CN110066829B - CRISPR/Cas9 gene editing system and application thereof - Google Patents

Abstract

The invention discloses a CRISPR/Cas9 gene editing system and an application thereof, belonging to the technical fields of genetic engineering and bioengineering. This system can achieve the purpose of gene editing of Mycobacterium aureus; the pML‑Cas9 plasmid and pJM‑sgRNA plasmid used in this system can stably exist and express in Mycobacterium The passage of mycobacteria is lost, and it will not be rejected by the protection mechanism of M. aureus itself; the CRISPR/Cas9 gene editing system of this system is used to edit the genes of M. aureus, and the knockout efficiency is high. Up to 50%.

Description

A kind of CRISPR/Cas9 gene editing system and its application

technical field

The invention relates to a CRISPR/Cas9 gene editing system and an application thereof, belonging to the technical fields of genetic engineering and bioengineering.

Background technique

Steroid drugs belong to hormone drugs, which are endogenous substances discovered in the study of mammalian endocrine system. aspects play an important role. These effects make steroid drugs become the second largest class of drugs after antibiotics in the chemical industry, and also make steroid drugs become one of the key points in the development of new drug resources in my country, and make steroid drugs and steroid drugs intermediate The body has become an important variety of my country's drug export.

However, due to the fact that there is still a certain gap between our country and the advanced countries in the world in terms of the production level of steroid drugs, the types of steroid drugs available in my country are only one-third of the steroid drugs that have been marketed abroad, and most of them are Middle and low-end products have lower profits. Therefore, there is an urgent need to improve the production level of steroid drugs in my country.

Androst-4-ene-3,17-dione (AD) and androst-1,4-diene-3,17-dione (ADD) are important steroid drug intermediates, which can be Modification and synthesis of almost all steroidal drugs plays an important role in the production of steroidal drugs. However, due to problems such as low substrate concentration, long fermentation conversion cycle, and insufficient knowledge of steroidal microbial metabolic mechanisms, my country Steroid microbial transformation production also lags behind the advanced countries in the world. Therefore, it is urgent to improve the production level of steroidal drug intermediate ADD in my country, and then improve the production level of steroidal drugs in my country.

Mycobacterium neoaurum can transform phytosterols into androst-4-ene-3,17-dione (AD) and androst-1,4-diene-3,17-dione (ADD), And has the advantage of fast growth, is a kind of ADD production bacterium with great potential, if can transform 3-sterone-Δ1-dehydrogenase, cholesterol oxidase, sterol in the process of synthetic ADD of Mycobacterium aureus Key enzymes such as ketone C27 monooxygenase and 3-sterone-9α-hydroxylase were studied, combined with metabolic engineering strategies to modify the ADD synthesis-related pathways of Mycobacterium neoaureus, it is very likely to obtain ADD that can efficiently accumulate The modified Mycobacterium aureus is undoubtedly an important breakthrough in improving the production level of steroidal drug intermediate ADD in China, and it also provides more possibilities for improving the production level of steroidal drugs in China.

However, due to the strong exclusiveness of the protective mechanism of M. aureus itself, it is difficult for foreign genes to stably exist and express in M. aureus, which makes it applicable to the gene editing method of M. aureus Less, for example, because the incidence of homologous recombination in mycobacteria is significantly lower than other bacteria, only 10 ^-6 ~ 10 ^-5 , and the gene editing of M. A resistance tag is brought into the genome. Therefore, the use of homologous recombination to edit the genes of Mycobacterium aureus has disadvantages such as low efficiency, long cycle, large amount of screening, and complicated operation; the suicide vector system (pNIL/pGOAL series plasmids), etc. Although other gene editing methods can perform gene editing on Mycobacterium neoaureus, their efficiency is not high, and they do not have any advantages over homologous recombination.

Therefore, gene editing is undoubtedly a difficulty in the metabolic engineering of M. aureus. It is urgent to improve the gene editing system for M. aureus in order to transform M. aureus more efficiently.

Contents of the invention

[technical problem]

The technical problem to be solved by the present invention is to provide a highly targeted and efficient gene editing system for Mycobacterium aureus.

[Technical solutions]

In order to solve the above problems, the present invention provides a CRISPR/Cas9 gene editing system, the CRISPR/Cas9 gene editing system comprises a pML-Cas9 plasmid and a pJM-sgRNA plasmid;

The pML-Cas9 plasmid comprises a pMV261 expression vector, a Cas9 gene, a Pj23119 promoter and an NHEJ repair gene; the Hsp60 promoter on the pMV261 expression vector is replaced with a Tac promoter; the NHEJ repair gene comprises an encoding DNA end binding protein The gene of mku and the gene encoding DNA ligase LigD; the Cas9 gene is located downstream of the Tac promoter of the pMV261 expression vector and upstream of the rrnB terminator of the pMV261 expression vector, and is expressed by the Tac promoter; the Pj23119 promoter is located in pMV261 Downstream of the KanR resistance gene of the expression vector; the NHEJ repair gene is located downstream of the Pj23119 promoter and upstream of the origin of replication (ori) of the pMV261 expression vector, driven by the Pj23119 promoter for expression;

The pJM-sgRNA plasmid sequentially comprises OriM replicon, pMB1 replicon, Pj23119 promoter, sgRNA sequence, rrnB terminator and aadA resistance gene, named as pJM-sgRNA plasmid; the sgRNA sequence is a targeting sequence, which can specifically Targeting target sequence; the target sequence refers to the nucleotide sequence of the gene to be edited.

In one embodiment of the present invention, the nucleotide sequence of the Cas9 gene is shown in SEQ ID NO:1.

In one embodiment of the present invention, the nucleotide sequence of the gene encoding the Pj23119 promoter in the pML-Cas9 plasmid and the pJM-sgRNA plasmid is shown in SEQ ID NO:2.

In one embodiment of the present invention, the nucleotide sequence of the gene encoding the DNA terminal collection protein mku is shown in SEQ ID NO:3.

In one embodiment of the present invention, the nucleotide sequence of the gene encoding the DNA ligase LigD is shown in SEQ ID NO:4.

In one embodiment of the present invention, the nucleotide sequence of the gene encoding the Tac promoter is shown in SEQ ID NO:5.

In one embodiment of the present invention, the nucleotide sequence of the gene encoding the OriM replicon is shown in SEQ ID NO:6.

In one embodiment of the present invention, the nucleotide sequence of the gene encoding the pMB1 replicon is shown in SEQ ID NO:7.

In one embodiment of the present invention, the nucleotide sequence of the gene encoding the rrnB terminator is shown in SEQ ID NO:8.

In one embodiment of the present invention, the nucleotide sequence of the aadA resistance gene is shown in SEQ ID NO:9.

The present invention also provides a gene editing method that can be used in Mycobacterium aureus, and the method uses the above-mentioned CRISPR/Cas9 gene editing system.

In one embodiment of the present invention, the method is to first introduce the pML-Cas9 plasmid in the above-mentioned CRISPR/Cas9 gene editing system into Mycobacterium aureus, so that the Cas9 gene on the pML-Cas9 plasmid, encoding The gene of DNA end-binding protein mku and the gene encoding DNA ligase LigD are expressed, and then the pJM-sgRNA plasmid in the above-mentioned CRISPR/Cas9 gene editing system is introduced into Mycobacterium aureus, so that the pJM-sgRNA plasmid is transcribed to generate sgRNA sequence, the Cas9 handle and Terminator in the sgRNA sequence will form a stem-loop structure, and the Cas9 gene will recognize this stem-loop structure to combine with the sgRNA sequence to form a complex, which will target the gene to be edited and anchor to the gene to be edited , after anchoring, the Cas9 gene will play an active role in cutting the gene to be edited so that the gene to be edited in M. The expressed DNA end-binding protein mku and DNA ligase LigD on the pML-Cas9 plasmid will repair the DSB break and complete the gene editing process of the new Mycobacterium aureus.

The present invention also provides a plasmid, the plasmid comprising pMV261 expression vector, Cas9 gene, Pj23119 promoter and NHEJ repair gene, named as pML-Cas9 plasmid; the Hsp60 promoter on the pMV261 expression vector is replaced by Tac promoter The NHEJ repair gene comprises the gene encoding DNA end-binding protein mku and the gene encoding DNA ligase LigD; the Cas9 gene is located downstream of the Tac promoter of the pMV261 expression vector and upstream of the rrnB terminator of the pMV261 expression vector, by Tac promoter drives expression; the Pj23119 promoter is positioned at the downstream of the KanR resistance gene of the pMV261 expression vector; the NHEJ repair gene is positioned at the downstream of the Pj23119 promoter and the upstream of the replication origin (ori) of the pMV261 expression vector, initiated by Pj23119 sub-driver expression.

In one embodiment of the present invention, the nucleotide sequence of the Cas9 gene is shown in SEQ ID NO:1.

In one embodiment of the present invention, the nucleotide sequence of the gene encoding the Pj23119 promoter is shown in SEQ ID NO:2.

In one embodiment of the present invention, the nucleotide sequence of the gene encoding the DNA terminal collection protein mku is shown in SEQ ID NO:3.

In one embodiment of the present invention, the nucleotide sequence of the gene encoding the DNA ligase LigD is shown in SEQ ID NO:4.

In one embodiment of the present invention, the nucleotide sequence of the gene encoding the Tac promoter is shown in SEQ ID NO:5.

The present invention also provides a plasmid, which comprises OriM replicon, pMB1 replicon, Pj23119 promoter, sgRNA sequence, rrnB terminator and aadA resistance gene in sequence, named pJM-sgRNA plasmid; the sgRNA sequence is The targeting sequence can specifically target the target sequence; the target sequence refers to the nucleotide sequence of the gene to be edited.

In one embodiment of the present invention, the nucleotide sequence of the gene encoding the OriM replicon is shown in SEQ ID NO:6.

In one embodiment of the present invention, the nucleotide sequence of the gene encoding the pMB1 replicon is shown in SEQ ID NO:7.

In one embodiment of the present invention, the nucleotide sequence of the gene encoding the Pj23119 promoter is shown in SEQ ID NO:2.

In one embodiment of the present invention, the nucleotide sequence of the gene encoding the rrnB terminator is shown in SEQ ID NO:8.

In one embodiment of the present invention, the nucleotide sequence of the aadA resistance gene is shown in SEQ ID NO:9.

The present invention also provides the above-mentioned CRISPR/Cas9 gene editing system or the above-mentioned gene editing method that can be used for Mycobacterium aureus, or the above-mentioned pML-Cas9 plasmid or the above-mentioned pJM-sgRNA plasmid in editing the new Golden Mycobacterium. Applications of mycobacterial genes.

[beneficial effect]

(1) The present invention provides a dual-plasmid-based CRISPR/Cas9 gene editing system, which can achieve the purpose of gene editing of Mycobacterium neogolden, and its working mechanism is as follows: first, the pML-Cas9 plasmid is introduced into the new In mycobacteria, the Cas9 gene on the pML-Cas9 plasmid, the gene encoding the DNA end-binding protein mku, and the gene encoding the DNA ligase LigD are expressed, and then the pJM-sgRNA plasmid is introduced into the new Mycobacterium aureus, so that the pJM- The sgRNA plasmid is transcribed to generate the sgRNA sequence. The Cas9 handle and Terminator in the sgRNA sequence will form a stem-loop structure. The Cas9 gene will recognize the stem-loop structure to combine with the sgRNA sequence to form a complex. This complex will target the gene to be edited and anchor On the gene to be edited, after anchoring, the Cas9 gene will play an active role in cutting the gene to be edited so that the gene to be edited in M. At this time, the expressed DNA end-binding protein mku and DNA ligase LigD on the pML-Cas9 plasmid will repair the DSB break and complete the editing process of the new Mycobacterium aureus gene;

(2) The pML-Cas9 plasmid and pJM-sgRNA plasmid used in the CRISPR/Cas9 gene editing system of the present invention can stably exist and express in Mycobacterium aureus, and will not be lost along with the passage of Mycobacterium aureus, Nor will it be rejected by the protective mechanism of Mycobacterium neoaureus itself;

(3) Using the CRISPR/Cas9 gene editing system of the present invention to edit the genes of Mycobacterium aureus, the knockout efficiency is high, up to 50%.

Description of drawings

Figure 1: Plasmid map of pML-Cas9.

Figure 2: PJM-sgRNA plasmid map.

Detailed ways

The present invention is further defined below in conjunction with specific examples.

The pMD18T vector, pMV261 expression vector, and ptrc99A expression vector involved in the following examples were purchased from Youbao Biology; the ClonExpress II One Step Cloning Kit involved in the following examples was purchased from Nanjing Nuoweizan Biotechnology Co., Ltd.; the following implementation The Mycobacterium neoaurum involved in the example was purchased from Beina Biology, and the resource number is ATCC 25795.

The medium involved in the following examples is as follows:

LB liquid medium: glucose 10g/L, yeast powder 5g/L, NaCl 10g/L.

Slope/solid medium: glucose 10g/L, tryptone 6g/L, yeast powder 5g/L, NaCl 10g/L, agar powder 20g/L, pH 7.5.

Seed medium: glucose 20g/L, tryptone 10g/L, yeast powder 6g/L, NaCl 10g/L, pH 7.5.

Fermentation medium: phytosterol 1g/L, glucose 20g/L, tryptone 10g/L, yeast powder 6g/L, NaCl 10g/L, K ₂ HPO ₄ 3g/L, MgSO ₄ 0.3g/L, MnCl ₂ 5× 10 ^-4 g/L, β-cyclodextrin 3g/L, pH 7.5.

Competent medium: glucose 20g/L, tryptone 10g/L, yeast powder 6g/L, NaCl 10g/L, Tween-800.2% (V/V), pH 7.5.

The detection methods involved in the following examples are as follows:

3-sterone-Δ1-dehydrogenase detection method:

The most suitable KSDD assay system (3mL) contains: crude enzyme solution (100μL), Tris-HC1 (pH 7.0) at a final concentration of 50mM, DCPIP at a final concentration of 40μM, PMS at a final concentration of 1.5mM, and steroidal substrate male at a final concentration of 500μM. Ste-4-ene-3,17-dione (AD) (dissolved in 100% isopropanol); react at 30°C for 2 hours, and detect the change in the absorbance value of the obtained reaction solution at 600nm;

The enzyme activity of 3-sterone-Δ1-dehydrogenase is defined as: the amount of enzyme required to reduce 1 μmol DCPIP in 1 min is defined as an enzyme activity unit, and the unit is U.

Detection method of gene knockout efficiency:

The formula for calculating the gene knockout efficiency is as follows: gene knockout efficiency=(number of colonies with successful knockout verification/total number of picked transformants)×100%.

The preparation method of neomycobacterium aureus JC-12 competent cell is as follows:

Inoculate the Mycobacterium neoaureus JC-12 in the frozen tube into 10mL LB liquid medium, and culture it on a shaker at 30°C for 48 hours to obtain the seed liquid; transfer the seed liquid to 50mL competent medium at an inoculum size of 1%. cultured on a shaker at 30°C for 4-6 hours to obtain the bacterial solution; put the bacterial solution, 10% glycerin, dry and sterile electric shock cup and sterilized 50mL centrifuge tube on ice for 30 minutes; Put the pre-cooled bacterial solution into a centrifuge tube, centrifuge at 8000rpm at 4°C for 5min, discard the supernatant, add 10mL of 10% glycerol and suspend the bacteria, centrifuge at 8000rpm at 4°C for 5min, repeat the previous step, discard Remove the supernatant, add 2mL of 10% glycerol and suspend the bacteria, and divide into EP tubes, 80 μL per tube, to obtain JC-12 competent cells.

The transformation method of Mycobacterium aureus JC-12 is as follows:

Add the plasmid to be transformed into the JC-12 competent cells in a sterile ultra-clean workbench (double-distilled sterile water), mix well, and place on ice for 30 minutes; after 30 minutes, transfer all the competent cells to the pre-cooled In the electric shock cup, quickly dry the outer wall of the cup, place it in the electroporator, 2200V, 5ms electric shock transformation; after the electric shock, add 1mL seed medium to the electric shock cup, mix well, transfer all to the EP tube, shake at 30°C The bed was recovered for 4-6 hours; after recovery, the cells were collected by centrifugation, and part of the supernatant was removed, spread on the slant/solid medium containing resistance, and cultured upside down in a 30°C incubator for 5-7 days to obtain the transformed new golden fraction. Mycobacterium JC-12.

Example 1: Construction and use of CRISPR/Cas9 gene editing system

Specific steps are as follows:

1. Construction of CRISPR/Cas9 gene editing system

(1) Replace the Hsp60 promoter on the pMV261 expression vector with the Tac promoter by restriction enzymes Xba I and BamH I and T4 ligase, and carry out digestion of the pMV261 expression vector by restriction enzymes XbaI and HindIII Linearization to obtain the basic skeleton;

(2) obtain the Cas9 gene (nucleotide sequence as shown in SEQ ID NO: 1) by chemical synthesis, namely Frag1;

(3) the gene (nucleotide sequence shown in SEQ ID NO: 3) and the gene (nucleotide sequence shown in SEQ ID NO: 4) of encoding DNA ligase LigD are obtained by chemical synthesis ; The gene encoding DNA end assembly protein mku obtained by chemical synthesis and the gene encoding DNA ligase LigD were used as templates, respectively using P1 primers, P2 primers, P3 primers, and P4 primers in Table 1 to clone to obtain linearized encoding The gene encoding DNA end assembly protein mku and the gene encoding DNA ligase LigD; the obtained linearized gene encoding DNA end assembly protein mku and the gene encoding DNA ligase LigD are fused by PCR with P1 and P4 primers to obtain the fusion fragment NHEJ Repair the gene; connect the obtained NHEJ repair gene to the pMD18T vector through ClonExpress II One Step Cloning KitC112 to obtain the recombinant pMD18T-LigD::mku vector; use the obtained recombinant pMD18T-LigD::mku vector as a template, use the The P5 primer and P6 primer were used for cloning to obtain the Pj23119-LigD::mku expression framework, namely Frag2;

(4) Assemble the basic skeleton obtained in (1) and the Frag1 obtained in (2) through ClonExpress II One StepCloning KitC112 (see Table 3 for the Gibson assembly ligation reaction system) to obtain a circular vector; The obtained circular vector was digested and linearized to obtain a linear vector; the obtained linear vector was assembled with Frag2 obtained in (3) through ClonExpress II One Step Cloning KitC112 (see Table 3 for the Gibson assembly ligation reaction system) to obtain pML-Cas9 Plasmid (see Figure 1 for the plasmid map), wherein, the Cas9 gene is located downstream of the Hsp60 promoter of the pMV261 expression vector and upstream of the rrnB terminator of the pMV261 expression vector, and is expressed by the Hsp60 promoter; the Pj23119 promoter is located at the KanR anti- The downstream of the sex gene; the NHEJ repair gene is located downstream of the Pj23119 promoter and upstream of the origin of replication (ori) of the pMV261 expression vector, and is expressed by the Pj23119 promoter;

(5) Using the pMV261 expression vector as a template, use the P9 primer and P10 primer in Table 1 to clone to obtain the OriM replicon, namely Frag3;

(6) Using the ptrc99A vector as a template, use the P7 primer and P8 primer in Table 1 to clone to obtain the pMB1 replicon and aadA resistance gene, namely Frag4;

(7) Using the sequence of the gene to be edited in Mycobacterium aureus as the target sequence, design a targeting sequence that can specifically target the target sequence, and obtain the sgRNA sequence; the sgRNA sequence was synthesized by Jinweizhi Biotechnology Company and connected to the pMD18T vector Above, obtain the recombinant pMD18T-sgRNA vector; use the obtained recombinant pMD18T-sgRNA vector as a template, use the P11 primer and P12 primer in Table 1 to clone, and obtain the Pj23119-sgRNA-rrnB expression framework, namely Frag5;

(8) Frag4 obtained in (6) and Frag5 obtained in (7) were assembled by ClonExpress II One Step Cloning Kit C112 (see Table 3 for the Gibson assembly connection reaction system) and then assembled with Frag3 obtained in (5) by ClonExpress II One Step Cloning Kit C112 ( The Gibson assembly ligation reaction system is shown in Table 3), and the pJM-sgRNA plasmid was obtained (see Figure 2 for the plasmid map), wherein the pJM-sgRNA plasmid sequentially contained the OriM replicon, pMB1 replicon, Pj23119 promoter, sgRNA sequence, rrnB terminator and aadA resistance gene.

2. Use of CRISPR/Cas9 gene editing system

The CRISPR/Cas9 gene editing system contains both the pML-Cas9 plasmid and the pJM-sgRNA plasmid. When using the CRISPR/Cas9 gene editing system, the pML-Cas9 plasmid must first be introduced into Mycobacterium aureus. After successful screening and verification, Then, the new Mycobacterium aureus transformants screened out were used as competent, and the pJM-sgRNA plasmid was introduced into the transformants.

Table 1 Primers and their sequences

Table 2 PCR amplification reaction system

<![CDATA[ddH₂0]]> up to 50μL 2×Phanta Max Master Mix 25 μL upstream primer 2μL downstream primer 2μL template DNA 1μL

Table 3 Gibson assembly ligation reaction system

linearized vector 90ng insert fragment 180ng 5×CE II Buffer 4μL Exnase II 2μL <![CDATA[ddH₂O]]> add to 20μL

Embodiment 2: Application of CRISPR/Cas9 gene editing system

Specific steps are as follows:

(1) Construction of CRISPR/Cas9 gene editing system

The ksdd gene of Mycobacterium aureus JC-12 is used as the gene to be knocked out, and the nucleotide sequence of the gene to be knocked out is used as the target sequence, and the sgRNA (nucleotide sequence) for specifically targeting the target sequence is designed according to the target sequence As shown in SEQ ID NO:24); According to Example 1, the sgRNA obtained according to the design is constructed to obtain the CRISPR/Cas9 gene editing system;

(2) Gene editing of neomycobacterium aureus JC-12

Using the new Mycobacterium aureus JC-12 that was not edited by the CRISPR/Cas9 gene editing system as a blank control, the pML-Cas9 plasmid in the CRISPR/Cas9 gene editing system obtained in (1) was transformed into the new Mycobacterium aureus JC- 12 Competent cells, spread on a slant/solid medium (containing 50 μg/mL kanamycin), culture at 30°C for 3 to 4 days; pick transformants, and use the P13 and P14 primers in Table 4 for PCR For preliminary verification, select the transformed transformants that have been successfully verified as competent, and add 0.5mM IPTG to induce at 30°C while preparing the competent, and use the pJM-sgRNA plasmid in the CRISPR/Cas9 gene editing system obtained in (1) Transform into competent cells of transformants, spread on slant/solid medium (containing 50 μg/mL kanamycin and 50 μg/mL streptomycin), and culture at 30°C for 3 to 4 days; pick transformants, and use The P13 primers and P14 primers in Table 4 were initially verified by PCR, and the transformants that were successfully verified were selected for sequencing verification.

The gene editing efficiency of Mycobacterium aureus JC-12 gene editing using the CRISPR/Cas9 gene editing system was tested, and the test results showed that the knockout efficiency of ksdd could reach 50%.

Single colonies of Mycobacterium aureus JC-12 in the blank control group and Mycobacterium aureus JC-12/ksdd after the ksdd gene was knocked out using the CRISPR/Cas9 gene editing system were picked and inoculated in the seed medium respectively. Cultivate at 30°C for 48 hours to obtain seed liquid; inoculate the seed liquid into the fermentation medium according to the inoculum amount of 10% and cultivate at 30°C and 160 rpm for 168 hours to obtain the fermentation liquid; centrifuge the fermentation liquid to collect the bacteria; break the bacteria through the cell Centrifuge the crude enzyme solution to obtain the precipitate to obtain 3-sterone-Δ1-dehydrogenase, detect the crude enzyme solution obtained from the fermentation of Mycobacterium aureus JC-12 in the blank control group and use CRISPR /Cas9 gene editing system Knockout the ksdd gene The enzyme activity of the crude enzyme solution obtained from the fermentation of Mycobacterium aureus JC-12/ksdd, the detection result is: the new Ksdd gene knockout after the CRISPR/Cas9 gene editing system was used The 3-sterone-Δ1-dehydrogenase activity of the crude enzyme solution fermented by Mycobacterium aureus JC-12/ksdd was higher than the 3-sterone-Δ1-dehydrogenase activity of the crude enzyme solution fermented by Mycobacterium aureus JC-12 in the blank control group. The activity of sterone-Δ1-dehydrogenase decreased by 80%.

Table 4 Primers and their sequences

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore The scope of protection of the present invention should be defined by the claims.

sequence listing

<110> Jiangnan University

<120> A CRISPR/Cas9 gene editing system and its application

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<170> PatentIn version 3.3

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tacctccagg aaatcttctc taatgagatg gcaaaggtgg atgactcctt tttccaccgc 300

ctcgaagagt ccttcctggt ggaagaggac aagaaacacg agcgccatcc tatcttcggc 360

aatattgtcg atgaagtcgc atatcatgaa aaatacccaa ccattacca tctccgtaaa 420

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atcaacgcat ctggcgtcga cgcaaaagcc atcctgtccg cccgtctctc caagtctcgt 660

cgcctcgaaa acctcattgc ccagctccct ggcgagaaga aaaacggtct gttcggcaat 720

ctgatcgccc tgtctctggg tctgacccca aatttcaaat ccaactttga tctcgcagaa 780

gatgccaagc tgcagctctc taaggacacc tacgatgatg acctggataa cctcctcgcc 840

cagatcggcg accagtacgc cgatctcttc ctcgcagcca agaacctctc tgacgcaatt 900

ctgctgtccg acatcctgcg cgtgaacacc gaaatcacta aggcaccact ctctgcctcc 960

atgattaagc gctacgacga gcatcatcag gatctcactc tcctcaaagc cctggtccgc 1020

cagcagctcc cagagaagta caaggaaatc tttttcgacc aatccaaaaa cggctacgca 1080

ggttacatcg atggcggcgc ctctcaggaa gagttttaca aattcattaa gccaatcctc 1140

gaaaagatgg acggcaccga ggaactgctg gtgaaactca accgtgaaga tctcctgcgc 1200

aaacagcgca ccttcgacaa cggttccatt cctcaccaga tccacctggg cgaactgcac 1260

gcaatcctcc gccgtcaaga ggacttctac ccattcctga aggacaaccg tgaaaagatc 1320

gaaaagattc tcaccttccg catcccttac tacgtgggtc ctctcgcccg tggcaattcc 1380

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aacctcccaa acgagaaagt gctgcctaag cattccctcc tgtacgagta ctttactgtc 1560

tacaatgagc tgactaaggt gaagtatgtc actgaaggca tgcgtaagcc agcctttctc 1620

tccggcgagc agaaaaaggc aatcgtcgac ctcctgttta aaaccaaccg caaagtgact 1680

gtgaaacagc tcaaggaaga ttactttaaa aaaatcgaat gcttcgattc tgtggaaatc 1740

tccggcgtgg aggatcgttt caacgcctcc ctgggtacct atcacgacct cctgaagatt 1800

atcaaggaca aagattttct ggataacgag gaaaacgagg attctcga ggacattgtg 1860

ctgaccctga ccctcttcga ggaccgcgag atgatcgagg agcgcctgaa gacctatgcc 1920

cacctctttg acgacaaggt catgaagcaa ctcaagcgcc gccgctatac cggttggggc 1980

cgtctctccc gtaagctcat caatggtatc cgcgacaagc aatccggcaa gactatcctg 2040

gactttctga agtctgacgg cttcgccaac cgcaatttta tgcaactgat ccacgacgat 2100

tccctgacct tcaaagagga catccagaaa gcccaagtgt ccggtcaagg cgactccctg 2160

cacgaacaca tcgccaatct ggcaggttcc ccagcaatca agaagggcat cctgcagacc 2220

gtcaaggtgg tggacgaact cgtcaaagtg atgggtcgcc acaaaccaga aaacatcgtc 2280

atcgagatgg cccgtgagaa ccagaccacc cagaaaggcc agaaaaactc ccgtgagcgc 2340

atgaagcgca ttgaagaagg cattaaagag ctcggctctc agatcctgaa agagcatcct 2400

gtcgagaaca cccaactgca gaatgagaag ctgtatctgt attatctcca gaacggccgc 2460

gacatgtacg tcgaccagga actggacatc aaccgtctct ctgattacga tgtggaccat 2520

atcgtccctc agtctttcct gaaagacgac tctattgaca acaaagtcct cacccgctcc 2580

gacaagaacc gcggcaagtc cgataacgtg ccatccgagg aggtcgtgaa gaagatgaag 2640

aactactggc gccagctgct caacgccaag ctgatcactc agcgcaagtt cgataacctg 2700

accaaggccg aacgtggtgg tctctccgag ctcgacaagg caggctttat caagcgccaa 2760

ctcgtggaga ctcgccaaat cactaaacac gtcgcccaga tcctcgactc ccgcatgaat 2820

accaagtacg atgaaaatga caagctcatc cgcgaagtga aagtcattac cctgaagtcc 2880

aaactggtct ctgactttcg caaggatttc cagttctaca aggtccgcga gattaataac 2940

tatcatcatg cacacgatgc atacctcaac gcagtcgtgg gtaccgcact gatcaagaag 3000

taccctaaac tggagtccga gttcgtctat ggcgactaca aggtgtacga cgtccgcaaa 3060

atgattgcca agtccgagca ggagatcggc aaagcaactg ccaaatattt cttttactcc 3120

aacatcatga acttcttcaa gaccgaaatc accctcgcca acggtgaaat ccgcaaacgt 3180

ccactcatcg agactaatgg tgaaaccggc gagatcgtct gggacaaggg ccgtgacttc 3240

gcaaccgtcc gcaaggtcct ctccatgcca caggtcaaca tcgtgaaaaa gaccgaggtg 3300

caaaccggcg gcttctccaa ggagtctatc ctgcctaaac gcaattccga taagctcatt 3360

gcacgcaaaa aggactggga ccctaaaaaa tacggcggtt tcgactcccc aactgtcgca 3420

tattctgtgc tcgtggtcgc caaagtggaa aagggcaaat ccaaaaagct caagtccgtc 3480

aaggaactcc tgggtatcac catcatggaa cgctcctcct ttgagaagaa ccctatcgat 3540

ttcctcgaag caaaaggtta caaggaggtg aagaaagatc tgatcatcaa gctccctaaa 3600

tactccctct tcgagctcga gaacggccgc aagcgtatgc tggcctccgc aggtgaactg 3660

caaaaaggta acgagctggc actcccatcc aagtatgtca actttctcta cctggcctcc 3720

cactacgaaa agctgaaagg ttccccagaa gacaacgagc agaaacagct gttcgtggag 3780

cagcacaagc actacctgga cgaaatcatc gagcagatct ccgagttctc taaacgcgtc 3840

attctggccg atgccaacct cgataaagtg ctctccgcct acaataagca tcgtgataag 3900

ccaatccgtg agcaggcaga gaacatcatt cacctgttca ctctcaccaa cctgggtgca 3960

ccagccgcct ttaagtactt cgacaccacc atcgaccgca agcgctatac ctccactaag 4020

gaggtgctcg atgcaaccct gatccaccag tctatcaccg gcctctacga gactcgcatc 4080

gatctctccc agctgggtgg cgactaa 4107

<210> 2

<211> 35

<212> DNA

<213> Artificial sequence

<400> 2

ttgacagcta gctcagtcct aggtataatg ctagc 35

<210> 3

<211> 822

<212>DNA

<213> Artificial sequence

<400> 3

atgcgagcca tttggacggg ttcgatcgcc ttcgggctgg tgaacgtgcc ggtcaaggtg 60

tacagcgcta ccgcagacca cgacatcagg ttccaccagg tgcacgccaa ggacaacgga 120

cgcatccggt acaagcgcgt ctgcgaggcg tgtggcgagg tggtcgacta ccgcgatctt 180

gcccgggcct acgagtccgg cgacggccaa atggtggcga tcaccgacga cgacatcgcc 240

agcttgcctg aagaacgcag ccgggagatc gaggtgttgg agttcgtccc cgccgccgac 300

gtggacccga tgatgttcga ccgcagctac tttttggagc ctgattcgaa gtcgtcgaaa 360

tcgtatgtgc tgctggctaa gacactcgcc gagaccgacc ggatggcgat cgtgcatttc 420

acgctgcgca acaagaccag gctggcggcg ttgcgcgtca aggatttcgg caagcgagag 480

gtgatgatgg tgcacacgtt gctgtggccc gatgagatcc gcgaccccga cttcccggtg 540

ctggaccaga aggtggagat caaacccgcg gaactcaaga tggccggcca ggtggtggac 600

tcgatggccg acgacttcaa tccggaccgc taccacgaca cctaccagga gcagttacag 660

gagctgatcg acaccaaact cgaaggtggg caggcattta ccgccgagga ccaaccgagg 720

ttgctggacg agcccgaaga cgtctccgac ctgctcgcca agctggaggc cagcgtgaag 780

gcgcgctcga aggccaactc aaacgtccca acgcctccgt ga 822

<210> 4

<211> 2280

<212> DNA

<213> Artificial sequence

<400> 4

atgggttcgg cgtcggagca acgggtgacg ctgaccaacg ccgacaaggt gctctatccc 60

gccaccggga ccacaaagtc cgatatcttc gactactacg ccggtgttgc cgaagtcatg 120

ctcggccaca tcgcgggacg gccggcgacg cgcaagcgct ggcctaacgg cgtcgaccaa 180

cccgcgttct tcgaaaagca gttggcgttg tcggcgccgc cttggctgtc acgtgcaacg 240

gtggcgcacc ggtccgggac gacgacctat ccgatcatcg atagcgcaac cgggctggcc 300

tggatcgccc aacaggcggc gctggaggtg cacgtgccgc agtggcggtt tgtcgccgag 360

cccggatcag gtgagttaaa tccgggcccg gcaacgcgtt tggtgttcga cctggacccg 420

ggcgaaggcg tgatgatggc ccagctggcc gaggtggcgc gcgcggttcg tgatcttctc 480

gccgatatcg ggttggtcac cttcccggtc accagcggca gcaagggatt gcatctgtac 540

acaccgctgg atgagccggt gagcagcagg ggagccacgg tgttggccaa gcgcgtcgcg 600

cagcgattgg agcaggcgat gcccgcgttg gtcacctcga ccatgaccaa aagcctgcgg 660

gccgggaagg tgtttgtgga ctggagccag aacagcggct cgaagaccac catcgcgccg 720

tactcactac gtggccggac gcatccgacc gtcgcggcgc cacgcacctg ggcggagctc 780

gacgaccccg cactgcgtca gctctcctac gacgaggtgc tgacccggat tgcccgcgac 840

ggcgatctgc tcgagcggct ggatgccgac gctccggtag cggaccggtt gacccgatac 900

cgccgcatgc gcgacgcatc gaaaactccc gagccgattc ccacggcgaa acccgttacc 960

ggagacggca atacgttcgt catccaggag catcacgcgc gtcggccgca ctacgatttc 1020

cggctggaat gcgacggcgt gctggtctcg tgggcggtac cgaaaaacct gcccgacaac 1080

acatcggtta accatctagc gatacacacc gaggaccacc cgctggaata cgccacgttc 1140

gagggcgcga ttcccagcgg ggagtacggc gccggcaagg tgatcatctg ggactccggc 1200

acttacgaca ccgagaagtt ccacgatgac ccgcacacgg gggaggtcat cgtgaatctg 1260

cacggcggcc ggatctctgg gcgttatgcg ctgattcgga ccaacggcga tcggtggctg 1320

gcgcaccgcc taaagaatca gaaagaccag aaggtgttcg agttcgacaa tctggcccca 1380

atgcttgcca cgcacggcac ggtggccggt ctaaaggcca gccagtgggc gttcgaaggc 1440

aagtgggacg gctaccggtt gctggttgag gctgaccacg gcgccgtgcg gctgcggtcc 1500

cgcagcgggc gcgatgtcac cgccgagtat ccgcaattgc gggcattggc ggaggatctc 1560

gccgatcacc acgtggtgct ggacggcgag gccgtcgtac ttgactcctc tggtgtgccc 1620

agcttcagcc agatgcagaa tcggggccgc gacacccgtg tcgagttctg ggcgttcgac 1680

ctgctctacc tcgacggccg cgcgctgcta ggcacccgct accaagaccg gcgtaagctg 1740

ctcgaaaccc tagctaacgc aaccagtctc accgttcccg agctgctgcc cggtgacggc 1800

gcccaagcgt ttgcgtgctc gcgcaagcac ggctgggagg gcgtgatcgc caagaggcgt 1860

gactcgcgct atcagccggg ccggcgctgc gcgtcgtggg tcaaggacaa gcactggaac 1920

acccaggaag tcgtcattgg tggctggcgc gccggggaag gcgggcgcag cagtggcgtc 1980

gggtcgctgc tcatgggcat ccccggtcca ggtgggctgc agttcgccgg gcgggtcggt 2040

accggcctca gcgaacgcga actggccaac ctcaaggaga tgctggcgcc gctgcatacc 2100

gacgagtccc ccttcgacgt accactgccc gcgcgtgacg ccaagggcat cacatatgtc 2160

aagccggcgc tggttgcaga ggtgcgctac agcgagtgga ctccggaggg ccggctgcgt 2220

caatcaagct ggcgtgggct gcggccggac aagaaaccca gtgaggtggt gcgcgaatga 2280

<210> 5

<211> 165

<212> DNA

<213> Artificial sequence

<400> 5

aattcgtgtc gctcaaggcg cactcccgtt ctggataatg ttttttgcgc cgacatcata 60

acggttctgg caaatattct gaaatgagct gttgacaatt aatcatcggc tcgtataatg 120

tgtggaattg tgagcggata acaatttcac acaggaaaca gaatt 165

<210> 6

<211> 1896

<212> DNA

<213> Artificial sequence

<400> 6

gtgagcccac cagctccgta agttcgggtg ctgtgtggct cgtacccgcg cattcaggcg 60

gcaggggtc taacgggtct aaggcggcgt gtacggccgc cacagcggct cttagcggcc 120

cggaaacgtc ctcgaaacga cgcatgtgtt cctcctggtt ggtacaggtg gttgggggtg 180

ctcggctgtc gctggtgttt catcatcagg gctcgacggg agagcggggg agtgtgcagt 240

tgtggggtgg cccctcagcg aaatatctga cttggagctc gtgtcggacc atacaccggt 300

gattaatcgt ggtttattat caagcgtgag ccacgtcgcc gacgaatttg agcagctctg 360

gctgccgtac tggtccctgg caagcgacga tctgctcgag gggatctacc gccaaagccg 420

cgcgtcggcc ctaggccgcc ggtacatcga ggcgaaccca acagcgctgg caaacctgct 480

ggtcgtggac gtagaccatc cagacgcagc gctccgagcg ctcagcgccc gggggtccca 540

tccgctgccc aacgcgatcg tgggcaatcg cgccaacggc cacgcacacg cagtgtgggc 600

actcaacgcc cctgttccac gcaccgaata cgcgcggcgt aagccgctcg catacatggc 660

ggcgtgcgcc gaaggccttc ggcgcgccgt cgatggcgac cgcagttact caggcctcat 720

gaccaaaaac cccggccaca tcgcctggga aacggaatgg ctccactcag atctctacac 780

actcagccac atcgaggccg agctcggcgc gaacatgcca ccgccgcgct ggcgtcagca 840

gaccacgtac aaagcggctc cgacgccgct agggcggaat tgcgcactgt tcgattccgt 900

caggttgtgg gcctatcttc ccgccctcat gcggatctac ctgccgaccc ggaacgtgga 960

cggactcggc cgcgcgatct atgccgagtg ccacgcgcga aacgccgaat ttccgtgcaa 1020

cgacgtgtgt cccggaccgc taccggacag cgaggtccgc gccatcgcca acagcatttg 1080

gcgttggatc acaaccaagt cgcgcatttg ggcggacggg atcgtggtct acgaggccac 1140

actcagtgcg cgccatgcgg ccatctcgcg gaagggcgca gcagcgcgca cggcggcgag 1200

cacagttgcg cggcgcgcaa agtccgcgtc agccatggag gcattgctat gagcgacggc 1260

tacagcgacg gctacagcga cggctacaac tggcagccga ctgtccgcaa aaagcggcgc 1320

gtgaccgccg ccgaaggcgc tcgaatcacc ggactatccg aacgccacgt cgtccggctc 1380

gtggcgcagg aacgcagcga gtggttcgcc gagcaggctg cacgccgcga acgcatccgc 1440

gcctatcacg acgacgaggg ccactcttgg ccgcaaacgg ccaaacattt cgggctgcat 1500

ctggacaccg ttaagcgact cggctatcgg gcgaggaaag agcgtgcggc agaacaggaa 1560

gcggctcaaa aggcccacaa cgaagccgac aatccaccgc tgttctaacg caattgggga 1620

gcgggtgtcg cgggggttcc gtggggggtt ccgttgcaac gggtcggaca ggtaaaagtc 1680

ctggtagacg ctagttttct ggtttgggcc atgcctgtct cgttgcgtgt ttcgttgcgc 1740

ccgttttgaa taccagccag acgagacggg gttctacgaa tcttggtcga taccaagcca 1800

tttccgctga atatcgggga gctcaccgcc agaatcggtg gttgtggtga tgtacgtggc 1860

gaactccgtt gtagtgcctg tggtggcatc cgtggc 1896

<210> 7

<211> 589

<212> DNA

<213> Artificial sequence

<400> 7

ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 60

agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt 120

cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt 180

caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc 240

tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa 300

ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac 360

ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg 420

gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga 480

gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact 540

tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaa 589

<210> 8

<211> 792

<212>DNA

<213> Artificial sequence

<400> 8

atgagggaag cggtgatcgc cgaagtatcg actcaactat cagaggtagt tggcgtcatc 60

gagcgccatc tcgaaccgac gttgctggcc gtacatttgt acggctccgc agtggatggc 120

ggcctgaagc cacacagtga tattgatttg ctggttacgg tgaccgtaag gcttgatgaa 180

acaacgcggc gagctttgat caacgacctt ttggaaactt cggcttcccc tggagagagc 240

gagattctcc gcgctgtaga agtcaccatt gttgtgcacg acgacatcat tccgtggcgt 300

tatccagcta agcgcgaact gcaatttgga gaatggcagc gcaatgacat tcttgcaggt 360

atcttcgagc cagccacgat cgacattgat ctggctatct tgctgacaaa agcaagagaa 420

catagcgttg ccttggtagg tccagcggcg gaggaactct ttgatccggt tcctgaacag 480

gatctatttg aggcgctaaa tgaaacctta acgctatgga actcgccgcc cgactgggct 540

ggcgatgagc gaaatgtagt gcttacgttg tcccgcattt ggtacagcgc agtaaccggc 600

aaaatcgcgc cgaaggatgt cgctgccgac tgggcaatgg agcgcctgcc ggcccagtat 660

cagcccgtca tacttgaagc tagacaggct tatcttggac aagaagaaga tcgcttggcc 720

tcgcgcgcag atcagttgga agaatttgtc cactacgtga aaggcgagat caccaaggta 780

gtcggcaaat aa 792

<210> 9

<211> 45

<212> DNA

<213> Artificial sequence

<400> 9

caaataaaac gaaaggctca gtcgaaagac tgggcctttc gtttt 45

<210> 10

<211> 29

<212> DNA

<213> Artificial sequence

<400> 10

atgggttcgg cgtcggagca acgggtgac 29

<210> 11

<211> 51

<212>DNA

<213> Artificial sequence

<400> 11

ctagtattct cctctttaat ctctagtatc attcgcgcac cacctcactg g 51

<210> 12

<211> 51

<212> DNA

<213> Artificial sequence

<400> 12

attaaagagg agaatactag atgcgagcca tttggacggg ttcgatcgcc t 51

<210> 13

<211> 29

<212> DNA

<213> Artificial sequence

<400> 13

tcacggaggc gttgggacgt ttgagttgg 29

<210> 14

<211> 87

<212>DNA

<213> Artificial sequence

<400> 14

tctagattga cagctagctc agtcctaggt ataatgctag ctactagaga aagaggagaa 60

atactagatg ggttcggcgt cggagca 87

<210> 15

<211> 48

<212> DNA

<213> Artificial sequence

<400> 15

ttcgaattct gcagcttcac ggaggcgttg ggacgtttga gttggcct 48

<210> 16

<211> 30

<212> DNA

<213> Artificial sequence

<400> 16

atgagggaag cggtgatcgc cgaagtatcg 30

<210> 17

<211> 28

<212> DNA

<213> Artificial sequence

<400> 17

tttccatagg ctccgccccc ctgacgag 28

<210> 18

<211> 28

<212> DNA

<213> Artificial sequence

<400> 18

gtgagcccac cagctccgta agttcggg 28

<210> 19

<211> 24

<212> DNA

<213> Artificial sequence

<400> 19

gccacggatg ccaccacagg cact 24

<210> 20

<211> 55

<212>DNA

<213> Artificial sequence

<400> 20

gcctgcaggt cgactctaga ggatccttga cagctagctc agtcctaggt ataat 55

<210> 21

<211> 42

<212>DNA

<213> Artificial sequence

<400> 21

catgattacg aattcaaaaa aagcaccgac tcggtgccac tt 42

<210> 22

<211> 22

<212>DNA

<213> Artificial sequence

<400> 22

cggctcattg agcaaccgat ac 22

<210> 23

<211> 19

<212>DNA

<213> Artificial sequence

<400> 23

cgcccgacac agcctttca 19

<210> 24

<211> 102

<212>DNA

<213> Artificial sequence

<400> 24

ctcgacagta gtcgttgaga gttttagagc tagaaatagc aagttaaaat aaggctagtc 60

cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tt 102

Claims (3)

1. A gene editing method for Mycobacterium aureus, characterized in that, the method uses a CRISPR/Cas9 gene editing system; The CRISPR/Cas9 gene editing system comprises a pML-Cas9 plasmid and a pJM-sgRNA plasmid; The pML-Cas9 plasmid comprises a pMV261 expression vector, a Cas9 gene, a Pj23119 promoter and an NHEJ repair gene; the Hsp60 promoter on the pMV261 expression vector is replaced with a Tac promoter; the NHEJ repair gene comprises an encoding DNA end binding protein The gene of mku and the gene encoding DNA ligase LigD; the Cas9 gene is located downstream of the Tac promoter of the pMV261 expression vector and upstream of the rrnB terminator of the pMV261 expression vector, and is expressed by the Tac promoter; the Pj23119 promoter is located in pMV261 Downstream of the KanR resistance gene of the expression vector; the NHEJ repair gene is located downstream of the Pj23119 promoter and upstream of the replication origin ( ori ) of the pMV261 expression vector, and is expressed by the Pj23119 promoter; The pJM-sgRNA plasmid sequentially comprises OriM replicon, pMB1 replicon, Pj23119 promoter, sgRNA sequence, rrnB terminator and aadA resistance gene, named as pJM-sgRNA plasmid; the sgRNA sequence is a targeting sequence, which can specifically Targeting target sequence; the target sequence refers to the nucleotide sequence of the gene to be edited; The nucleotide sequence of the Cas9 gene is shown in SEQ ID NO:1; The nucleotide sequence of the gene encoding the Pj23119 promoter in the pML-Cas9 plasmid and the pJM-sgRNA plasmid is shown in SEQ ID NO: 2; The nucleotide sequence of the gene encoding the DNA end-binding protein mku is shown in SEQ ID NO:3; The nucleotide sequence of the gene encoding the DNA ligase LigD is shown in SEQ ID NO:4. 2. a kind of gene editing method that is used for neomycobacterium aureus as claimed in claim 1, is characterized in that, described method is first the pML in a kind of CRISPR/Cas9 gene editing system described in claim 1 -The Cas9 plasmid is introduced into the new Mycobacterium aureus, so that the Cas9 gene on the pML-Cas9 plasmid, the gene encoding the DNA end-binding protein mku and the gene expression encoding the DNA ligase LigD, then a kind of CRISPR described in claim 1 The pJM-sgRNA plasmid in the /Cas9 gene editing system is introduced into Mycobacterium aureus, so that the pJM-sgRNA plasmid is transcribed to generate the sgRNA sequence, and the Cas9 handle and Terminator in the sgRNA sequence will form a stem-loop structure, and the Cas9 gene will recognize the stem-loop The structure is combined with the sgRNA sequence to form a complex. This complex will target the gene to be edited and be anchored on the gene to be edited. After anchoring, the Cas9 gene will play an active role in cutting the gene to be edited so that the gene to be edited in the new Mycobacterium aureus can be edited. The gene is knocked out, and the knockout of the gene to be edited will cause a DSB break in the M. aureus gene. At this time, the DNA end-binding protein mku and DNA ligase LigD expressed on the pML-Cas9 plasmid will repair the DSB break. Complete the editing process of the new Mycobacterium aureus gene. 3. The application of a gene editing method for Mycobacterium aureus described in claim 1 or 2 in editing the Mycobacterium aureus gene.

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