Streptomyces mini-gene editing system based on coupled TnpB and reconstructed NHEJ as well as construction method and application of streptomyces mini-gene editing system

Abstract

The invention discloses a streptomyces mini-gene editing system based on coupled TnpB and reconstructed NHEJ as well as a construction method and application of the streptomyces mini-gene editing system. The system comprises TnpB nuclease which can be expressed in prokaryotic microorganisms through codon optimization, a reRNA guide box of the TnpB nuclease, and a LigD gene expression box, the prokaryotic microorganism itself does not have NHEJ or has a defective NHEJ pathway (such as streptomyces); the reRNA guide box is used for guiding TnpB protein to move towards a target sequence; and the LigD gene expression cassette is used for expressing ligase LigD and reconstructing the prokaryotic microorganism DNA repair system NHEJ. According to the system disclosed by the invention, small InDels can be efficiently introduced near a target site under the action of a reconstructed streptomyces repair system, so that editing of a target gene is realized, and a gene large fragment deletion phenomenon introduced by self-defect repair is avoided.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology and relates to a Streptomyces mini-gene editing system based on coupling TnpB and reconstructed NHEJ, as well as a construction method and application thereof; in particular, it relates to a Streptomyces mini-gene editing tool based on coupling transposition-associated nuclease TnpB and reconstructed non-homologous end joining (NHEJ) system, as well as a construction method and application thereof. Background Art

[0002] Streptomyces is the largest genus in the Actinobacteria. 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 current mainstream idea of ​​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 operations 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 1200, which means that the number of nucleotides (pairs) encoding this effector protein is greater than 3600. 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] 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 and applying novel gene-editing techniques, such as base editing and prime editing. Furthermore, researchers are exploring and developing compact Cas proteins to develop novel CRISPR gene-editing 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 endogenous gene editing in multiple species. However, compared to Cas9, TnpB and other reported compact gene-editing tools generally suffer from low editing efficiency. More importantly, existing mini-gene editors based on TnpB have not yet been reported for editing in Streptomyces. Summary of the Invention

[0005] The purpose of the present invention is to address the current situation of the lack of mini gene editing tools in Streptomyces and the general low efficiency of current mini gene editing tools. The present invention deeply analyzes the core elements and their mechanism of action of the small-volume, programmable TnpB nuclease for gene editing, designs and repairs the defective DNA DSB (Double-strandedbreak, DSB) repair system of Streptomyces, systematically develops an efficient mini gene editing system suitable for Streptomyces, and then inactivates the target gene, thereby providing a Streptomyces mini gene editing system based on coupling TnpB and reconstructing NHEJ, as well as its construction method and application.

[0006] The present invention guides the TnpB nuclease to the target gene site and cuts double-stranded DNA through a guide RNA box, and then couples the reconstructed Streptomyces DNA double-strand break (DSB) repair system to repair DNA double-strand breaks through non-homologous end joining. In Streptomyces, a gene editing event similar to that of eukaryotic cells is achieved by introducing small InDels (Insertion and Delations) around the DSB site to cause the target gene to produce a frameshift mutation, thereby inactivating the target gene. The present invention can efficiently achieve efficient in vivo genome editing of prokaryotic microorganisms that do not have NHEJ or have defective NHEJ pathways, such as the vast majority of Streptomyces, and has great promotion and application value.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] In the first aspect, the present invention provides a prokaryotic microbial mini gene editing system based on coupling TnpB and reconstructing the NHEJ repair system; the system comprises: a codon-optimized TnpB nuclease and its reRNA guide box, and a LigD gene expression box that can be expressed in a prokaryotic microorganism; the prokaryotic microorganism itself does not have NHEJ or has a defective NHEJ pathway; the reRNA guide box is used to guide the TnpB protein to move to the target sequence; the LigD gene expression box is used to express the ligase ligD, thereby reconstructing the prokaryotic microbial DNA repair system NHEJ.

[0009] As an embodiment of the present invention, the reRNA guide box includes the following core elements in the direction from 5' to 3': a guide RNA base sequence of the TnpB protein, a base sequence that can target a target DNA fragment (eg, a protospacer sequence), and a hepatitis delta virus (HDV) ribozyme.

[0010] As an embodiment of the present invention, the LigD gene expression cassette includes the following core elements suitable for the prokaryotic microorganism in the direction from 5' to 3': promoter ermE*, ligase encoding gene ScaligD and terminator t0.

[0011] As one embodiment of the present invention, the prokaryotic microorganism includes Streptomyces.

[0012] As one embodiment of the present invention, the amino acid sequence of the TnpB nuclease is shown in SEQ ID NO. 1, and the gene sequence is shown in SEQ ID NO. 2.

[0013] As one embodiment of the present invention, the base sequence of the guide RNA of the TnpB protein in the reRNA guide box is shown as SEQ ID NO.3.

[0014] As one embodiment of the present invention, the base sequence of the target DNA fragment is a nucleic acid fragment with a length of 12-40 bp following the TAM sequence (5'TTGAT) on the target gene. In some implementation examples, the key gene SCO5087 (actI) for the synthesis of actinomycetoma in the model strain Streptomycin coelicolor A3 (2) is selected as the endogenous gene target, and the base sequence of the target DNA fragment in the reRNA guide box corresponding to the target is 5'-gtagtcgatgtccgtcgcgt.

[0015] As an embodiment of the present invention, the gene sequence of the hepatitis delta virus (HDV) ribozyme is shown as SEQ ID NO.4.

[0016] As one embodiment of the present invention, the sequence of the LigD gene expression cassette is shown in SEQ ID NO. 5. After the addition of the LigD gene expression cassette, the editing efficiency of TnpB is improved, and the accuracy is also improved (no large fragment deletion).

[0017] In a second aspect, the present invention provides a recombinant expression plasmid vector, wherein the recombinant plasmid vector expresses the gene editing system;

[0018] In a third aspect, the present invention provides a method for constructing an efficient mini gene editing system for Streptomyces, the method comprising: constructing a gene editing plasmid containing a plasmid-related resistance (ampramycin resistance) screening marker; the plasmid is based on the Escherichia coli-Streptomyces shuttle plasmid, and the codon-optimized TnpB nuclease and its reRNA guide box, and the LigD gene expression box are respectively inserted.

[0019] In a fourth aspect, the present invention provides a method for gene editing a target gene in a recipient bacterium, Streptomyces, using the above-mentioned gene editing system, the method comprising:

[0020] S1. Based on the E. coli-Streptomyces shuttle plasmid, the codon-optimized TnpB nuclease and its reRNA guide cassette, and the LigD gene expression cassette were inserted respectively; a gene editing plasmid containing a plasmid-associated resistance (ampramycin resistance) selection marker was constructed;

[0021] S2. Under non-inducing conditions, the gene-editing plasmid is transformed into the target host, and reRNA is used to guide TnpB to generate a DSB in the target gene. The DSB is then coupled with a reconstructed NHEJ repair system for error-prone repair, resulting in gene deletion or gene knock-in or replacement events near the DSB site of the target gene, causing a frameshift mutation in the target gene, thereby inactivating the target gene; transformants carrying the gene knock-out / knock-in plasmid are obtained through antibiotic screening for plasmid-related resistance;

[0022] S3. The transformants are streaked onto a culture medium plate containing plasmid resistance antibiotics and a promoter inducer (such as thiostrepton), and cultured at a constant temperature until single colonies are visible; the single colonies are randomly selected, and colony PCR is used to verify the strain with target gene mutation.

[0023] Compared with the existing technology, the present invention provides a Streptomyces mini gene editing system based on a reconstruction repair system, as well as its construction method and application. Under the action of the reconstruction Streptomyces repair system, the system can efficiently introduce small InDels near the target site to achieve editing of the target gene, avoiding the large gene fragment deletion phenomenon introduced by its own defective repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] 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:

[0025] Figure 1 is the pTnpB-reRNA and pTnpB-reRNA-ligD plasmid spectrum;

[0026] Figure 2 The figure shows the results of editing the endogenous gene of Streptomyces by pTnpB-reRNA and pTnpB-reRNA-ligD (DNA electrophoresis diagram);

[0027] Figure 3 Comparison of the editing efficiency of endogenous genes in Streptomyces fungi between pTnpB-reRNA and pTnpB-reRNA-ligD;

[0028] Figure 4 A diagram showing the sequencing results. DETAILED DESCRIPTION

[0029] 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.

[0030] Example 1: Construction of an efficient mini gene editing tool in Streptomyces

[0031] 1.1 Plasmid design and construction

[0032] The amino acid sequence of TnpB used in the experiment is shown in SEQ ID NO. 1, and the gene sequence is shown in SEQ ID NO. 2. Both were synthesized by GenScript Biotech Co., Ltd. This sequence is codon-optimized for the TnpB nuclease from Deinococcus radiodurans ISDra2 based on the codon preference of Streptomyces. It shares 79.74% DNA similarity with the TnpB nuclease from Deinococcus radiodurans ISDra2.

[0033] The reRNA guide box for guiding 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 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.

[0034] The LigD gene expression cassette (SEQ ID NO. 5) is used to repair the defective non-homologous end joining pathway in Streptomyces. This expression cassette includes the following core elements suitable for Streptomyces, in the 5' to 3' direction: the promoter ermE* (SEQ ID NO. 6), the ligase ScaligD (SEQ ID NO. 7), and the terminator t0 (SEQ ID NO. 8). The LigD gene expression cassette used in the examples was synthesized by GenScript Biotech Co., Ltd.

[0035] 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) to obtain the plasmid pTnpB-reRNA (as Figure 1 ). The LigD gene expression cassette was then inserted into the plasmid pTnpB-reRNA to obtain the plasmid pTnpB-reRNA-ligD (eg Figure 1 ).

[0036] The plasmids constructed in the present invention were subjected to Sanger sequencing to ensure that the sequences were completely correct.

[0037] Example 2: Application of the Streptomyces Efficient Ultra-mini Editing System

[0038] 2.1 Conversion

[0039] 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 pTnpB-reRNA or pTnpB-reRNA-ligD 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 The cells were collected by centrifugation at 5000 rpm for 5 minutes at a concentration of about 0.4; 20 mL of antibiotic-free LB liquid medium was added, and the cells were centrifuged at 5000 rpm for 5 minutes, and the supernatant was discarded. This step was repeated twice; finally, the cells were resuspended in 2 mL of LB liquid medium.

[0040] 2.2 Binding transfer and resistance screening

[0041] 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 the 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.

[0042] Example 3: Evaluation of gene editing efficiency

[0043] Single clones from step 2.2 were randomly picked and streaked onto ISP2 solid medium supplemented with 25 μg / mL apramycin and 0.5 μg / mL thiostrepton. The cells were inverted and cultured at 30°C until visible bacteria were observed. A small amount of bacteria was 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 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 is shown in Table 1, the PCR reaction system is shown in Table 2, and the PCR reaction procedure is shown in Table 3.

[0044] Table 1 PCR amplification primers targeting act

[0045] serial number Primers Sequence (5'-3') SEQ ID NO.9 SCO5087-LH-F atgattccggaactccggt SEQ ID NO.10 SCO5087-R accacagcttgcggaact

[0046] Table 2 PCR reaction system

[0047] 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

[0048] Table 3 PCR reaction program

[0049]

[0050]

[0051] The PCR amplification products were subjected to 1% agarose gel electrophoresis. Figure 2 As shown, WT is a strain of S. coelicolor A3 (2) without any transformation, 1-4 represent different single clones randomly picked after E. coli containing pTnpB-reRNA plasmid was combined with S. coelicolor A3 (2) and transferred; 5-8 represent different single clones randomly picked after E. coli containing pTnpB-reRNA-ligD plasmid was combined with S. coelicolor A3 (2) and transferred. The above PCR products were 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 single clones that did not obtain PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for whole genome resequencing analysis. The results showed that the editing efficiency of the target gene by the TnpB-reRNA gene editing system combined with the reconstructed DNA repair system (i.e., pTnpB-ZH-ligD, which completes the defective DNA repair pathway through the LigD gene expression cassette) is about twice that of the system combined with the Streptomyces' own defective DNA repair system (i.e., pTnpB-ZH). Figure 3); Sanger sequencing and whole genome resequencing analysis revealed that under the reconstructed DNA repair system, small InDels (Insertion and Deletions) can be introduced near the target site (such as Figure 4 ). The above results fully demonstrate that the Streptomyces mini gene editing tool based on the reconstruction repair system obtained in the present invention can achieve editing of the target gene by introducing small InDels near the target site.

[0052] Example 4: Inactivation of target genes using a Streptomyces mini-gene editing tool based on coupled TnpB and reconstructed NHEJ repair system

[0053] Under non-inducing conditions, pTnpB-ZH-ligD 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 containing plasmid-resistant antibiotics (50 ng / mL apramycin) and thiostrepton (0.5 μg / mL), inverted, cultured at 30°C, and pigment secretion was observed. In the embodiment of the present invention, SCO5087 (actI), a key gene 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 shown in Example 3. The results of the inactivation of target genes by the Streptomyces mini gene editing tool based on the reconstruction repair system are shown in Figure 4 After culturing the target gene mutation strain on the above-mentioned resistance plate for 2 weeks, no blue pigment was secreted, further indicating that the Streptomyces mini-editing system combined with the reconstructed Streptomyces repair system 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.

[0054] Example 5. Various culture media involved in each embodiment

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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 prokaryotic microbial mini-gene editing system based on coupling TnpB and reconstructing the NHEJ repair system; the system comprises: TnpB nuclease and its reRNA guide cassette, and LigD gene expression cassette that have been codon-optimized for expression in prokaryotes; The prokaryotic microorganism itself does not have NHEJ or has a defective NHEJ pathway; the reRNA guide box is used to guide the TnpB protein to move to the target sequence; the LigD gene expression box is used to express the ligase ligD, reconstructing the prokaryotic microorganism DNA repair system NHEJ.

2. The mini gene editing system according to claim 1, characterized in that The reRNA guide box includes the following core elements in sequence from the 5' end to the 3' end: a guide RNA base sequence of the TnpB protein, a base sequence that can target a target DNA fragment, and a hepatitis D virus ribozyme.

3. The mini gene editing system according to claim 1, characterized in that The LigD gene expression cassette includes the following core elements suitable for the prokaryotic microorganism in the direction from 5' to 3': promoter ermE*, ligase encoding gene ScaligD and terminator t0.

4. The mini gene editing system according to any one of claims 1 to 3, characterized in that The prokaryotic microorganisms include Streptomyces.

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

2.

6. The mini gene editing system according to claim 4, characterized in that: The guide RNA base sequence of the TnpB protein in the reRNA guide box is shown in SEQ ID NO.3; the base sequence of the target DNA fragment is a nucleic acid fragment with a length of 12-40bp after the TAM sequence 5'TTGAT on the target gene; the gene sequence of the hepatitis D virus ribozyme is shown in SEQ ID NO.

4.

7. The mini gene editing system according to claim 4, characterized in that: The LigD gene expression cassette sequence is shown in SEQ ID NO.

5.

8. A recombinant expression plasmid vector, characterized in that: The recombinant plasmid vector expresses the mini gene editing system according to any one of claims 1 to 7.

9. A method for constructing a mini gene editing system according to any one of claims 1 to 7, characterized in that: The method comprises: constructing a gene editing plasmid containing a plasmid-related resistance screening marker; the plasmid is based on an Escherichia coli-Streptomyces shuttle plasmid, and a codon-optimized TnpB nuclease and its reRNA guide box and a LigD gene expression box are respectively inserted.

10. A method for gene editing a target gene in a recipient bacterium Streptomyces using the mini gene editing system according to any one of claims 1 to 7, characterized in that: The method comprises: S1. Based on the E. coli-Streptomyces shuttle plasmid, the codon-optimized TnpB nuclease and its reRNA guide cassette, and the LigD gene expression cassette were inserted respectively to construct a gene editing plasmid containing a plasmid-related resistance selection marker; S2. Under non-inducing conditions, the gene-editing plasmid is transformed into the target host, and reRNA is used to guide TnpB to generate a DSB in the target gene. The DSB is then coupled with a reconstructed NHEJ repair system for error-prone repair, resulting in gene deletion or gene knock-in or replacement events near the DSB site of the target gene, causing a frameshift mutation in the target gene, thereby inactivating the target gene; transformants carrying the gene knock-out / knock-in plasmid are obtained through antibiotic screening for plasmid-related resistance; S3. The transformants are streaked onto a culture medium plate containing plasmid resistance antibiotics and promoter inducers, and cultured at a constant temperature until single colonies are visible; the single colonies are randomly selected, and colony PCR is performed to verify the target gene mutation strain.

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