TnpB gene editing system suitable for streptomyces as well as construction and application of TnpB gene editing system

Abstract

The invention discloses a TnpB gene editing system suitable for streptomyces as well as construction and application of the TnpB gene editing system. The system comprises engineered TnpB nuclease and a reRNA guide box, wherein the engineered TnpB nuclease has amino acid difference relative to an amino acid sequence shown in SEQ ID NO.1. Further, the amino acid difference is located at one of the 188, 217, 125, 179, 388, 27, 25, 8, 96, 240, 208, 279, 254, 110, 267, 286, 186, 356, 162, 210, 332, 385, 9, 200, 111, 333, 57 . The engineered TnpB nuclease and the effector protein thereof disclosed by the invention have higher activity, and the gene editing efficiency of the engineered TnpB nuclease and the effector protein thereof in a streptomyces body is more excellent; the editing efficiency of the TnpB (such as D333V, S57R and the like) mutant on the endogenous gene SCO5087 of streptomyces is close to 100%.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology and relates to a TnpB gene editing system suitable for Streptomyces, its construction, and application; in particular, to an engineered TnpB nuclease, a TnpB gene editing system, and a gene editing method suitable for Streptomyces. The engineered TnpB nuclease comprises a mutation based on a reference TnpB nuclease. The engineered TnpB and TnpB gene editing system of the present invention can achieve efficient and precise gene editing in Streptomyces; and compared to unengineered TnpB gene editing systems, the engineered TnpB nuclease and TnpB gene editing system of the present invention can significantly improve the editing efficiency of target genes. Background Art

[0002] Streptomyces is the largest genus in the Actinobacteria phylum. Its ability to produce structurally rich, widely active, and highly applicable secondary metabolites, combined with its potential to encode a vast array of new compounds, presents broad application prospects in fields such as medicine, health, food, industry, and agriculture. However, due to the characteristics of Streptomyces itself, such as the high GC content of its genome, the linear nature of its chromosomes, and the fact that most of its secondary metabolite biosynthetic gene clusters are silent and unexpressed under experimental conditions, the development of new valuable secondary metabolites or the increase in production of known active secondary metabolites through systematic metabolic engineering using traditional homologous double crossover methods has been greatly limited. The current mainstream approach to secondary metabolite research and development is to utilize synthetic biology for efficient bottom-up exploration based on the genome, and possessing a powerful genetic operating system is the foundation for developing and improving existing secondary metabolites in Streptomyces.

[0003] However, the Streptomyces genome is large (8-10Mb) and has a high GC content (generally over 70%). Compared with other microorganisms, it is very difficult to complete genetic operations such as gene editing in Streptomyces, and the means of genetic manipulation are very limited. With the development of CRISPR-Cas9 technology and its application in Streptomyces, the problem of time-consuming and inefficient gene editing in Streptomyces has been alleviated to a great extent, effectively breaking the bottleneck of the lack of efficient genetic operations in the field of Streptomyces, and becoming the main gene editing tool currently applicable to Streptomyces. Although the CRISPR-Cas9 series of gene editing tools play an important role in Streptomyces gene editing, there are still many problems to be improved. For example, the size of CRISPR-Cas9 is too large, which affects the efficiency of plasmid construction and delivery; the high GC PAM sequence leads to potential high off-target effects (Off-target effects) and other problems.

[0004] To overcome the limitations of proteins like Cas9 due to their large size, researchers have systematically optimized and modified key components of the CRISPR-Cas system, including using orthologous Cas9 enzymes, modifying Cas9, and optimizing guide RNAs. Furthermore, they are exploring the application of novel gene-editing techniques, such as base editing and prime editing. Furthermore, researchers are continuously exploring and developing compact CRISPR proteins to develop novel CRISPR systems. TnpB, a recently discovered programmable nuclease approximately one-third the size of Cas9 (approximately 400 amino acids), is guided by a long noncoding RNA and can cleave DNA sequences 5' to the TTGAT region, attracting considerable attention from researchers both domestically and internationally. To date, TnpB has been successfully applied to edit endogenous genes in multiple species. However, compared to Cas9, TnpB and other reported compact gene-editing tools generally suffer from lower editing efficiency. More importantly, existing small gene editors have not yet been reported for editing in Streptomyces. Summary of the Invention

[0005] The object of the present invention is to provide a TnpB gene editing system suitable for Streptomyces and its construction and application. In view of the current situation that mini gene editing tools are still lacking in Streptomyces and the common problem of low efficiency in editing with current mini gene editing tools, the present invention constructs a Streptomyces mini gene editing system between TnpB. On this basis, through in-depth analysis and rational design of the TnpB-DNA-RNA three-dimensional crystal structure, the TnpB nuclease is optimized and transformed, and an efficient Streptomyces mini gene editing system between TnpB nuclease is developed, which achieves precise and efficient editing of the Streptomyces genome in vivo, thereby enriching the Streptomyces gene manipulation tool library, promoting the study of the gene function of the Streptomyces silencing gene cluster, and providing a powerful enabling tool for precise modification of metabolic pathways and intelligent biomanufacturing.

[0006] To overcome the low editing efficiency of current mini-gene editors and further enrich the Streptomyces gene editing tool library, the present invention uses TnpB nuclease (only about 400 amino acids) as a basis to construct a Streptomyces mini-gene editing system based on TnpB. By analyzing the three-dimensional crystal structure of TnpB-DNA-RNA and rationally designing and engineering the TnpB nuclease, an efficient Streptomyces gene editing system based on the engineered TnpB nuclease and the corresponding gene editing methods and processes using this system were developed. The purpose of the present invention is to achieve this through the following technical solutions:

[0007] In a first aspect, the present invention provides an engineered TnpB nuclease, wherein the engineered TnpB nuclease has amino acid differences relative to the amino acid sequence of an unmodified TnpB nuclease (such as that shown in SEQ ID NO. 1).

[0008] As one embodiment of the present invention, the amino acid difference is located at one of positions 188, 217, 125, 179, 388, 27, 25, 8, 240, 208, 96, 279, 254, 110, 267, 286, 186, 356, 162, 210, 332, 385, 9, 200, 111, 333, and 57.

[0009] As one embodiment of the present invention, the amino acid difference is preferably located at one of positions 356, 162, 210, 332, 385, 9, 200, 111, 333, and 57.

[0010] In some preferred embodiments, the engineered TnpB nuclease is characterized by replacing amino acid residue A at position 188 with V; amino acid residue S at position 217 with K; amino acid residue N at position 125 with G; amino acid residue Y at position 179 with P; amino acid residue Y at position 388 with A; amino acid residue S at position 27 with C; amino acid residue L at position 25 with F; amino acid residue V at position 8 with K; amino acid residue G at position 240 with R; amino acid residue H at position 208 with K; amino acid residue T at position 96 with R; amino acid residue H at position 279 with T; and amino acid residue V at position 254 with S. ; amino acid residue R at position 110 is replaced by K; amino acid residue S at position 267 is replaced by R; amino acid residue R at position 286 is replaced by L; amino acid residue F at position 186 is replaced by K; amino acid residue E at position 356 is replaced by A; amino acid residue I at position 162 is replaced by N; amino acid residue Q at position 210 is replaced by N; amino acid residue H at position 332 is replaced by S; amino acid residue H at position 385 is replaced by C; amino acid residue V at position 9 is replaced by Y or F, preferably F; amino acid residue V at position 200 is replaced by L; amino acid residue K at position 111 is replaced by S; amino acid residue D at position 333 is replaced by V; amino acid residue S at position 57 is replaced by R.

[0011] In some preferred embodiments, the engineered TnpB nuclease is characterized by replacing amino acid residue E at position 356 with A; amino acid residue I at position 162 with N; amino acid residue Q at position 210 with N; amino acid residue H at position 332 with S; amino acid residue H at position 385 with C; amino acid residue V at position 9 with Y or F, preferably F; amino acid residue V at position 200 with L; amino acid residue K at position 111 with S; amino acid residue D at position 333 with V; and amino acid residue S at position 57 with R.

[0012] In a second aspect, the present invention provides a system for realizing efficient mini gene editing in Streptomyces, which comprises the above-mentioned engineered TnpB nuclease and its RNA guide box.

[0013] As one embodiment of the present invention, the reRNA guide box includes the following core components in sequence according to the direction of the gene editing target gene: the guide RNA base sequence of the TnpB protein, the gene targeting segment, and the hepatitis delta virus (HDV) ribozyme, which are used to guide the TnpB protein to move toward the target sequence DNA.

[0014] As one embodiment of the present invention, the base sequence of the guide RNA of the TnpB protein is shown as SEQ ID NO.3.

[0015] As one embodiment of the present invention, the gene targeting segment is located at the 3' end of the guide RNA base sequence, and is a nucleic acid fragment with a length of 12-40 bp following the 5'TTGAT of the TAM sequence on the target gene.

[0016] In a third aspect, the present invention provides a recombinant expression plasmid vector for expressing the aforementioned Streptomyces mini gene editing system.

[0017] In a fourth aspect, the present invention provides a method for constructing the aforementioned Streptomyces mini gene editing system, the method comprising constructing a gene editing plasmid containing an apramycin resistance screening marker; the gene editing plasmid is based on the Escherichia coli-Streptomyces shuttle plasmid, and the engineered TnpB nuclease and its reRNA guide box are inserted.

[0018] In a fifth aspect, the present invention provides an application of the aforementioned Streptomyces mini gene editing system for Streptomyces genome editing based on double-strand breaks and Streptomyces' own repair system.

[0019] In a sixth aspect, the present invention provides a gene editing method suitable for Streptomyces, and the gene editing method is specifically as follows: under non-induction conditions, the above-mentioned gene editing system is transferred into the target Streptomyces host by conjugation transfer, so that gene deletion or gene knock-in or replacement occurs near the target site of the host, resulting in frameshift mutation, thereby inactivating the target gene.

[0020] As an embodiment of the present invention, the gene editing method comprises the following steps:

[0021] S1. Under non-inducing conditions, the gene-editing plasmid constructed by the above method is transformed into the target host. Through homologous end ligation of Streptomyces itself, gene deletion, insertion, or replacement occurs near the target site, causing frameshift mutations, thereby inactivating the target gene; transformants carrying the target gene mutation are obtained through antibiotic screening related to plasmid resistance;

[0022] S2. The transformant is streaked onto a medium containing a plasmid resistance antibiotic and a promoter inducer (e.g., thiostrepton), and cultured at a constant temperature until a single colony is visible;

[0023] S3. Randomly select the single clones in step S2 and verify by colony PCR to obtain the target gene mutant strain.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The engineered TnpB nuclease and its effector protein of the present invention have higher activity. Compared with existing conventional TnpB gene editing tools, the engineered TnpB nuclease of the present invention has better gene editing efficiency in Streptomyces; for example, the editing efficiency of multiple TnpB mutants (such as D333V, S57R, etc.) in the examples of the present invention for the endogenous gene SCO5087 of Streptomyces is close to 100%. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments with reference to the following drawings:

[0027] Figure 1 is the editing efficiency of the engineered TnpB nuclease; Note: WT is the unengineered TnpB nuclease; the dotted bar graph indicates that the editing efficiency is 2 times or more than that of the unengineered TnpB nuclease;

[0028] Figure 2 The figure shows the electrophoresis results (TnpB(S57V));

[0029] Figure 3 Figure 2 shows the sequencing results (TnpB(S57V));

[0030] Figure 4 The figure shows the results of target gene inactivation by engineered TnpB nuclease (TnpB(S57V)). DETAILED DESCRIPTION

[0031] The present invention will be described in detail below with reference to the examples. The following examples will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that those skilled in the art may make several adjustments and improvements without departing from the scope of the present invention. These all fall within the scope of protection of the present invention.

[0032] Example 1: Engineering TnpB nuclease

[0033] 1.1 Plasmid design and construction

[0034] Based on the codon preference of Streptomyces, the TnpB nuclease from Deinococcus radiodurans ISDra2 was codon-optimized and synthesized by GenScript Biotech Co., Ltd. The amino acid sequence of TnpB is shown in SEQ ID NO. 1, and the gene sequence is shown in SEQ ID NO. 2. In this invention, it is designated as wild-type TnpB and shares 79.74% DNA similarity with the TnpB nuclease from Deinococcus radiodurans ISDra2.

[0035] The reRNA guide box for guiding the wild-type TnpB nuclease to perform double-stranded cutting near the endogenous target site of Streptomyces includes the following core elements in the direction from 5' to 3': the guide RNA base sequence of the TnpB protein (SEQ ID NO.3), the gene targeting segment, and the hepatitis D virus (HDV) ribozyme (SEQ ID NO.4). The gene targeting segment is located at the 3' end of the RNA backbone and is a nucleic acid fragment with a length of 12-40 bp following the TAM sequence (5'TTGAT) on the target gene. In the embodiment of the present invention, the key gene SCO5087 (actI) for the synthesis of actinomycetin in the model strain Streptomycin coelicolor A3 (2) (genome sequence number: GeneBank: GCA_008931305.1) is selected as the endogenous gene target of the cell. The base sequence of the gene targeting segment in the reRNA guide box corresponding to the target is 5'-gtagtcgatgtccgtcgcgt. The reRNA guide cassette used in the examples was synthesized by GenScript Biotech Co., Ltd.

[0036] The synthesized wild-type TnpB and its reRNA guide cassette were used to replace the Cas9 and sgRNA fragments on the Streptomyces-Escherichia coli shuttle plasmid vector pCRISPR-Cas9 (https: / / doi.org / 10.1021 / acssynbio.5b00038), respectively, to obtain the plasmid pTnpB-ZH.

[0037] Based on the crystal structure of the Deinococcus radiodurans ISDra2-derived TnpB nuclease bound to its guide RNA and substrate DNA, 80 single-point mutations of TnpB were designed (Table 1). Using pTnpB-ZH as a template, mutations were introduced by inverse PCR (Table 2) to achieve site-directed mutagenesis of TnpB. The primers are shown in Table 3. The 2× MegaPfu Premix (with dye) was purchased from Tolo Biotech (Cat. No. 21809). The inverse PCR product was then purified (the kit was purchased from Nanjing Novozymes Biotech Co., Ltd.). Gel DNA Extraction Mini Kit, DC301); then, the purified PCR product was transferred into competent cells E. coli DH5α, and its own DNA recombination and repair system was used to achieve in vivo circularization of the linear DNA, thereby obtaining the TnpB optimized plasmid system.

[0038] The plasmids constructed in the present invention were subjected to Sanger sequencing to ensure complete accuracy.

[0039] Table 1 TnpB nuclease single point mutation sequence

[0040]

[0041]

[0042] Table 2 Reverse PCR reaction system

[0043]

[0044]

[0045] Table 3 Reverse PCR primers and annealing temperature

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054] Example 2: Application of the Optimized Streptomyces Mini-Editing System

[0055] 2.1 Conversion

[0056] The TnpB optimized target plasmids were transferred into the homemade competent cells E. coli ET12567 / pUZ8002 (https: / / doi.org / 10.1016 / 0378-1119(92)90549-5) respectively. The specific operation method was as follows: 200 ng of TnpB optimized plasmids were added into 100 μL of thawed E. coli ET12567 / pUZ8002 competent cells were gently mixed; after standing on ice for 30 minutes, placed in a 42°C water bath for 45 seconds, and then immediately placed on ice for 2 minutes; then, added to 400 μL of LB liquid medium without antibiotics, and cultured at 37°C, 200 rpm for 1 hour; then 100 μL was spread on an LB solid plate containing kanamycin (25 μg / mL), chloramphenicol (12.5 μg / mL) and apramycin (50 μg / mL); after overnight culture at 37°C, single clones were picked and cultured in 20 mL of LB liquid medium containing kanamycin (25 μg / mL), chloramphenicol (12.5 μg / mL) and apramycin (50 μg / mL) until the OD 600 When the cell density was about 0.4, the cells were collected by centrifugation (5000 rpm, 5 minutes); an equal volume of antibiotic-free LB liquid medium was added to wash the cells twice, and finally the cells were resuspended in 2 mL of LB liquid medium.

[0057] 2.2 Binding transfer and resistance screening

[0058] Under non-inducing conditions, the plasmid in step 2.1 was transferred into S. coelicolor A3 (2) by conjugation transfer. The specific operation method is as follows: take the previously collected Streptomyces spores, centrifuge (5000 rpm, 5 minutes), discard the supernatant, and then resuspend in 2 mL 2×YT liquid culture medium; heat shock at 50℃ for 10 minutes, and then pre-germinate in a 30℃ shaker at 200 rpm for 30 minutes; take 500 μL of the E. coli culture collected in step 2.1 and 200 μL of the Streptomyces spore suspension in a 1.5 mL centrifuge tube and mix them evenly; take 100 μL of the mixture and spread it on an MS plate; incubate the MS plate in an incubator at 30℃ for 18 hours, cover the surface of the culture medium with apramycin (1 mg / mL) and nalidixic acid (1 mg / mL), wait for the surface to dry, and continue to incubate the MS plate in an inverted position at 30℃ until the conjugate grows (about 5 days). At this time, the successfully edited conjugate can be obtained.

[0059] Example 3: Evaluation of gene editing efficiency of the engineered TnpB system

[0060] The resistant conjugates in step 2.2 were randomly picked and streaked onto ISP2 solid culture medium containing plasmid resistance antibiotics (50 ng / mL apramycin) and thiostrepton (0.5 μg / mL), inverted, and cultured at 30°C until bacteria were visible; a small amount of bacteria was picked into a PCR tube containing 20 μL DMSO, and the cells were lysed in a metal bath (100°C, 15 minutes) to obtain cell lysate; the editing efficiency of the optimized TnpB system was systematically evaluated by colony PCR. The specific operation was as follows: the target site fragment was PCR amplified using the 2× Rapid Taq Master Mix (purchased from Nanjing Novozymes Biotech Co., Ltd., catalog number: P222-01) kit. The primer design is shown in Table 4, the PCR reaction system is shown in Table 5, and the PCR reaction procedure is shown in Table 6.

[0061] Table 4 PCR amplification primers targeting SCO5087 (actI)

[0062] serial number Primers Sequence (5'-3') SEQ ID NO.5 SCO5087-LH-F atgattccggaactccggt SEQ ID NO.6 SCO5087-R accacagcttgcggaact

[0063] Table 5 PCR reaction system

[0064] system 15 μL <![CDATA[ddH2O]]> 5.25 μL Forward primer (10 μM, T-SCO5087-F) 0.75μL Reverse primer (10 μM, T-SCO5087-R) 0.75μL 2× Rapid Taq Master Mix 7.50 μL Cell lysis buffer (containing DMSO) 0.75μL

[0065] Table 6 PCR reaction program

[0066]

[0067]

[0068] This embodiment uses a combination of 2% agarose gel electrophoresis detection and Sanger sequencing or whole genome resequencing to determine whether the randomly selected binders are edited. The TnpB system cooperates with the defective non-homologous end joining of Streptomyces itself to achieve editing of the target gene, such as gene knockout, insertion, replacement, etc. If successfully edited, the band size of the above-mentioned PCR amplification product after 2% agarose gel electrophoresis is different from that of the wild type; for samples that cannot be judged by gel electrophoresis, the Weizan plasmid purification kit (item number: DC201) is used to purify and recover samples that cannot be judged by gel electrophoresis to determine whether they are edited and sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for Sanger sequencing analysis; for samples with no amplification products after PCR, the bacteria are collected and sent to Sangon Biotech (Shanghai) Co., Ltd. for whole genome resequencing to further evaluate the gene editing situation. The results show that ( Figure 1-3), using the target gene SCO5087 (actI) in the model strain S. ceolicolor A3 (2) as the target, and after single point mutations in the TnpB site (A188V, S217K, N125G, Y179P, Y388A, S27C, L25F, V8K, G240R, H208K, T96R, H279T, V254S, R110K, S267R, R286L, F186K, E356A, I162N, Q210) relative to the wild-type TnpB system. N, H332S, H385C, V9Y, V200L, K111S, V9F, D333V, S57R) showed high gene editing efficiency, with an increase of at least 2 times in gene editing ability. Among them, the TnpB nuclease contained mutations at one of the amino acids at positions 356, 162, 210, 332, 385, 200, 111, 9, 333 and 57, and the editing efficiency was increased by 3.0-4.3 times.

[0069] Example 4: Inactivation of target genes using engineered TnpB enzymes

[0070] Under non-inducing conditions, the TnpB optimized plasmid system (S57R) was transferred into S. coelicolor A3 (2) by conjugation transfer, as in step 2.2. About 5 days later, resistant conjugates were randomly picked and streaked onto ISP2 solid culture medium with plasmid resistance antibiotics (50 ng / mL apramycin) and thiostrepton (0.5 μg / mL), inverted, cultured at 30°C, and pigment secretion was observed. In the present embodiment, the key gene SCO5087 (actI) for actinomycin synthesis was used as the endogenous gene target of the cell. When the target gene mutated, S. coelicolor A3 (2) could not secrete blue pigment. The target gene was detected by combining colony PCR or whole genome resequencing to determine whether the randomly selected conjugate was edited, as detailed in Example 3. The results of the target gene inactivation by the engineered TnpB nuclease (TnpB (S57V)) are shown in Figure 4 ; The strains with target gene mutations were cultured on the above-mentioned resistance plates for 2 weeks, and no blue pigment was secreted. This further illustrates that the engineered TnpB system combined with Streptomyces' own non-homologous end joining (NHEJ) repair method caused gene deletion or gene knock-in and replacement near the target site, resulting in a frameshift mutation in the target gene, thereby inactivating the target gene.

[0071] Example 5: Various culture media involved in various implementations

[0072] LB medium: Weigh 10g tryptone, 5g yeast extract, and 10g sodium chloride, dissolve in 1L ddH2O, sterilize at 115°C for 30 minutes, and store at room temperature until ready to use. If preparing solid medium, add 2% agar powder.

[0073] MS medium: Weigh 10 g soybean cake powder, 10 g tryptone, and 10 g agar powder, dissolve in 500 mL tap water, and sterilize at 115°C for 30 min.

[0074] ISP2 medium: Weigh 10 g malt extract, 4 g yeast extract, and 4 g glucose, dissolve in 1 L ddH2O, adjust pH to 7.4, sterilize at 115°C for 30 min, and store at 4°C until used. If preparing solid medium, add 2% agar powder.

[0075] 2×YT medium: Weigh 16 g tryptone, 10 g malt extract, and 5 g sodium chloride, dissolve in 1 L ddH2O, adjust the pH to 7.0, sterilize at 115°C for 30 min, and store at 4°C until use.

[0076] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various variations or modifications within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. An engineered TnpB nuclease for a Streptomyces mini-gene editing system, characterized in that: The engineered TnpB nuclease has amino acid differences relative to the TnpB nuclease having the amino acid sequence shown in SEQ ID NO.

1.

2. The engineered TnpB nuclease according to claim 1, wherein The amino acid difference is located at one of positions 188, 217, 125, 179, 388, 27, 25, 8, 240, 208, 96, 279, 254, 110, 267, 286, 186, 356, 162, 210, 332, 385, 9, 200, 111, 333, and 57 of the amino acid sequence shown in SEQ ID NO.

1.

3. The engineered TnpB nuclease according to claim 1 or 2, wherein: The engineered TnpB nuclease has one of the following mutations in the amino acid sequence shown in SEQ ID NO. 1: A188V, S217K, N125G, Y179P, Y388A, S27C, L25F, V8K, G240R, H208K, T96R, H279T, V254S, R110K, S267R, R286L, F186K, E356A, I162N, Q210N, H332S, H385C, V9Y or V9F, V200L, K111S, D333V, S57R.

4. A Streptomyces mini gene editing system, characterized in that: The system comprises the engineered TnpB nuclease and a reRNA guide cassette according to any one of claims 1 to 3.

5. The Streptomyces mini gene editing system according to claim 4, characterized in that The reRNA guide box comprises a guide RNA base sequence of the TnpB protein, a gene targeting segment and a gene sequence of the hepatitis D virus ribozyme.

6. The Streptomyces mini gene editing system according to claim 5, characterized in that The gene targeting segment is located at the 3' end of the guide RNA base sequence and is a nucleic acid fragment with a length of 12-40 bp following the 5' TTGAT of the TAM sequence on the target gene.

7. A method for constructing a Streptomyces mini-gene editing system according to any one of claims 4 to 6, characterized in that: The method comprises constructing a gene editing plasmid containing an apramycin resistance selection marker; the gene editing plasmid is based on an Escherichia coli-Streptomyces shuttle plasmid, and the engineered TnpB nuclease and its reRNA guide box are inserted.

8. A recombinant expression plasmid vector for expressing the Streptomyces mini-gene editing system according to any one of claims 4 to 6.

9. An application of the Streptomyces mini gene editing system according to any one of claims 4 to 6 in Streptomyces genome editing based on double-strand breaks and Streptomyces self-repair system.

10. A gene editing method applicable to Streptomyces, characterized in that: Under non-inducing conditions, the Streptomyces mini gene editing system according to any one of claims 6 to 8 is transferred into the target Streptomyces host by conjugative transfer, so that gene deletion or gene knock-in or replacement occurs near the target site of the host, causing frameshift mutation, thereby inactivating the target gene.

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