Efficient gene editing system for streptomyces as well as construction method and application of efficient gene editing system

Abstract

The invention discloses an efficient gene editing system for streptomyces as well as a construction method and application of the efficient gene editing system. The system comprises a TnpB nuclease which can be expressed in streptomyces and a guide RNA, wherein the guide RNA comprises an RNA skeleton, a gene targeting section and a gene sequence of hepatitis delta virus (HDV) ribozyme. Wherein the gene targeting segment is located at the 3'end of the RNA skeleton and is a nucleic acid fragment with the length of 12-40bp after a TAM sequence (5 'TTGAT) on a target gene; the hepatitis D virus (HDV) ribozyme is used for stabilizing the RNA skeleton-gene targeting section structure. According to the streptomyces mini-gene editing tool provided by the invention, in the presence of a homologous repair template, the gene editing efficiency can reach 70-100% in streptomyces, and the gene editing efficiency under the action of the guide RNA after rational design can reach 100%.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology and relates to an efficient gene editing system for Streptomyces, its construction method, and its application. In particular, it relates to an optimized guide RNA, a TnpB gene editing system, its construction method, and its application in Streptomyces. By optimizing the guide RNA of the TnpB system, the present invention systematically develops a novel mini-gene editing tool suitable for Streptomyces. The Streptomyces mini-gene editing tool of the present invention can achieve precise and efficient editing of the Streptomyces genome in vivo and has great value for promotion and application. Background Art

[0002] Streptomyces is the largest genus of actinomycetes. It is considered to be a group with great development value because it can produce a large number of valuable active secondary metabolites and its genome also contains rich silent biosynthetic gene clusters. Starting from genomic information, the "bottom-up" efficient and high-throughput development of new active secondary metabolites based on the concept of synthetic biology is the mainstream idea of ​​current natural product drug research and development, and the key lies in having an efficient, accurate and convenient genetic manipulation system. However, due to its large genome (8-10Mb) and high GC content (generally GC content exceeds 70%), Streptomyces is very difficult to perform genetic manipulations such as gene editing compared to other microorganisms, and the means of genetic manipulation are very limited.

[0003] With the development of the CRISPR-Cas gene editing system 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 manipulation in the field of Streptomyces. It has become the main gene editing tool currently applicable to Streptomyces, achieving efficient and precise gene editing of the Streptomyces genome, and has been well applied in exploring new secondary metabolites of Streptomyces, increasing secondary metabolic production, and transforming metabolic pathways. However, there are still many problems to be solved in the current gene editing based on the CRISPR-Cas system. For example, the number of amino acids in the Cas9 or Cas12a protein exceeds 1000, that is, the number of nucleotides (pairs) encoding this effector protein is greater than 3000. It is difficult to effectively package so many nucleotides into some delivery systems, thereby affecting plasmid construction and transformation efficiency. The large size of Cas9 / Cas12a also limits the modification space of subsequent editing systems.

[0004] In order to break through the application limitations caused by the large size of proteins such as Cas9, researchers have conducted research and exploration from different directions: through systematic optimization and transformation of the CRISPR-Cas system to overcome shortcomings, such as modifying Cas9, optimizing guide RNA, using Cas9 orthologous enzymes and other measures; exploring the application of new gene editing technologies, such as developing prime editing; developing new CRISPR systems, and exploring and developing compact CRISPR proteins, including CasX (about 980 amino acids), Cas12f (400-700 amino acids), Cas12i (about 1000 amino acids), Cas12Φ (700-800 amino acids), Cas12m (604 amino acids), Cas12I (about 860 amino acids), Casλ (about 800 amino acids), etc. The TnpB protein is a programmable nuclease recently discovered in transposon systems. It may be an evolutionary ancestor of Cas12. It is guided by a long noncoding RNA of approximately 150 nt to cleave a DNA sequence near the 5' end of TTGAT. Its size is only about one-third that of the Cas protein (approximately 400 amino acids), making it a highly sought-after target for research both domestically and internationally. To date, TnpB nucleases have been successfully applied to endogenous gene editing in a variety of species, including human cells, mouse embryos, and monocotyledonous and dicotyledonous plants. Researchers have also conducted large-scale, systematic research and study on the widespread distribution of TnpB nucleases in organisms, identifying multiple TnpBs with targeted editing activity. However, compared to Cas9, TnpB and other compact gene editing tools suffer from lower editing efficiency. More importantly, existing small editors have not yet been reported for gene editing in Streptomyces. Summary of the Invention

[0005] The present invention aims to provide an efficient gene editing tool for Streptomyces, its construction method, and its application. This invention addresses the current lack of mini-gene editing tools in Streptomyces and the widespread low editing efficiency of current mini-gene editing tools. By deeply analyzing the core components and mechanisms of action of the small, programmable TnpB nuclease for gene editing, the invention designs RNA sequences that guide TnpB nuclease to achieve double-strand cleavage. This method systematically develops an efficient mini-gene editing tool suitable for Streptomyces, thereby achieving efficient and precise editing of the Streptomyces genome based on double-strand break and targeted repair.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] In a first aspect, the present invention provides a guide RNA for guiding TnpB protein to move to a target sequence. The guide RNA comprises (from 5' to 3' end) an RNA backbone, a gene targeting segment, and a gene sequence of a hepatitis delta virus (HDV) ribozyme.

[0008] In one embodiment, the nucleotide sequence of the RNA backbone is SEQ ID NO.3.

[0009] In one embodiment, the nucleotide sequence of the RNA backbone is a rationally designed RNA nucleotide sequence based on in-depth research and analysis of the guide RNA of TnpB in the present invention, and the nucleotide sequence thereof is SEQ ID NO.6.

[0010] In the present invention, the system targets genes located at or near the TAM sequence (5'TTGAT). In one embodiment, the gene-targeting segment, located at the 3' end of the RNA backbone, is a 12-40 bp nucleic acid fragment following the TAM sequence (5'TTGAT) on the target gene. The hepatitis delta virus (HDV) ribozyme is used to stabilize the RNA backbone-gene-targeting segment structure.

[0011] In a second aspect, the present invention provides a TnpB-mediated Streptomyces mini-gene editing system; the system comprises: a TnpB nuclease that can be expressed in Streptomyces and the guide RNA.

[0012] In one embodiment, the TnpB nuclease is a codon-optimized TnpB nuclease that can be expressed in Streptomyces (DNA similarity to its wild-type TnpB from Deinococcus radiodurans ISDra2 is 79.74%). The amino acid sequence of the TnpB nuclease is shown in SEQ ID NO. 1. The gene sequence encoding the TnpB nuclease is shown in SEQ ID NO. 2.

[0013] In a third aspect, the present invention provides a recombinant expression plasmid vector for expressing the Streptomyces mini gene editing system.

[0014] In a fourth aspect, the present invention provides a method for constructing the Streptomyces (efficient) mini gene editing system, comprising the following steps:

[0015] The construction includes a gene knockout / knockin plasmid containing an apramycin resistance selection marker; the gene knockout / knockin plasmid is based on the Escherichia coli-Streptomyces shuttle plasmid, and the codon-optimized TnpB protein and its guide RNA and the gene knockout / knockin box are respectively inserted.

[0016] In some implementation examples, the codon-optimized TnpB protein and its guide RNA are used to replace the Cas9 and sgRNA fragments on the Streptomyces-Escherichia coli shuttle plasmid, respectively, to obtain plasmid A; the plasmid A is enzymatically digested to obtain a linearized plasmid B; and the gene knockout / knock-in cassette is inserted to obtain the gene knockout / knock-in plasmid.

[0017] In one embodiment, the gene knockout plasmid is based on the Escherichia coli-Streptomyces shuttle plasmid, and a TnpB gene editing system (TnpB protein and its guide RNA) and a gene knockout box suitable for Streptomyces are inserted.

[0018] In one embodiment, the gene knockout cassette includes the following core elements in the direction of the gene editing target gene: an upstream homologous arm of the target gene and a downstream homologous arm of the target gene; the upstream and downstream homologous sequences of the target gene are used to provide a homologous recombination template when gene editing occurs.

[0019] In one embodiment, the gene knock-in plasmid is based on the Escherichia coli-Streptomyces shuttle plasmid, and a TnpB gene editing system (TnpB protein and its guide RNA) and a gene knock-in box suitable for Streptomyces are inserted.

[0020] In one embodiment, the gene knock-in cassette includes the following core elements in the direction of the gene editing target gene: an upstream homology arm of the target gene, an inserted gene, and a downstream homology arm of the target gene.

[0021] In a fifth aspect, the present invention provides an application of the above-mentioned Streptomyces mini gene editing system in the precise editing of the Streptomyces genome based on double-strand breaks and directed repair.

[0022] In a sixth aspect, the present invention provides a method for gene editing a target gene in a recipient bacterium, Streptomyces, using the Streptomyces mini gene editing system. The method comprises:

[0023] ① Under non-induction conditions, the gene knockout / insertion plasmid constructed using the above method is transformed into the target host. The gene is knocked out (inserted) by homologous exchange of the homologous arm sequence of the gene knockout / insertion cassette with the upstream and downstream homologous sequences of the target gene. Transformants carrying the gene knockout / insertion plasmid are obtained through antibiotic screening for plasmid-related resistance.

[0024] ② The transformants from step ① were streaked onto a culture medium plate containing plasmid resistance antibiotics and promoter inducers, and cultured at a constant temperature until a single colony was visible;

[0025] ③ Randomly select single clones from step ② and verify by colony PCR to obtain a traceless gene knockout / in strain.

[0026] In some embodiments of the present invention, the promoter inducing agent is selected from thiostrepton.

[0027] Compared with the existing technology, the Streptomyces mini gene editing tool provided by the present invention can achieve a gene editing efficiency of 70-100% in Streptomyces in the presence of a homologous repair template, among which the gene editing efficiency can reach 100% under the action of a rationally designed guide RNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] 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:

[0029] Figure 1 The plasmid maps of HpTnpB-reRNA-ZH and HpTnpB-reRNA-ZJ are shown;

[0030] Figure 2 Schematic diagram of the genome structure of the strain in which the target gene (SCO5087) was theoretically successfully edited;

[0031] Figure 3 This is a diagram showing the results of the TnpB system (HpTnpB-reRNA-ZJ) of the present invention on the endogenous gene editing of Streptomyces (DNA electrophoresis);

[0032] Figure 4 Comparison of the editing efficiency of HpTnpB-reRNA-ZH and HpTnpB-reRNA-ZJ in Streptomyces endogenous genes;

[0033] Figure 5 This is the Sanger sequencing result diagram. DETAILED DESCRIPTION

[0034] 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.

[0035] The present invention uses the model strain Streptomycin coelicolor A3(2) as the target strain, and the genome sequence number of the strain is: GeneBank: GCA_008931305.1. The present invention uses the key gene SCO5087 (actI) for actinomycin synthesis in the above strain as the endogenous gene target, and aims at gene knockout to test the editing efficiency of the present invention.

[0036] Example 1: Construction of an efficient mini gene editing tool in Streptomyces

[0037] 1.1 Plasmid design and construction

[0038] The TnpB gene used is a codon-optimized TnpB protein gene, which was codon-optimized according to the codon preference of Streptomyces. Its amino acid sequence is shown in SEQ ID NO. 1, and its nucleotide sequence is shown in SEQ ID NO. 2. A guide RNA, designated reRNA-ZH, comprises the following core elements, from the 5' to the 3' end: an RNA backbone (SEQ ID NO. 3), a gene-targeting region (SEQ ID NO. 4 when targeting SCO5087 as described above), and a hepatitis delta virus (HDV) ribozyme gene sequence (SEQ ID NO. 5). The RNA backbone is an RNA nucleotide sequence reported in the literature (https: / / doi.org / 10.1038 / s41586-021-04058-1) that can guide TnpB to cleave double-stranded DNA near the target gene. Another guide RNA, named reRNA-J, consists of the following core elements, in order from the 5' to 3' direction: an RNA backbone (SEQ ID NO. 6), a gene-targeting region (SEQ ID NO. 4 for SCO5087 as described above), and the hepatitis delta virus (HDV) ribozyme gene sequence (SEQ ID NO. 5). The RNA backbone (SEQ ID NO. 6) is a rationally designed RNA nucleotide sequence of the present invention. All of these gene segments were synthesized by GenScript Biotech Co., Ltd.

[0039] The codon-optimized TnpB gene and reRNA-ZH 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-reRNA-ZH.

[0040] The codon-optimized TnpB gene and reRNA-J 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-reRNA-ZJ.

[0041] The plasmids pTnpB-reRNA-ZH and pTnpB-reRNA-ZJ were then digested with SphI endonuclease to obtain linearized pTnpB-reRNA-ZH and linearized pTnpB-reRNA-ZJ. The knockout cassette consisting of the upstream and downstream homology arms of the target gene was then seamlessly cloned and assembled with the linearized plasmid vectors pTnpB-reRNA-ZH and pTnpB-reRNA-ZJ (the kit was purchased from Vazyme, ClonExpress Ultra One Step Cloning Kit), obtaining plasmid HpTnpB-reRNA-ZH (the mass spectrum of the plasmid is shown in FIG. 1 ). Figure 1 ) and HpTnpB-reRNA-ZJ (plasmid spectrum as shown Figure 1 When SCO5087 (actI) as described above is used as the endogenous gene target, the upstream homology arm sequence is shown in SEQ ID NO. 7, and the downstream homology arm sequence is shown in SEQ ID NO. 8. The constructed plasmids of the present invention were all subjected to Sanger sequencing to ensure complete accuracy.

[0042] Example 2: Application of the Streptomyces Efficient Mini-Editing System

[0043] 2.1 Conversion

[0044] The target plasmid was transformed into E. coli ET12567 / pUZ8002 (https: / / doi.org / 10.1016 / 0378-1119(92)90549-5) as follows: 200 ng of plasmid HpTnpB-reRNA-ZH or HpTnpB-reRNA-ZJ was added to 100 μL of thawed homemade competent cells E. coli ET12567 / pUZ8002, gently tap the tube wall to mix; after standing on ice for 30 minutes, in a 42°C water bath for 45 seconds, and then immediately place on ice for 2-3 minutes; then add antibiotic-free LB liquid medium, 200rpm, 37°C for 1 hour; then centrifuge at 5000rpm for 5 minutes, discard 900μL supernatant; resuspend the bacteria in the remaining medium, add to the LB solid plate containing 25μg / mL kanamycin, 12.5μg / mL chloramphenicol and 50μg / mL apramycin and gently spread it with a sterile coating stick; after overnight incubation at 37°C, pick a single colony and culture it in 20mL LB medium containing kanamycin (25μg / mL), chloramphenicol (12.5μg / mL) and apramycin (50μg / mL), and culture it at OD 600 When the p-value is about 0.4, collect the bacteria by centrifugation at 5000 rpm for 5 minutes; add 20 mL of antibiotic-free LB liquid medium, centrifuge at 5000 rpm for 5 minutes, discard the supernatant, repeat this step twice; finally, resuspend the bacteria in 2 mL of LB liquid medium.

[0045] 2.2. Binding transfer and resistance screening

[0046] Under non-inducing conditions, the plasmid in step 2.1 was transferred into Streptomyces coelicolorA3(2) by conjugation. The specific method was as follows: the previously collected Streptomyces spores were centrifuged at 5000 rpm for 5 minutes, the supernatant was discarded, and the bacteria were resuspended in 2 mL of 2×YT liquid medium. The centrifuge tube containing the spores was placed in a 50°C water bath for heat shock for 10 minutes, and then pre-germination was carried out in a shaker at 200 rpm and 30°C for 30 minutes; 500 μL of the E. coli culture collected in step 2.1 was placed in a 1.5 mL centrifuge tube containing 200 μL of Streptomyces spore suspension, and the mixture was vortexed and evenly spread on an MS plate; the MS plate was inverted and incubated in a 30°C incubator. After 18 hours, 1 mL of antibiotic premix (1 mg / mL apramycin and 1 mg / mL nalidixic acid) was added. After the surface was dry, the MS plate was inverted and incubated at 30°C until conjugates grew (about 5 days). At this point, the result is a successfully edited conjugate.

[0047] Example 3: Evaluation of gene editing efficiency

[0048] Single clones from step 2.2 were picked and streaked onto ISP2 solid medium supplemented with 25 μg / mL apramycin and 0.5 μg / mL thiostrepton. The cells were incubated inverted at 30°C until visible cells were observed. A small amount of cells were transferred to a PCR tube containing 20 μL DMSO, incubated at 100°C for 15 minutes, and then refrigerated at -20°C for 30 minutes. This step was repeated twice to fully lyse the cells and obtain a cell lysate. The target site fragment was then amplified by PCR using the Novozymes 2× Phanta Flash Master Mix (Cat. No. P510-01) kit. The primer design, PCR reaction system, and PCR procedure are shown in Table 1, Table 2, and Table 3, respectively.

[0049] Table 1 PCR amplification primers targeting SCO5087 (actI)

[0050] serial number Primers Sequence (5'-3') SEQ ID NO.9 SCO5087-LH-F atgattccggaactccggt SEQ ID NO.10 SCO5087-R accacagcttgcggaact

[0051] Table 2 PCR reaction system

[0052] system 15 μL <![CDATA[ddH2O]]> 5.25 μL Forward primer (10 μM, SCO5087-LH-F) 0.75μL Reverse primer (10 μM, SCO5087-R) 0.75μL 2×Phanta Flash Master Mix 7.50 μL Cell lysis buffer (containing DMSO) 0.75μL

[0053] Table 3 PCR reaction procedure

[0054]

[0055] The PCR amplification products were subjected to 1% agarose gel electrophoresis, and some of the results were as follows: Figure 3 As shown, Figure 3 1-21 represent different single clones randomly picked after E. coli containing HpTnpB-reRNA-ZJ plasmid was combined with S. coelicolor A3 (2) for transfer. If the plasmid HpTnpB-reRNA-ZH or HpTnpB-reRNA-ZJ containing the homologous recombination repair template successfully knocks out the target gene SCO5087 (actI), the theoretical PCR band size should be 1329 bp ( Figure 2 ). PCR results of randomly selected colonies grown in the solid resistance screening medium showed PCR products of the same size as the theoretical values, indicating that both HpTnpB-reRNA-ZH and HpTnpB-reRNA-ZJ plasmids can successfully knock out the target gene SCO5087 (actI) in the Streptomyces model strain S.ceolicolor A3(2); under the action of unoptimized guide RNA, the knockout efficiency of the plasmid system (HpTnpB-reRNA-ZH) for the target gene was 69.5%, while under the action of optimized guide RNA, the gene knockout efficiency of the plasmid system (HpTnpB-reRNA-ZJ) was as high as 100% ( Figure 4 ), indicating that after optimizing the guide RNA sequence, the knockout efficiency of the plasmid system is significantly improved. Homologous recombination is one of the main methods for achieving gene knockout and gene knock-in. Both use exogenous DNA fragments (i.e., upstream and downstream homologous arms of the target gene) and homologous sequences on the target genome to undergo a recombination reaction in vivo, thereby achieving the knockout or replacement of the target gene (i.e., inserting the exogenous gene into the position of the target gene, thereby achieving gene knock-in). Since both gene knock-in and gene knockout are based on the mechanism of homologous recombination, it is confirmed that the ultra-mini gene editor of the present invention is also suitable for knocking out / in other target genes.

[0056] The PCR product was then purified and recovered using the WeiZan Plasmid Purification Kit (Cat. No. DC201) and sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for Sanger sequencing analysis. The sequencing results further confirmed the gel electrophoresis results ( Figure 5 The above results fully demonstrate that the novel ultra-mini gene editor HpTnpB-reRNA-ZJ obtained in the present invention can effectively mediate Streptomyces genome editing.

[0057] Example 4: Various culture media involved in various implementations

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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. A guide RNA for guiding TnpB protein to move to a target sequence, characterized in that The guide RNA comprises an RNA backbone, a gene targeting segment and a gene sequence of hepatitis D virus ribozyme; the nucleotide sequence of the RNA backbone is SEQ ID NO.3 or SEQ ID NO.

6.

2. The guide RNA according to claim 1, characterized in that 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 5' TTGAT of the TAM sequence on the target gene.

3. A TnpB-mediated Streptomyces mini-gene editing system, characterized in that: The system comprises TnpB nuclease expressible in Streptomyces and the guide RNA according to claim 1 or 2.

4. The Streptomyces mini gene editing system according to claim 3, characterized in that The amino acid sequence of the TnpB nuclease is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.

2.

5. A recombinant expression plasmid vector, characterized in that: Used to express the Streptomyces mini gene editing system as described in claim 3 or 4.

6. A method for constructing a Streptomyces mini-gene editing system according to claim 3 or 4, characterized in that: The method comprises constructing a gene knockout / knockin plasmid containing an apramycin resistance selection marker; the gene knockout / knockin plasmid is based on an Escherichia coli-Streptomyces shuttle plasmid, and the TnpB nuclease and its guide RNA that can be expressed in Streptomyces and a gene knockout / knockin box are inserted.

7. The construction method according to claim 6, characterized in that: The gene knockout cassette includes the following core elements in the direction of the gene editing target gene: the upstream homologous arm of the target gene and the downstream homologous arm of the target gene; the upstream and downstream homologous sequences of the target gene are used to provide a homologous recombination template when gene editing occurs.

8. The construction method according to claim 6, characterized in that: The gene knock-in cassette includes the following core elements in the direction of the gene editing target gene: upstream homology arm of the target gene, inserted gene, and downstream homology arm of the target gene.

9. An application of the Streptomyces mini-gene editing system as described in claim 3 or 4 in the precise editing of the Streptomyces genome based on double-strand break and directed repair.

10. A method for gene editing a target gene in a recipient bacterium Streptomyces using the Streptomyces mini gene editing system according to claim 3 or 4, characterized in that: The method comprises the following steps: S1. Under non-inducing conditions, the gene knockout / in plasmid constructed by the method of claim 6 is transformed into the target host, and the gene knockout / in is achieved by homologous exchange of the homologous arm sequence of the gene knockout / in cassette with the upstream and downstream homologous sequences of the target gene. Transformants carrying the gene knockout / in plasmid are obtained by screening for antibiotics associated with the plasmid resistance; S2. The transformant is streaked onto a culture medium containing a plasmid resistance antibiotic and a promoter inducer, and cultured at a constant temperature until a single colony is visible; S3. Randomly select single clones from step S2 and verify by colony PCR to obtain a traceless gene knockout / in strain.

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