further includes a terminator T. rrnB .

[0087] In other embodiments, the first cistron and the second cistron are located in the same carrier.

[0088] In some specific implementations, the promoter P11F comprises the sequence shown in SEQ ID NO:9, and the terminator T rrnB Contains the sequence shown in SEQ ID NO:10:

[0089] P11F(SEQ ID NO:9):

[0090] TTTTTCCCACATAAGCTGGCAATGTTGCGACGCAACAGGTACAGTGTAATTCA

[0091] TrrnB(SEQ ID NO:10):

[0092] CCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTTTGTTTATTTTTCTAAATACATTCAA. In some embodiments, the pRecT plasmid contains a recompensated per gene, the per gene sequence of which is shown in SEQ ID NO:12:

[0093]

[0094] As shown. Specifically, the pRecT-1 plasmid containing the complemented expression of the per gene is shown in SEQ ID NO:13.

[0095] SEQ ID NO:13: pRecT-1: (The bold part is the recT gene sequence, and the italic part is the per gene sequence)

[0096]

[0097]

[0098] In some implementations, the resistance gene of the gene editing system is not a chloramphenicol resistance gene, but a resistance gene that is more conducive to plasmid stability so that the plasmid is less likely to be lost during passage; for example, the resistance gene includes a zizomycin resistance gene.

[0099] In some implementations, the target nucleic acid sequence comprises a mutated sequence at the site to be edited, and upstream and downstream homologous arms of the site to be edited. Specifically, the target nucleic acid sequence includes a codon sequence of the target mutation, which may be one or more consecutive sequences corresponding to the site to be edited in the target gene. Specifically, the total length of the upstream and downstream homologous arm sequences of the site to be edited is 40bp to 150bp, preferably 50bp to 140bp, more preferably 70bp to 130bp, and even more preferably 67bp to 127bp, for example, 67bp, 70bp, 73bp, 76bp, 79bp, 82bp, 85bp, 88bp, 91bp, 94bp, 97bp, 100bp, 103bp, 106bp, 109bp, 112bp, 115bp, 118bp, 121bp, 124bp, 127bp, and 130bp.

[0100] <Polynucleotides, vectors, recombinant cells>

[0101] This disclosure provides isolated polynucleotides comprising the gene editing system described above. In some embodiments, the polynucleotides may be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or synthetically produced DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. Polynucleotides encoding mutants of this disclosure include: a coding sequence encoding only the mutant; a coding sequence of the mutant and various additional coding sequences; a coding sequence of the mutant (and optional additional coding sequences) and a non-coding sequence.

[0102] This disclosure provides vectors comprising the isolated polynucleotides described above. In some embodiments, the vectors include expression vectors that can be expressed in prokaryotes.

[0103] This disclosure provides recombinant cells comprising the gene editing system, polynucleotide, or vector described above. In some alternative embodiments, the recombinant cells comprise prokaryotic cells; exemplaryly, the recombinant cells comprise *Escherichia coli* or *Corynebacterium glutamicum*; further, the recombinant cells are *Corynebacterium glutamicum* strain ATCC13032, *Corynebacterium glutamicum* strain ATCC13869, *Corynebacterium glutamicum* strain ATCC 14067, or derivatives of any of the above.

[0104] Methods for editing target nucleotides

[0105] This disclosure also provides a method for editing a target nucleotide, the method comprising: causing the gene editing system described above to bind to the target nucleotide and edit the target nucleotide.

[0106] In some implementations, the target nucleic acid is located within a cell. For example, the gene editing system, polynucleotide, or vector described above is introduced into a cell (i.e., the cell to be edited) to construct the recombinant cell described above. The recombinant cell is then cultured in a culture system to achieve the editing of the target nucleic acid.

[0107] In some specific embodiments, the culture system includes TSB, LBG, BHI, or CGIII media. Further, the TSB medium contains 2–6 g / L glucose, 2–6 g / L yeast extract, 5–10 g / L soybean peptone, 2–5 g / L urea, 0.2–0.7 g / L succinic acid, 0.5–1.5 g / L K₂HPO₄·3H₂O, 0.05–0.5 g / L MgSO₄·7H₂O, 0.005–0.05 mg / L biotin, 0.05–0.5 mg / L vitamin B1, and 15–30 g / L MOPS. The LBG medium contains 2–6 g / L glucose, 2–6 g / L yeast extract, 5–15 g / L tryptone, and 5–15 g / L NaCl. The BHI medium contains 35–40 g / L brain and heart extract. The CGIII culture medium contains 2–6 g / L glucose, 5–15 g / L yeast extract, 5–15 g / L tryptone, and 20–30 g / L NaCl.

[0108] In some preferred embodiments, the LBG medium consists of: glucose, 5 g / L; yeast extract, 5 g / L; tryptone, 10 g / L; NaCl, 10 g / L; the BHI medium consists of: brain heart extract, 37.1 g / L; the CGIII medium consists of: glucose, 5 g / L; yeast extract, 10 g / L; tryptone, 10 g / L; NaCl, 25 g / L; and the TSB plate medium consists of: glucose, 5 g / L; yeast extract, 5 g / L; soybean peptone, 9 g / L; urea, 3 g / L; succinic acid, 0.5 g / L; K2HPO4·3H2O, 1 g / L; MgSO4·7H2O, 0.1 g / L; biotin, 0.01 mg / L; vitamin B1, 0.1 mg / L; MOPS, 20 g / L; and agar powder, 15 g / L.

[0109] Preferably, the culture system contains TSB, LBG, or BHI medium to ensure that editing efficiency is maintained at a high level.

[0110] <Composition, Reagent or Kit>

[0111] This disclosure also provides a composition comprising at least one of the gene editing system, polynucleotide, vector, and recombinant cells described above.

[0112] This disclosure provides a reagent or kit comprising at least one of the gene editing systems, polynucleotides, vectors, recombinant cells, and compositions described above.

[0113] In some embodiments, the composition, reagent, or kit described herein is a composition, reagent, or kit that can be used for target nucleic acid modification or editing.

[0114] Applications of gene editing systems

[0115] This disclosure provides the application of the gene editing systems, polynucleotides, vectors, recombinant cells, compositions, reagents, or kits described above in nucleotide editing at any location on the genome. For example, they can be used for the construction of mutant strains, the construction of gene mutant libraries, etc.

[0116] <Methods for constructing mutant libraries>

[0117] In some embodiments, the construction method includes the steps of designing the gene editing system described above based on the target gene and transforming it into host cells, and culturing the host cells. Optionally, it also includes the step of editing nucleotides at any position on the genome of the host cells.

[0118] In some optional implementations, there is a step of extracting the edited genome from the host cell.

[0119] In some implementations, the host cell includes prokaryotic cells, such as Escherichia coli, Corynebacterium glutamicum, etc.

[0120] In some embodiments, the nucleotide sequence includes any gene coding and non-coding sequence; the non-coding sequence includes DNA elements and ncRNA, and may further be a promoter, and / or DNA elements and ncRNA such as RBS.

[0121] In some optional embodiments, the gene includes any gene that can be expressed in prokaryotic cells, such as genes that can be expressed in *Escherichia coli*, *Corynebacterium glutamicum*, and especially genes that can be expressed in *Corynebacterium glutamicum*. Exemplarily, the gene includes a gene encoding green fluorescent protein.

[0122] Example

[0123] Other objects, features, and advantages of this disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific embodiments (although illustrating specific implementations of this disclosure) are given for illustrative purposes only, as various changes and modifications that can be made within the spirit and scope of this disclosure will become apparent to those skilled in the art upon reading this detailed description.

[0124] Unless otherwise specified, the experimental techniques and methods used in this embodiment are conventional techniques and methods. For example, experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in the embodiments can be obtained through legitimate commercial channels.

[0125] Example 1. Editing test of different types of Retron systems in Corynebacterium glutamicum.

[0126] This disclosure selects to perform editing tests on the rpsL gene of Corynebacterium glutamicum ribosomal protein S12. A mutation at amino acid position 43 of the protein encoded by this gene can confer streptomycin resistance in Corynebacterium glutamicum, namely rpsL. K43R (The codon is mutated from AAG to CGC), and this mutation site is used as the target for editing tests. The Retron system consists of ncRNA and reverse transcriptase (RT). When a 70bp fragment containing the mutation site and upstream and downstream homologous arms is inserted into the coding sequence of the ncRNA, transcription and translation by RT occur. RT specifically recognizes the conserved region of the ncRNA secondary structure, and finally the 70bp is reverse transcribed into a large amount of ssDNA. This disclosure selects four Retron systems from Escherichia coli: Retron-Ec86, Retron-Ec73, Retron-Ec107, and Retron-Ec48. The editing function is further achieved by intracellularly generated 70nt ssDNA mediated by the recombinase RecT (this disclosure selects the pRecT plasmid disclosed in patent CN112111469B to perform this function, and the pRecT plasmid is incorporated herein by reference).

[0127] This embodiment first constructs the Corynebacterium glutamicum Retron editing vector. Sequences of four Retron systems were artificially synthesized by a sequencing company. Based on the pXMJ19 plasmid backbone, P... tacThe promoter (SEQ ID NO:1: TTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGCTCACAATTTCACACAGGAA ACAGAATT) simultaneously initiates the expression of ncRNA and RT. The specific construction is as follows: Taking Ec48 as an example, using the synthesized Ec48 gene sequence as a template, two retrotron fragments were amplified using primers Ec48-1 / 2 and Ec48-3 / 4, respectively. A 70bp segment with a mutation site was then used for rpsL editing.

[0128] ( The fragment containing three mutated bases and the 33 bp before and 34 bp after the mutation site was ligated into ncRNA using primers. Using the pXMJ19 plasmid (disclosed in Jakoby, M; Ngouoto-Nkili, CE; Burkovski, A. Construction and application of new Corynebacterium glutamicum vectors, incorporated herein by reference) as a template, the pXMJ19 plasmid backbone was amplified using primers pXMJ-1 and pXMJ-2, respectively. The two retrotron fragments of Ec48 obtained were cloned and ligated to the plasmid backbone using a one-step recombination kit from Novizan to obtain pRetron-48.

[0129] Similarly, the pRetron-86, pRetron-107, and pRetron-73 editing vectors were obtained using the above method. The primer sequences used are shown in Table 1.

[0130] The final Retron phylogenetic sequence is as follows (italicized ncRNA sequence; bold italicized rpsL edited 70bp sequence; underlined RBS sequence; normal font RT sequence):

[0131] Retron-Ec48: SEQ ID NO:2

[0132]

[0133]

[0134] Retron-Ec86: SEQ ID NO:3

[0135]

[0136] Retron-Ec107: SEQ ID NO:4

[0137]

[0138] Retron-Ec73: SEQ ID NO:5

[0139]

[0140] Table 1

[0141]

[0142]

[0143] In this embodiment, the four successfully constructed Retron editing vectors and pRecT were co-transformed into Corynebacterium glutamicum ATCC 13032. The transformed strains were plated on TSB solid plates containing 25 μg / mL kanamycin and 5 μg / mL chloramphenicol, and transformant strains containing two plasmids were obtained respectively (one plasmid was pRecT, and the other plasmid was Retron-48 editing vector / Retron-86 editing vector / Retron-107 editing vector / Retron-73 editing vector). The TSB plate medium consists of: glucose, 5 g / L; yeast extract, 5 g / L; soybean peptone, 9 g / L; urea, 3 g / L; succinic acid, 0.5 g / L; K2HPO4·3H2O, 1 g / L; MgSO4·7H2O, 0.1 g / L; biotin, 0.01 mg / L; vitamin B1, 0.1 mg / L; MOPS, 20 g / L; and agar powder, 15 g / L.

[0144] Transformants containing two plasmids were transferred into 24-well plates for induction culture. The plates were incubated at 30°C using 1 ml of TSB medium (containing 25 μg / mL kanamycin, 5 μg / mL chloramphenicol, and 0.5 mM IPTG). When the OD... 600 When the culture reaches level 5, it is transferred again to a 24-well plate (10 μL). 1 ml of TSB medium is added, and 25 μg / mL kanamycin, 5 μg / mL chloramphenicol, and 0.5 mM IPTG are added for induction. This step is repeated three times to obtain the bacterial culture for detection. Further, using rpsl-1 / 2 primers (Table 1) and the third passage bacterial culture as a template, PCR amplification of the fragment containing the mutation site is performed. Next-generation sequencing is performed on the amplified fragment, and the editing efficiency is obtained by calculating the percentage of GCG mutant reads in the total number of reads. In this example, the editing efficiencies were obtained by performing next-generation sequencing on the above four Retron systems (Table 2), with Retron-Ec73 showing the highest editing efficiency at 3.6383 ± 0.95%.

[0145] Table 2

[0146] Types of editing systems Retron-Ec86 Retron-Ec73 Retron-Ec107 Retron-Ec48 Editing efficiency 0.0013±0.00％ 3.6383±0.95％ 0.0329±0.00％ 0.0820±0.01％

[0147] In Retron-Ec73, the nucleotide sequence of the ncRNA is shown in SEQ ID NO:6:

[0148] SEQ ID NO:6:

[0149]

[0150] An edit template is inserted between the underlined sequence CCGTCGTT and the bolded sequence ATCGAC (i.e., between 87bp and 88bp of SEQ ID NO:6).

[0151] The amino acid sequence of RT (reverse transcriptase) is shown in SEQ ID NO:7:

[0152] SEQ ID NO:7:

[0153] MRIYSLIDSQTLMTKGFASEVMRSPEPPKKWDIAKKKGGMRTIYHPSSKVKLIQYWLMNNVFSKLP

[0154] MHNAAYAFVKNRSIKSNALLHAESKNKYYVKIDLKDFFPSIKFTDFEYAFTRYRDRIEFTTEYDKELL

[0155] QLIKTICFISDSTLPIGFPTSPLIANFVARELDEKLTQKLNAIDKLNATYTRYADDIIVSTNMKGASKLIL

[0156] DCFKRTMKEIGPDFKINIKKFKICSASGGSIVVTGLKVCHDFHITLHRSMKDKIRLHLSLLSKGILKDE

[0157] DHNKLSGYIAYAKDIDPHFYTKLNRKYFQEIKWIQNLHNKVE

[0158] The amino acid sequence of the RecT is shown in SEQ ID NO:8:

[0159] SEQ ID NO:8:

[0160] MTKQPPIAKADLQKTQGNRAPAAVKNSDVISFINQPSMKEQLAAALPRHMTAERMIRIATTEIRKVP

[0161] ALGNCDTMSFVSAIVQCSQLGLEPGSALGHAYLLPFGNKNEKSGKKNVQLIIGYRGMIDLARRSGQI

[0162] ASLSARVVREGDEFSFEFGLDEKLIHRPGENEDAPVTHVYAVARLKDGGTQFEVMTRKQIELVRSLS

[0163] KAGNNGPWVTHWEEMAKKTAIRRLFKYLPVSIEIQRAVSMDEKEPLTIDPADSSVLTGEYSVIDNSEE Example 2. Optimizing the Retron-Ec73 system to improve editing efficiency

[0164] (1) Optimization of pRetron-73 plasmid: Bicistronic structure for expression of RT and ncRNA

[0165] This disclosure expresses the two parts of Retron-73 separately through a bicistronic structure. In Example 1, P tac Based on the expression RT, the promoter P11F (SEQ ID NO:9: TTTTCTCCACATAAGCTGGCAATGTTGCGACGCAACA GGTACAGTG TAATTCA) and the terminator T are used. rrnB (Shown SEQ ID NO:10) ncRNA transcription and expression were performed. Here, the pCas9gRNA-proB2 plasmid (disclosed in patent CN112111469B and incorporated herein by reference) was used as a template to amplify two portions of the plasmid backbone using primers pgj-1 / pgj-2 and pgj-3 / pgj-4, respectively; and the pRetron-Ec73 plasmid from Example 1 was used as a template, with ncRNA-1 / 2 and RT-1 / 2 as primers to amplify ncRNA (containing rpsL) respectively. K43R The above four fragments were cloned and ligated using the Novizan one-step recombination kit, and the correct plasmid obtained was pRetronEc73-1.

[0166] pRetronEc73-1 plasmid sequence: SEQ ID NO:11: (The lowercase sequence is a two-part plasmid backbone; the uppercase and bolded part is the RT gene; the uppercase, bolded and underlined part is the RBS; the uppercase and italicized part is the ncRNA)

[0167]

[0168]

[0169] In this embodiment, the successfully constructed editing vector pRetronEc73-1 and the pRecT plasmid used in Example 1 were co-transformed into Corynebacterium glutamicum ATCC 13032. The resulting culture was spread onto TSB agar plates supplemented with 25 μg / mL kanamycin and 5 μg / mL chloramphenicol. After culturing, a single clone containing the Retron Ec73-1 editing system was obtained. The single clone was then transferred into 24-well plates for induction culture, and the culture conditions and mutation efficiency detection methods were the same as in Example 1. The results are shown in Table 4. When bicistronic expression of RT and ncRNA was performed, the editing efficiency increased by 1.8-fold, to 10.37 ± 2.94%.

[0170] (2) Adding the per gene improves the stability of the pRecT plasmid.

[0171] Literature reports that removing the per gene from the pRecT plasmid is beneficial for post-editing plasmid loss. [3] To increase the stability of the plasmid, this disclosure adds the per gene to the pRecT plasmid. Using the pRecT plasmid used in Example 1 as a template, the plasmid backbone fragment was amplified using primers RecT-1 / 2. Using the pEC-XK99E plasmid as a template, the per gene fragment (gene sequence as shown in SEQ ID NO:12) was amplified using per-1 / 2 primers.

[0172] The two fragments are shown in the image. The two fragments were cloned and ligated using a one-step recombination kit from Novizan, and the resulting plasmid was named pRecT-1 (SEQ ID NO: 13).

[0173] The successfully constructed editing vector pRecT-1 and the pRetronEc73-1 constructed in Example 2 were co-transformed into Corynebacterium glutamicum ATCC 13032. The transformed samples were plated on TSB agar plates containing 25 μg / mL kanamycin and 5 μg / mL chloramphenicol, and monoclonal strains containing the RetronEc73-2 editing system were obtained after culturing. These monoclonal strains were then transferred to 24-well plates for induction culture, and the culture conditions and mutation efficiency detection methods were the same as in Example 1. The results are shown in Table 4. After the per gene was reintroduced, the editing efficiency was further improved to 18.22 ± 0.43%.

[0174] Table 3

[0175] Primers nucleotide sequence pgj-1 AGCTTGGCTGTTTTGGCGG pgj-2 TGAATTACACTGTACCTGTTG pgj-3 GTTTTAGAGCTAGAAATAGC pgj-4 GTGTGAAATTGTGAGCGCTCAC ncRNA-1 CAACAGGTACAGTGTAATTCACAGAGCCAAACCTAGCATT ncRNA-2 TGCTATTTCTAGCTCTAAAACAGAGCCAAACCTACTTGAG RT-1 GAGCGCTCACAATTTCACACAGAAAGGAAGTACATGCGCA RT-2 TCCGCCAAAACAGCCAAGCTTTATTATTCAACCTTGTTGT RecT-1 CGCTGATGCTTCAGGCCAGT RecT-2 TGGAGGATCGCATCAGCTGC per-1 GCAGCTGATGCGATCCTCCACTACTGCGCGTCCCTCCTGGC per-2 ACTGGCCTGAAGCATCAGCGGTGGATCTCTCGATGTCGC pRetron-1 TTTTTTTAAGGCAGTTATTGGTGCCCTTCG pRetron-2 GCCAGGGTGGTTTTTCTTTTCACCAGTGAG spe-1 CAATAACTGCCTTAAAAAAATTATTTGCCGACTACCTTGGTG spe-2 AAAAGAAAAACCACCCTGGCCGAGCCGTTCCATACAGAAGC

[0176] (3) Replace the chloramphenicol resistance gene in the pRetronEc73-1 plasmid with the zithromycin resistance gene.

[0177] When using chloramphenicol resistance genes and corresponding working concentrations of antibiotics, plasmids are easily lost during multiple passages. To address this issue, this disclosure modifies the resistance gene in the pRetronEc73-1 plasmid to a zizomycin resistance gene, thereby improving plasmid stability. Using the pRetronEc73-1 plasmid constructed in Example 2 as a template, the plasmid backbone fragment was amplified using primers pRetron-1 / 2. Furthermore, using a synthetically produced zizomycin gene as a template, the zizomycin resistance gene fragment was amplified using primers spe-1 / 2. (The zizomycin resistance gene is shown in SEQ ID NO:14.)

[0178] TTATTTGCCGACTACCTTGGTGATCTCGCCTTTCACGTAGTGGACAAATTCTTCCAACTGATCTGC

[0179] GCGCGAGGCCAAGCGATCTTCTTCTTGTCCAAGATAAGCCTGTCTAGCTTCAAGTATGACGGGCT

[0180] GATACTGGGCCGGCAGGCGCTCCATTGCCCAGTAGGCAGCGACATCCTTCGGCGCGATTTTGCCG

[0181] GTTACTGCGCTGTACCAAATGCGGGACAACGTAAGCACTACATTTCGCTCATCGCCAGCCCAGTC

[0182] GGGCGGCGAGTTCCATAGCGTTAAGGTTTCATTTAGCGCCTCAAATAGATCCTGTTCAGGAACCG

[0183] GATCAAAGAGTTCCTCCGCCGCTGGACCTACCAAGGCAACGCTATGTTCTCTTGCTTTTGTCAGC

[0184] AAGATAGCCAGATCAATGTCGATCGTGGCTGGCTCGAAGATACCTGCAAGAATGTCATTGCGCTG

[0185] CCATTCTCCAAATTGCAGTTCGCGCTTAGCTGGATAACGCCACGGAATGATGTCGTCGTGCACAA

[0186] CAATGGTGACTTCTACAGCGCGGAGAATCTCGCTCTCCAGGGGAAGCCGAAGTTTCCAAAAG

[0187] GTCGTTGATCAAAGCTCGCCGCGTTGTTTCATCAAGCCTTACGGTCACCGTAACCAGCAAATCAA

[0188] TATCACTGTGTGGCTTCAGGCCGCCATCCACTGCGGAGCCGTACAAATGTACGGCCAGCAACGT

[0189] CGGTTCGAGATGGCGCTCGATGACGCCAACTACCTCTGATAGTTGAGTCGATACTTCGGCGATCACCGCTTCCCTCAT).

[0190] The two fragments were cloned and ligated using the Novozymes one-step recombination kit, and the resulting plasmid was named pRetronEc73-2. The pRetronEc73-2 plasmid sequence is SEQ ID NO:15: (Bold text indicates the RT sequence, underline indicates ncRNA, underlined and bold text indicates the 70bp edited fragment, and italic text indicates the zithromycin gene sequence):

[0191]

[0192]

[0193] The successfully constructed editing vector pRetronEc73-2 and the pRecT-1 constructed in Example 2 were co-transformed into Corynebacterium glutamicum ATCC 13032. The transformed strains were plated on TSB agar plates containing 25 μg / mL kanamycin and 100 μg / mL zimuth subtilis to obtain a single clone containing the RetronEc73-3 editing system. The culture conditions and mutation efficiency detection methods were the same as in Example 1. The results are shown in Table 4. Replacing the resistance with zimuth subtilis increased the editing efficiency to 33.00 ± 0.46%.

[0194] Table 4

[0195] Editing system Retron-Ec73 RetronEc73-1 RetronEc73-2 RetronEc73-3 Editing efficiency 3.63±0.95％ 10.37±2.94％ 18.22±0.43％ 33.00±0.46％

[0196] Example 3. Optimizing the culture medium to improve editing efficiency

[0197] To test the effect of different culture media on editing efficiency, this disclosure selected four culture media: TSB, LBG, BHI, and CGIII for subculturing the editing strain. The LBG medium consisted of: glucose, 5 g / L; yeast extract, 5 g / L; tryptone, 10 g / L; and NaCl, 10 g / L. The BHI medium consisted of: brain and heart extract, 37.1 g / L. The CGIII medium consisted of: glucose, 5 g / L; yeast extract, 10 g / L; tryptone, 10 g / L; and NaCl, 25 g / L.

[0198] All four culture media were supplemented with kanamycin to a final concentration of 25 μg / mL, zithromycin to a final concentration of 100 μg / mL, and IPTG to a final concentration of 0.5 mM. Subsequently, strain RetronEc73-3 was transferred to 24-well plates for induction culture. The culture conditions and mutation efficiency detection methods were the same as in Example 1. The results are shown in Table 5. Among the four culture media, the Retron editing system cultured in LBG medium exhibited the highest editing efficiency, at 42.64 ± 0.25%.

[0199] Table 5

[0200] Types of culture media TSB LBG BHI CGIII Editing efficiency 33.00±0.46％ 42.64±0.25％ 36.70±1.21％ 22.86±0.34％

[0201] Example 4. Optimizing the length of the editing template to improve editing efficiency

[0202] To further improve editing efficiency, this example tested the effect of the length of the editing template carried in the ncRNA. Using plasmid pRetronEc73-2 from Example 3 as a template, short backbone fragments were amplified using primers GK-F / 50-1, GK-F / 90-1, GK-F / 110-1, and GK-F / 130-1, respectively; and long backbone fragments were amplified using primers 50-2 / GK-R, 90-2 / GK-R, 110-2 / GK-R, and 130-2 / GK-R, respectively. The ncRNA carrying the rpsL... K43R (The underlined sequence represents the mutation codon) 50bp 90bp 110bp and 130bp The edited fragments were introduced into the plasmids using primers. The corresponding long and short backbone fragments were then cloned and ligated using Novizan's one-step recombination kit, resulting in the correct plasmids pRetronEc73-3, pRetronEc73-4, pRetronEc73-5, and pRetronEc73-6.

[0203] The successfully constructed editing vectors pRetronEc73-3 / 4 / 5 / 6 and pRecT-1 constructed in Example 2 were co-transformed into Corynebacterium glutamicum ATCC 13032. The transformed vectors were plated on TSB agar plates containing 25 μg / mL kanamycin and 100 μg / mL zizomycin, yielding strains RetrotronEc73-4, RetrotronEc73-5, RetrotronEc73-6, and RetrotronEc73-7, respectively. These strains were then transferred to 24-well plates for induction culture, with the culture conditions and mutation efficiency detection methods identical to those in Example 1. The results are shown in Table 7. The 70bp level corresponds to the LBG medium in Table 5. It was found that the editing efficiency was highest (59.86 ± 0.71%) when the template fragment increased to 130bp.

[0204] Table 6

[0205]

[0206] Table 7

[0207] Edit template length 50bp 70bp 90bp 110bp 130bp Editing efficiency 8.66±0.65％ 42.64±0.25％ 36.58±1.38％ 35.91±2.02％ 59.86±0.71％

[0208] Example 5. Editing test of multiple different genes on the genome

[0209] To facilitate rapid and efficient construction of editing plasmids, this disclosure first constructs the tool plasmid pRetron-2-ccdB, and simultaneously introduces the type II restriction endonuclease BspQI (5'…GCTCTTC(N)1…3') restriction site. Using plasmid pRetronEc73-2 from Example 3 as a template, and pRetron-3 / 4 as primers, the plasmid backbone is amplified; pCas9gRNA-proB2 is then used as the amplification site. [1] Using the plasmid as a template, the ccdB fragment was amplified using ccdB-1 / ccdB-2 primers. These two fragments were then cloned and ligated using Novizan's one-step recombination kit to obtain the correct plasmid pRetron-2-ccdB. Figure 1 This basic plasmid can be efficiently cloned using a golden gate to create a recombination template for mutation. The constructed plasmid is similar to pRetronEc73-2, except that the recombination template is replaced with the sequence used for the corresponding target site.

[0210] To further test the versatility of the optimized editing system, this embodiment further tested the editing efficiency of multiple different gene mutations. Mutations in three genes were selected: V150H (codon GTG to CAC) on the proB gene, F383V (codon TTT to GTT) on the ilvA gene, and T271I (codon ACC to ATC) on the mdh gene. Editing templates of 70bp and 130bp were designed for the three gene targets, and two complementary primers were synthesized accordingly. All primers are listed in Table 8. An annealing reaction system (10μL) was then prepared, with 4μL of 10mM stock solution added to each of the two complementary primers. The remaining volume was made up with deionized water. The mixture was denatured at 98℃ for 10min and then naturally cooled to room temperature to anneal into double strands. The double-stranded fragments were then used with the plasmid pRetron-2-ccdB to construct an editing plasmid using the Golden Gate method, i.e., replacing the 70bp or 130bp editing template with the aforementioned rpsL gene editing plasmid.

[0211] The six successfully constructed editing plasmids were co-transformed with pRecT-1 constructed in Example 2 into Corynebacterium glutamicum ATCC 13032, and plated on TSB solid plates containing 25 μg / mL kanamycin and 100 μg / mL zizomycin to obtain the corresponding strains. The obtained strains were then transferred to 24-well plates for culture and induction of editing, under the same culture and induction conditions as in Example 1. Further, using primers proB-1 / 2, ilvA-1 / 2, and mdh-1 / 2 (Table 8), and with the final edited bacterial culture as a template, PCR amplification of fragments containing the mutation sites was performed. Next-generation sequencing was performed on the amplified fragments, and the statistical efficiency of the proportion of mutant reads to the total number of reads was calculated to obtain the proB... V150H ,ilvA F383V ,mdh T271I Gene mutation editing efficiency.

[0212] The results are shown in Table 9. Similar to the results of rpsL gene mutation editing, increasing the length of the editing template is beneficial to improving the editing efficiency. The editing efficiency of the 130bp template length for the three target sites is higher than that of their respective 70bp template lengths. Different gene mutations can achieve high editing efficiency, indicating that the editing technology disclosed in this paper can edit nucleotide mutations at different positions on the genome.

[0213] Table 8

[0214]

[0215] Table 9

[0216]

[0217] Example 6. Multi-gene editing efficiency test

[0218] This disclosure further tests the application of the optimized editing system in simultaneous multi-gene editing. In this embodiment, two mdh sites were selected. T271I With ilvA F383V For testing, the 70bp editing templates corresponding to the two gene mutations were ligated into a single template, and two complementary primers were synthesized (Table 10). Then, an annealing reaction system (10 μL) was prepared, with 4 μL of 10 mM stock solution added to each of the two complementary primers, and the remaining volume made up with deionized water. The mixture was then denatured at 98 °C for 10 min, followed by natural cooling to room temperature for annealing to obtain double strands. The double-stranded fragment was further used to construct an editing plasmid using the Golden Gate method with the plasmid pRetron-2-ccdB.

[0219] The successfully constructed editing plasmids were co-transformed with pRecT-1, constructed in Example 2, into Corynebacterium glutamicum ATCC 13032. The plasmids were then plated on TSB agar plates containing 25 μg / mL kanamycin and 100 μg / mL zidimecrolimus to obtain the corresponding bacterial strains. These strains were then transferred to 24-well plates and cultured and induced for editing under the same conditions as in Example 1. The edited bacterial suspensions were diluted and plated on TSB agar plates containing 25 μg / mL kanamycin and 100 μg / mL zidimecrolimus. Twenty clones were randomly selected, and fragments of both genes were amplified using primers mdh-1 / 2 and ilvA-1 / 2, respectively, and sequenced. The editing efficiency was determined by calculating the percentage of clones with mutations at both sites among the 20 clones. The results showed that the dual-gene editing efficiency reached 25%.

[0220] Table 10

[0221]

[0222] Example 7. Application of the Retron editing system in the construction of deep mutant libraries of gfp genes on the genome.

[0223] To avoid the difficulty in objectively assessing the editing efficiency of the library due to the impact of gene mutations on cell growth, this disclosure selects the gfp gene (encoding green fluorescent protein) as the editing test gene. First, the gfp gene (SEQ ID NO:16) fragment is expressed using promoter P11F, RBS2 (CCTGTTTTAGAGCTAGAAAGGAG, SEQ ID NO:17) and inserted into the genome of Corynebacterium glutamicum ATCC 13032. The insertion position is between the Cgl1180 and Cgl1181 genes, and finally strain ATCC13032-gfp is obtained.

[0224] (1) Construction of GFP gene editing plasmid library

[0225] The gfp gene encodes a protein containing 239 amino acids, and its gene sequence is shown in SEQ ID NO:16. A 70nt fragment (totaling 14220 fragments) was designed, with each of the 237 amino acids (after removing the start and stop codons) undergoing 60 codon mutations (excluding 3 stop codons and the wild-type codon at that site). To facilitate the amplification of the primer pool fragment, YW-1 and BspQI restriction enzyme sites were added to the 5' end of the 70nt sequence. Add YW-2 and BspQI restriction sites to the 3' end of the 70bp sequence. Taking the second codon AGT in GFP as an example: In the lag sequence, select the 34 bases to the left of AGT and the 33 bases to the right of AGT, and replace AGT with 60 codons other than the three stop codons (TGA, TAA, TAG). The position corresponding to AGT is replaced with N NN, i.e. ATCCTGTTTTAGAGCTAGAA AGGAGTTGAGAATG NNN AAAGGAGAAGAACTTTT C ACTGGAGTTGTCCCA Based on this, the YW-1 and BspQI restriction site sequences and the YW-2 and BspQI restriction site sequences mentioned above were added to the 5' and 3' ends of the 70nt sequence, respectively, resulting in the following sequence (total 129nt): This process is repeated to complete the primer design for each site mutation, and then the company synthesizes a primer pool for whole-genome deep mutation scanning and editing of the gene (e.g., the gfp gene).

[0226] SEQ ID NO:16: gfp

[0227] ATGagtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaa

[0228] aacttacccttaaatttatttgcactactggaaaactacctgttccatggccaacacttgtcactactttcgcgtatggtcttcaatgctttgcgagatacccagatcatatgaaacagcatgactt

[0229] tttcaagagtgccatgcccgaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcga

[0230] gttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaaacaaaagaatggaatcaaagttaacttc

[0231] aaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgcc

[0232] ctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataa

[0233] This disclosure further uses the synthesized primer pool as a template and YW-3 / YW-4 as primers to amplify the mutant library fragment. The obtained fragment is then used to construct a mutant plasmid library with the constructed pRetron-2-ccdB via the Golden Gate method. The reaction system is 15 μL: pRetron-2-ccdB, 100 ng; mutant library fragment, 30 ng; T7 ligase, 1 μL; T7 buffer, 4.25 μL; BspQI, 1 μL; NE buffer. TM 3.1 (BspQI enzyme buffer), 1.5 μL; BSA (bovine serum albumin), 3.75 μL. The reaction program was: (43℃, 10 min; 25℃, 10 min) * 12 cycles; 80℃, 5 min. A library of approximately 500,000 *E. coli* transformants was obtained, and a mutant plasmid library was obtained through plasmid extraction.

[0234] Using the obtained plasmid library as a template, ncRNA fragments from the plasmid library were amplified using GK-1 / 2 primers. The purified fragments were then recovered and subjected to PE150 next-generation sequencing. Analysis of the 10G sequencing data revealed that the library covered 99.2% of the designed codon mutations at all sites, and also covered 99.6% of the designed amino acid mutations at all sites (Table 12). Figure 2 The primer sequences used above are shown in Table 11.

[0235] Table 11

[0236] Primers nucleotide sequence YW-3 GACATGCGCTTGGCGCATC YW-4 ACTGGCTGGTCTGTCCAC GK-1 CCATGAGTCATGGTTTCGCC GK-2 CAGAGCCAAACCTACTTGAGC gfp-1 ATCGCACCGGAAATCCTGTT gfp-2 GTCAGCACACGTGAAGTCGAGC

[0237] Table 12

[0238] Statistical parameters codon mutation Amino acid mutation Types of theories 14220 4740 Actual species 14112 4725 Coverage 99.2％ 99.6％

[0239] (2) Construction of a genome editing library for the GFP gene

[0240] This disclosure further constructs a Corynebacterium glutamicum genome editing library, and electroporates the obtained library plasmid into Corynebacterium glutamicum ATCC 13032-gfp (containing plasmid pRecT-1), yielding approximately 6 × 10⁶ pegs. 6 One transformant, covering approximately 400-fold of the theoretical library, was enriched in LBG medium containing 25 μg / mL kanamycin and 100 μg / mL zizomycin resistance. When OD... 600 When it reaches 4, the initial value is OD. 600 =0.2% was transferred to 24-well plates in LBG medium (containing 25 μg / mL kanamycin, 100 μg / mL zizomycin and 0.5 mM IPTG) and induced to OD. 600 The cells were transferred again to 24-well plates (10 μL transfer) for induction culture, using LBG medium supplemented with 25 μg / mL kanamycin, 100 μg / mL zizomycin, and 0.5 mM IPTG. This step was repeated three times. Finally, the genome of the edited library was extracted, and using the genome as a template, the GFP fragment was amplified using GFP-1 / 2 primers and then subjected to PE150 next-generation sequencing.

[0241] The results are shown in Table 13. This disclosure demonstrates that the GFP whole-genome library can be constructed using the Retron editing method. Analysis of 20G second-generation sequencing data after GFP gene editing revealed that the genome-edited library can cover 95% of the designed codon mutations at all sites, and also covers 99.1% of the designed amino acid mutations at all sites (Table 13). Figure 3 Further flow cytometry analysis of the fluorescence intensity distribution of cells before and after GFP gene editing revealed significant changes in the fluorescence intensity distribution of the mutant library. Figure 4 The presence of more cells in the high-fluorescence region indicates the presence of fluorescently enhanced mutants in the library. These findings demonstrate that constructing high-coverage mutant editing libraries can lay an important foundation for screening advantageous mutants.

[0242] Table 13

[0243]

[0244]

[0245] References:

[0246] [1] Zheng Ping; Liu Jiao; Wang Yu; Sun Jibin; Liu Moshi; Zhou Wenjuan; Guo Xuan; Ma Yanhe. γ-Glutamyl kinase mutants and their applications. CN112111469B.

[0247] [2]Jakoby, M; Ngouoto-Nkili, CE; Burkovski, A. Construction and application of new Coryne bacterium glutamicum vectors. Biotechnology Techniques, 1999, 13(6), 437-441.

[0248] [3]Nesvera J,Pátek M.Plasmid pGA1 from Corynebacterium glutamicumcodes for a gene p roduct that positively influences plasmid copy number.JBacteriol.1997Mar;179(5):1525-32.

[0249] All technical features disclosed in this specification can be combined in any way. Each feature disclosed in this specification can also be replaced by other features that have the same, equivalent, or similar function. Therefore, unless otherwise specified, each disclosed feature is merely an example of a series of equivalent or similar features.

[0250] Furthermore, from the above description, those skilled in the art can readily understand the key features of this disclosure. Without departing from the spirit and scope of this disclosure, many modifications can be made to the invention to suit various different uses and conditions. Therefore, such modifications are also intended to fall within the scope of the appended claims.

Claims

1. A novel Corynebacterium glutamicum gene editing system, said gene editing system comprising a plasmid expressing Retron and the recombinase RecT, wherein, The Retron is Retron-73, which includes non-coding RNA (ncRNA) and reverse transcriptase (RT).

2. The gene editing system as described in claim 1, wherein, Bicistronic structures were used to express ncRNA and RT.

3. The gene editing system as described in claim 1 or 2, wherein, The nucleotide sequence of the ncRNA includes the nucleotide sequence shown in SEQ ID NO:6, or a sequence that has more than 90% homology with the sequence shown in SEQ ID NO:6; The amino acid sequence of the RT includes the amino acid sequence shown in SEQ ID NO:7, or a sequence that has more than 90% homology with the sequence shown in SEQ ID NO:

7. The amino acid sequence of the recombinase RecT includes that shown in SEQ ID NO:8, or a sequence that has more than 90% homology with the sequence shown in SEQ ID NO:

8.

4. The gene editing system as described in claim 3, wherein, The ncRNA sequence contains an editing template sequence; the editing template sequence includes a mutation sequence of the site to be edited, and upstream and downstream homologous arm sequences of the site to be edited; preferably, the total length of the upstream and downstream homologous arm sequences is 40bp to 150bp, more preferably 50bp to 140bp, and more preferably 70bp to 130bp.

5. The gene editing system as described in claim 4, characterized in that, The bicistronic structure includes: a) First cistron: Contains a promoter operatively linked to the polynucleotide encoding the RT; b) Second cistron: Contains a promoter and a terminator operatively linked to the polynucleotide encoding the ncRNA; Optionally, use P tac The promoter expression RT uses the P11F promoter and the terminator T. rrnB Express ncRNA; Preferably, the promoter P tac The sequence is shown in SEQ ID NO:1; Preferably, the sequence of the P11F promoter is shown in SEQ ID NO:9; Preferably, the terminator T rrnB As shown in SEQ ID NO:

10.

6. The gene editing system as described in claim 5, wherein, The plasmid expressing Retron also contains a resistance gene that can increase plasmid stability; Preferably, the resistance gene is a zithromycin resistance gene; Preferably, the nucleotide sequence of the zebumycin resistance gene is shown in SEQ ID NO:

14.

7. The gene editing system according to any one of claims 1 to 6, wherein, The plasmid expressing the recombinase RecT contains the paraphrased per gene, as shown in SEQ ID NO:12; Optionally, the sequence of the RecT plasmid expressing the recombinase is shown in SEQ ID NO:

13.

8. The gene editing system according to any one of claims 1 to 7, wherein, The plasmid sequence expressing Retron is shown in SEQ ID NO:11 or 15.

9. Any one of (A) to (C): (A) Encoding a polynucleotide of the gene editing system as described in any one of claims 1 to 8; (B) A carrier containing the polynucleotide described in (A); (C) Recombinant cells containing (A) or (B); Optionally, the recombinant cells include prokaryotic cells; alternatively, the recombinant cells include Escherichia coli and Corynebacterium glutamicum, preferably Corynebacterium glutamicum.

10. A method for editing target nucleic acids, wherein, The method includes: causing the gene editing system according to any one of claims 1 to 8 to bind to a target nucleotide and edit a target nucleic acid; optionally, the target nucleic acid is a single target nucleic acid or multiple target nucleic acids.

11. The method according to claim 10, wherein, The method includes the steps of introducing the gene editing system according to any one of claims 1 to 8 into a host cell, constructing a recombinant cell, and then culturing the recombinant cell in a culture system; Optionally, the culture system includes TSB, LBG, BHI, or CGIII medium; Preferably, the culture system contains TSB, LBG, or BHI medium; More preferably, the LBG culture medium consists of: glucose, 5 g / L; yeast extract, 5 g / L; tryptone, 10 g / L; and NaCl, 10 g / L. More preferably, the BHI culture medium consists of: brain and heart extract, 37.1 g / L; More preferably, the CGIII culture medium consists of: glucose, 5 g / L; yeast extract, 10 g / L; tryptone, 10 g / L; and NaCl, 25 g / L.

12. The use of the gene editing system as described in any one of claims 1 to 8, the polynucleotide as described in claim 9, the vector, or the recombinant cell in nucleotide editing at any position on the genome; Optionally, the nucleotide sequence includes any gene coding and non-coding sequence; Optionally, the non-coding sequence includes promoters, DNA elements such as RBS, and ncRNA; Optionally, the gene coding sequence includes a gene encoding green fluorescent protein.

13. A method for constructing a genome mutant library, the method comprising the steps of converting the gene editing system according to any one of claims 1 to 8 into a host cell and editing nucleotides at any position on the genome of the host cell; Optionally, the nucleotide sequence includes any gene coding and non-coding sequence; Optionally, the non-coding sequence includes promoters, DNA elements such as RBS, and ncRNA; Optionally, the gene coding sequence includes a gene encoding green fluorescent protein.

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