CN104673816A - PCr-NHEJ (non-homologous end joining) carrier as well as construction method of pCr-NHEJ carrier and application of pCr-NHEJ carrier in site-specific knockout of bacterial genes - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering, and in particular relates to a pCr-NHEJ vector, a construction method thereof and an application for targeted knockout of bacterial genes. The sequence of the pCr-NHEJ vector of the present invention has the sequence shown in SEQ ID NO: 1. Application of the pCr-NHEJ vector in site-directed knockout of bacterial genes. The technical principle of the present invention is: the joint application of NHEJ and CRISPR-Cas9, in order to realize that after the CRISPR-Cas9 system cuts the bacterial genome DNA and causes DNA breaks, the NHEJ system can automatically connect the DNA ends, so that the bacteria can survive without manual introduction and Double-stranded DNA with homology to the target sequence repairs DNA breaks, thereby simplifying the operation steps of CRISPR-Cas9 technology.

Description

A kind of pCr-NHEJ carrier and its construction method and its application to bacterial gene site-directed knockout

technical field

[ The present invention relates to the technical field of genetic engineering, in particular to a pCr-NHEJ vector and its construction method and its application for bacterial gene knockout.

Background technique

"Science" published in 2013 reported a simple and efficient gene-directed editing technology - CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat; CRISPR-associated, CRISPR-Cas9). The technology system consists of a Cas9 nuclease from Streptococcus, a sequence-constant tracrRNA (trans-activating crRNA) and a 20nt-crRNA (CRISPR RNAs) specifically complementary to the target gene. The advanced structure formed by the 3' end of tracrRNA binds and activates Cas9, the 5' end of tracrRNA is complementary to the 3' end of crRNA, and the 5' end of crRNA is complementary to the target gene, thereby guiding Cas9 to reach and cut off the target sequence. Now two small RNAs have been fused into one RNA chain, referred to as sgRNA (single guide RNA), and it is more convenient to use sgRNA to operate. Because this technology only needs to design a specific crRNA sequence for the target gene, it can quickly realize the targeted knockout of the gene. This technology has not only completely changed the concept of traditional genome editing technology, but has been successfully applied to genome knockout, knockin or mutation of various organisms in just two years.

The sgRNA molecule is composed of a spacer sequence (spacer) and a trancrRNA sequence: 1) The spacer sequence is 20nt in length and is complementary to the target site of the gene. It is located at the 5' end of the PAM (Protospacer-Adjacent Motif) sequence (NGG). Individually designed according to the target gene sequence; 2) The tracrRNA sequence is constant and can bind and activate the Cas9 protein. Under the guidance of the spacer, the sgRNA brings the activated Cas9 protein to the target site of the gene, and cuts the DNA double strand of the target gene between 3-8 bases upstream of the PAM sequence, forming a blunt end break. In eukaryotic cells, after Cas9 cuts double-stranded DNA, the cell can autonomously produce homologous recombination (homologous recombination, HR) or non-homologous end joining (Non-homologous End Joining, NHEJ) two mechanisms to repair damaged DNA, resulting in Insertion mutation, frameshift mutation, gene knock-in, etc.

sgRNA and Cas9 genes and sgRNA-encoding DNA can be located in the same or different plasmids, and expressed in cells under the control of corresponding promoters. In addition, sgRNA can also be synthesized by in vitro transcription and co-transform host cells with the Cas9 plasmid.

Before the invention of CRISPR-Cas9 technology, gene knockout in bacteria mainly relied on homologous recombination technology. This technique has disadvantages such as low efficiency, complex operation process, and long time for screening positive clones. Studies have shown that compared with the above-mentioned homologous recombination technology, CRISPR-Cas9 technology has the following advantages: 1) High efficiency, CRISPR-Cas9 technology knockout target gene is 1000 times that of homologous recombination technology; 2) The same CRISPR-Cas9 technology can be used The Cas9 vector knocks out multiple targets at the same time or successively 3) It is easy to operate, only need to design one or two sgRNAs according to the target gene, clone the coding DNA of the sgRNA into the vector, and then transform the bacteria to achieve gene knockout.

However, since most bacteria do not have an autonomous non-homologous end-joining system, the DSB caused by Cas9 protein cutting bacterial genomic DNA will directly lead to bacterial death, and the gene knockout operation will also fail. Therefore, in the prior art, in order to avoid bacterial death, it is necessary to introduce linear double-stranded DNA with high homology to the target sequence into the bacteria through electroporation transformation at the same time, thereby increasing the operation steps and costs. Since the construction of homologous linear double-stranded DNA requires multiple PCRs and ligation of PCR products, the operation steps and costs are further increased. Therefore, the CRISPR-Cas9 system of the prior art has the disadvantages of cumbersome operation, high difficulty of operation, and insufficient gene knockout efficiency.

Contents of the invention

One of the objectives of the present invention is to provide a pCr-NHEJ vector for the deficiencies of the prior art, which can be used for bacterial gene knockout and has the advantage of high gene knockout efficiency.

The second object of the present invention is to provide a method for constructing a pCr-NHEJ vector in view of the deficiencies in the prior art. The method for constructing a pCr-NHEJ vector has the advantages of simple operation and low operation difficulty.

The third object of the present invention is to provide the application of pCr-NHEJ vector for site-directed knockout of bacterial genes in view of the deficiencies in the prior art.

In order to achieve one of the above objects, the present invention adopts the following technical solutions:

A pCr-NHEJ vector is provided, the sequence of the pCr-NHEJ vector has the sequence shown in SEQ ID NO: 1.

In order to achieve the above-mentioned object two, the present invention adopts the following technical solutions:

The application of the aforementioned pCr-NHEJ vector in site-directed knockout of bacterial genes is provided.

In order to achieve the above object three, the present invention adopts the following technical solutions:

A method for constructing a pCr-NHEJ carrier is provided, comprising the steps of:

(1) The broad host plasmid _P BBR1MCS-2 (GI: 773412) of Gram-negative bacteria was used as the basic vector; cas9

(2) Using the upstream and downstream primers with prokaryotic promoter PLtetO-1 for mRNA transcription to amplify the following three protein genes: cas9 (GI:674296984), ku (GI:444893469) and ligD (GI:444893469 ) to obtain cas9 amplification products, ku amplification products and ligD amplification products respectively; among them, the open reading frames of the three protein genes of cas9 , ku and ligD are all located downstream of the independent PLtetO-1, so that cas9 , ku and ligD The expression of the three protein genes of ligD is regulated by the promoter; there is a 15nt base overlap between the cas9 amplification product, the ku amplification product and the ligD amplification product;

(3) Between the 3' end of the cas9 amplification product and the 5' end of the ku amplification product, and between the 3' end of the ku amplification product and the 3' end of the ligD amplification product, there are There is a 15nt base overlap, and the cas9 amplification product, the ku amplification product and the ligD amplification product are connected by in-fusion PCR to obtain a first connection;

(4) Double-digest the base vector _P BBR1MCS-2 with restriction endonucleases KpnI and HindIII to obtain the digested product of the basic vector; the digested product is subjected to gel electrophoresis, gel cutting and DNA recovery kit purifying the digested product; then using in-fusion PCR to connect the purified digested product and the first linker to obtain the vector pNHEJ;

(5) Using NotI and SpeI to double-digest the vector pNHEJ to obtain the digested product of the vector pNHEJ;

(6) Simultaneously introduce two adjacent inverted BsaI restriction sites and two adjacent inverted BsmAI restriction sites into the vector pNHEJ for transcription to form a gene that can simultaneously bind to two positions of the target gene sgRNA; wherein, the DNA fragment with two adjacent inverted BsaI restriction sites is DBsaI, and the DNA fragment with two adjacent inverted BsmAI restriction sites is DBsmAI;

(7) Linking DBsaI and DBsmAI by in-fusion PCR to obtain the second linking product;

(8) Ligate the second ligation product and the digested product of the vector pNHEJ by in-fusion PCR to obtain the CRISPR-Cas9 system for knocking out the target gene, namely the pCr-NHEJ vector.

In the above technical scheme, in the step (2), the cas9 protein gene is amplified using the primer PPLtetO-1-cas9U having the sequence shown in SEQ ID NO: 2 and the primer PCas9D having the sequence shown in SEQ ID NO: 3 , to obtain the cas9 amplification product;

Using primer PPLtetO-1-KuU having the sequence shown in SEQ ID NO: 4 and primer PkuD having the sequence shown in SEQ ID NO: 5 to amplify the ku protein gene to obtain ku amplification products;

The ligD protein gene was amplified by using the primer PLtetO-1-ligDU having the sequence shown in SEQ ID NO: 6 and the primer PligDD having the sequence shown in SEQ ID NO: 7 to obtain the ligD amplification product.

In the above technical scheme, in the step (6), the construction of the DNA fragment DBsaI with two adjacent inverted BsaI restriction sites is as follows:

First directly synthesize the DNA sequence BsaIU with the sequence shown in SEQ ID NO: 8 and the DNA sequence BsaID with the sequence shown in SEQ ID NO: 9, wherein the 3' end of BsaIU and the 5' end of BsaID have a 15nt overlapping sequence; Wherein, BsaID contains tracrRNA sequence;

Connect BsaIU and BsaID by in-fusion PCR; Then adopt primer PBsaIU with sequence shown in SEQ ID NO: 10 and primer PBsaID with sequence shown in SEQ ID NO: 11, PCR amplification, promptly obtain DNA fragment DBsaI with two inverted BsaI restriction sites.

In the above technical scheme, in the step (6), the construction of the DNA fragment DBsmAI with two adjacent inverted BsmAI restriction sites is as follows:

First directly synthesize the DNA sequence BsmAIU with the sequence shown in SEQ ID NO: 12 and the DNA sequence BsmAID with the sequence shown in SEQ ID NO: 13; wherein, BsmAID contains the tracrRNA sequence;

Connect the DNA sequence BsmAIU and the DNA sequence BsmAID by in-fusion PCR; then use primer PBsmAIU with the sequence shown in SEQ ID NO: 14 and primer PBsmAID with the sequence shown in SEQ ID NO: 15 to carry out PCR Amplify to obtain the DNA fragment DBsmAI with two adjacent inverted BsmAI restriction sites.

Application of the pCr-NHEJ vector constructed by the above-mentioned construction method of a pCr-NHEJ vector in site-directed knockout of bacterial genes.

Compared with the prior art, the present invention has beneficial effects as follows:

(1) A pCr-NHEJ vector of the present invention, the pCr-NHEJ vector can be used for bacterial gene knockout, and has the advantage of high gene knockout efficiency.

(2) A method for constructing a pCr-NHEJ vector of the present invention, the method for constructing a pCr-NHEJ vector has the advantages of simple operation and low operation difficulty.

(3) The present invention provides the application of the pCr-NHEJ vector for targeted knockout of bacterial genes, which has the advantages of simple technical operation, low operation difficulty and high gene knockout efficiency.

(4) In order to save the connection steps of PCR and PCR products, the present invention constructs a pCr-NHEJ vector suitable for gene knockout of various Gram-negative bacteria on the basis of a broad host plasmid _P BBR1MCS-2, and The non-homologous end joining system (Non-homologous end joining, NHEJ) of Mycobacterium tuberculosis—ku and ligD genes—was introduced into the pCr-NHEJ vector. Among them, Ku can bind to DNA breaks and recruit DNA ligase LigD, and LigD can directly connect the ends of broken DNA without relying on template DNA. This application uses the NHEJ system of Mycobacterium tuberculosis to connect the broken end formed by Cas9 cutting DNA, so that the genomic DNA of the knocked-out bacteria can restore the circular structure and allow the bacteria to survive (see Figure 2 for the technical principle).

(5) Using the technology of the present invention, it is only necessary to construct the CRIPR-Cas9 plasmid of the target gene, and carry out plasmid transformation to construct the pCr-NHEJ vector, without simultaneously transforming linear homologous DNA, the knockout of the bacterial genome can be realized. The technical operation of the invention is simpler, the operation difficulty is greatly reduced, and the gene knockout efficiency reaches 87.5%-100%.

(6) In the method for constructing a pCr-NHEJ vector of the present invention, in step (2), since there is a base overlap of 15 nt between the cas9 amplification product, the ku amplification product and the ligD amplification product, it is convenient to use In-fusion PCR ligation made cas9 amplification products, ku amplification products and ligD amplification products all under the regulation of independent promoters.

(7) The technical principle of the present invention lies in the joint application of NHEJ and CRISPR-Cas9 to realize that after the CRISPR-Cas9 system cuts the bacterial genomic DNA and causes DNA breaks, the NHEJ system can automatically connect the broken ends of the DNA so that the bacteria can survive without Artificially introducing double-stranded DNA with homology to the target sequence to repair DNA breaks, thereby simplifying the operation steps of CRISPR-Cas9 technology.

Description of drawings

Figure 1 is a physical map of the broad host plasmid _P BBR1MCS-2 of Gram-negative bacteria. In the figure, MCS, multiple cloning sites, multiple cloning sites; KmR, kanamycin resistance gene; mob, the sequence required for plasmid binding transfer; rep, the plasmid autonomous replication protein gene.

Fig. 2 is a technical schematic diagram of the present invention.

Fig. 3 is a physical map of the vector pCr-NHEJ of the present invention. In Figure 3, the multiple cloning site of pBBR1MCS is constructed; double BsaI and double BsmAI are spacer DNA sequence insertion sites.

Fig. 4 is an electrophoresis diagram during the construction of the vector pNHEJ of the present invention. In Fig. 4, A, B and C are the electrophoresis diagrams of PCR products of cas9 , ku and li gD genes, and lane 1 is DNA marker, which are 2000, 1000, 750, 500, 250 and 100 from top to bottom; D, lane 2 is the PCR product after joining cas9 , ku and li gD genes, and lane 3 is the double enzyme digestion result of KpnI and HindIII of the plasmid pBBR1MCR; E, the electrophoresis result of the plasmid pNHEJ, the size of the DNA band indicated by the arrow is 7500bp.

Fig. 5 is an electrophoresis diagram during the construction of the vector pCr-NHEJ of the present invention. In Fig. 5, A, swimming lanes 1-7 are the PCR products of BsaID, BsaIU, DBsaI, DBsmAI, BsmAIU and BsmAID respectively; B, are the products connected with DBsaI and DBsmAI; C, the electrophoresis figure of plasmid pCr-NHEJ.

Figure 6 is the PCR identification result of knockout efficiency. In Fig. 6, A, B and C are respectively the colony PCR identification results of Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii; the number of identified clones is 11, 11 and 8 in sequence; the swimming lanes in A and B 12 is a positive control, lane 5 in C is a positive control, and the positive rates of gene knockout are 100%, 100% and 87.5%, respectively.

Detailed ways

In order to make the technical problems, technical solutions and beneficial effects solved by the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.

Among them, the pCr-NHEJ vector constructed by the present invention has two sgRNA backbone sequences, and can construct a gene-directed knockout vector by inserting spacer DNA fragments at BsaI or BsmAI sites successively.

Wherein, in the method for constructing a pCr-NHEJ vector of the present invention, in step (2), the primer PPLtetO-1-cas9U ( gctctagaactagtg GA GATTGA CATCCCTATCAGTGATAGA GATACT GAGCACAAGGAGTATACC ATGGATAAGAAATACTCAATAGG) having the sequence shown in SEQ ID NO: 2 is used, wherein, The underline at the 5' end of primer PPLtetO-1-cas9U is 15 nt identical to the end of the HindIII digested product of pBBR1MCS-2, so as to facilitate in-fusion PCR ligation; the region in bold is the promoter PLtetO-1-1, the underlined base The base regions are promoter -33 and -10 regions.

Wherein, in the method for constructing a pCr-NHEJ vector of the present invention, in step (2), the primer cas9D ( agggatgtcaatctc TCAGTCACCTCTAGCTGACTC) having the sequence shown in SEQ ID NO: 3 is used, wherein the underlined region at the 5' end of the primer cas9D is aligned with The 5' end of the primer PPLtetO-1-KuU has the same 15nt, so as to be connected by in-fusion PCR.

Wherein, in the construction method of a pCr-NHEJ vector of the present invention, in step (2), the primer PPLtetO-1-KuU ( gctctagaactagtg GA GATTGA CATCCCTATCAGTGATAGA GATACT GAGCACAAGGAGTATACC ATGCGAGCCATTTGGACGGGTT) having the sequence shown in SEQ ID NO: 4 is used, wherein, The part in bold is the promoter PLtetO-1-1 sequence, the region in bold is the promoter PLtetO-1-1, and the underlined base region is the promoter-33 and -10 regions.

Wherein, in the construction method of a pCr-NHEJ vector of the present invention, in step (2), the primer PLtetO-1-ligDU ( gctctagaactagtg GA GATTGA CATCCCTATCAGTGATAGA GATACT GAGCACAAGGAGTATACC ATGGGTTCGGCGTCGGAG) having the sequence shown in SEQ ID NO: 6 is used, wherein, The underlined region at the 5' end of the primer PLtetO-1-ligDU is 15 nt identical to the 5' end of PkuD for in-fusion PCR connection; the bold region is the promoter PLtetO-1-1, and the underlined base region is the promoter Sub-33 and -10 areas.

Wherein, in the method for constructing a pCr-NHEJ vector of the present invention, in step (2), the primer PligDD ( GGGAACAAAGCTGG CAAAAAACCCCTCAAGACCCGTTTAGAGGCCCCAAGGGGTTATGCTAG TCATTCGCGCACCACCTCACT) having the sequence shown in SEQ ID NO: 7 is used, wherein the underlined region at the 5' end of the primer PligDD The end of the KpnI digested product of pBBR1MCS-2 has 15 nt identical to facilitate connection by in-fusion PCR; the italic part is T7-transcription termination factor.

Among them, in the construction method of a pCr-NHEJ vector of the present invention, in the process of constructing the DNA fragment DBsaI with two adjacent inverted BsaI restriction sites, the synthesized DNA sequence BsaIU ( GA GATTGA CATCCCTATCAGTGATAGA GATACT GAGCAC gagacc aaa GGTCTC gttttagagctaGAAAtagcaagttaa, SEQ ID NO: 8), where the bold font is the PLtetO-1-2 promoter sequence for RNA transcription.

Wherein, in the construction method of a pCr-NHEJ vector of the present invention, in the process of constructing a DNA fragment DBsaI with two adjacent inverted BsaI restriction sites, the synthesized DNA sequence BsaID (GAAAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTC, SEQ ID NO: 9, tracrRNA sequence), wherein, the 3' end of BsaIU and the 5' end of BsaID have a 15nt overlapping sequence, so that the two gene fragments of BsaIU and BsaID can be connected by In-fusion PCR.

Among them, in the construction method of a pCr-NHEJ vector of the present invention, in the process of constructing the DNA fragment DBsmAI with two adjacent inverted BsmAI restriction sites, the synthesized DNA sequence BsmAIU ( GA GATTGA CATCCCTATCAGTGATAGA GATACT GAGCAC gagac aaa gtctcg ttttagagctaGAAAtagcaagttaa, SEQ ID NO: 12), wherein, the bold font is the PLtetO-1-2 promoter sequence for RNA transcription.

Among them, in the construction method of a pCr-NHEJ vector of the present invention, in the process of linking the digested product of DBsaI, DBsmAI and vector pNHEJ by In-fusion PCR, since the 5' end of PBsaID and PBsmAIU has a 15nt overlapping sequence , so that the two gene fragments can be connected by In-fusion PCR. The 5' end of PBsaIU has a 15 base overlapping sequence with the downstream of the pNHEJ restriction site NotI, and the 5' end of PBsmAID has a 15 base overlapping sequence with the upstream of the pNHEJ restriction site SpeI , so that DBsaI and DBsmAI can be connected with NotI and SpeI double-digested pNHEJ fragments by In-fusion PCR, so as to obtain the CRISPR-Cas9 system for knocking out the target gene, that is, the pCr-NHEJ vector.

Wherein, in the construction method of a pCr-NHEJ vector of the present invention, the constructed pCr-NHEJ vector has two sgRNA backbone sequences, therefore, the pCr-NHEJ vector can successively insert the spacer DNA fragment at the BsaI or BsmAI site Construction of vectors for targeted knockout of genes.

Example 1.

A pCr-NHEJ carrier, the sequence of the pCr-NHEJ carrier has the sequence shown in SEQ ID NO: 1. The pCr-NHEJ vector of this example can be used for bacterial gene knockout, and has the advantage of high gene knockout efficiency.

Example 2.

Application of a pCr-NHEJ vector in Example 1 in site-directed knockout of bacterial genes.

Example 3.

The construction method of a kind of pCr-NHEJ carrier of embodiment 1, comprises the steps:

(1) The broad host plasmid _P BBR1MCS-2 (GI: 773412) of Gram-negative bacteria was used as the basic vector; cas9

(2) Using the upstream and downstream primers with prokaryotic promoter PLtetO-1 for mRNA transcription to amplify the following three protein genes: cas9 (GI:674296984), ku (GI:444893469) and ligD (GI:444893469 ) to obtain cas9 amplification products, ku amplification products and ligD amplification products respectively; among them, the open reading frames of the three protein genes of cas9 , ku and ligD are all located downstream of the independent PLtetO-1, so that cas9 , ku and ligD The expression of the three protein genes of ligD are all regulated by the promoter; there is a 15nt base overlap between the cas9 amplification product, the ku amplification product and the ligD amplification product;

(3) There is a base overlap of 15 nt between the 3' end of the cas9 amplification product and the 5' end of the ku amplification product, and the 3' end of the ku amplification product and the 3' end of the ligD amplification product. In-fusion PCR connects the cas9 amplification product, the ku amplification product and the ligD amplification product to obtain the first connection;

(4) Digest the base vector _P BBR1MCS-2 with restriction endonucleases KpnI and HindIII to obtain the digested product of the base vector; the digested product is subjected to gel electrophoresis, gel cutting, and DNA recovery kit to purify the enzyme Then use in-fusion PCR to connect the purified digestion product and the first linker to obtain the vector pNHEJ;

(5) Use NotI and SpeI to double-digest the vector pNHEJ to obtain the digested product of the vector pNHEJ;

(6) Simultaneously introduce two adjacent inverted BsaI restriction sites and two adjacent inverted BsmAI restriction sites into the vector pNHEJ for transcription and formation of sgRNA that can simultaneously bind to two positions of the target gene; Wherein, the DNA fragment with two adjacent inverted BsaI restriction sites is DBsaI, and the DNA fragment with two adjacent inverted BsmAI restriction sites is DBsmAI;

(7) Linking DBsaI and DBsmAI by in-fusion PCR to obtain the second linking product;

(8) The second ligation product and the restriction product of the vector pNHEJ were connected by in-fusion PCR to obtain the CRISPR-Cas9 system for knocking out the target gene, that is, the pCr-NHEJ vector.

Wherein, in step (2), the cas9 protein gene is amplified by using the primer PPLtetO-1-cas9U having the sequence shown in SEQ ID NO: 2 and the primer PCas9D having the sequence shown in SEQ ID NO: 3 to obtain the cas9 amplification product;

Using the primer PPLtetO-1-KuU having the sequence shown in SEQ ID NO: 4 and the primer PkuD having the sequence shown in SEQ ID NO: 5 to amplify the ku protein gene to obtain the ku amplification product;

The ligD protein gene was amplified by using the primer PLtetO-1-ligDU having the sequence shown in SEQ ID NO: 6 and the primer PligDD having the sequence shown in SEQ ID NO: 7 to obtain the ligD amplification product.

Wherein, in step (6), the DNA fragment DBsaI with two adjacent inverted BsaI restriction sites is constructed as follows:

First directly synthesize the DNA sequence BsaIU with the sequence shown in SEQ ID NO: 8 and the DNA sequence BsaID with the sequence shown in SEQ ID NO: 9, wherein the 3' end of BsaIU and the 5' end of BsaID have a 15nt overlapping sequence; Wherein, BsaID contains tracrRNA sequence;

Connect BsaIU and BsaID by in-fusion PCR; Then adopt primer PBsaIU with sequence shown in SEQ ID NO: 10 and primer PBsaID with sequence shown in SEQ ID NO: 11, PCR amplification, promptly obtain DNA fragment DBsaI with two inverted BsaI restriction sites.

Wherein, in step (6), the DNA fragment DBsmAI with two adjacent inverted BsmAI restriction sites is constructed as follows:

First directly synthesize the DNA sequence BsmAIU with the sequence shown in SEQ ID NO: 12 and the DNA sequence BsmAID with the sequence shown in SEQ ID NO: 13; wherein, BsmAID contains the tracrRNA sequence;

The DNA sequence BsmAIU and the DNA sequence BsmAID are connected by in-fusion PCR; then adopt the primer PBsmAIU with the sequence shown in SEQ ID NO: 14 and the primer PBsmAID with the sequence shown in SEQ ID NO: 15 to carry out PCR amplification, namely A DNA fragment DBsmAI with two adjacent inverted BsmAI restriction sites was obtained.

Example 4.

The application of the pCr-NHEJ carrier knockout bacterial gene of embodiment 1 is as follows:

In order to verify the efficiency of the carrier for knocking out genes of different Gram-negative bacteria, the present invention applies the carrier to gene knockout for Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii. Three efflux pump genes tolC (GI: 224015931), RND family drug transporter (locus_tag="A1S_1773") and RND-type multi-drug resistance efflux pump for Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii, respectively Gene mexA (locus_tag="OU9_00435") designed a pair of spacer DNA sequences respectively. The 20nt spacer sequence was designed by the following method:

1) Use the online software (http://crispr.dbcls.jp/) to design the spacer DNA for the first 250nt of the ORF of the above three genes, and compare the spacer DNA with the genomic DNA of the corresponding bacteria by BLAST on PUBMED to remove the The 12nt repeat spacer DNA in the PAM region obtained the spacer sequence with the lowest possibility of off-target.

2) In order to clone two 20bp spacer coding DNA sequences, it is necessary to synthesize the forward and reverse sequences of gcaagcacgccttagtaaccspacer DNA at the same time, and add the sequence "AGCAC" to the 5' end of the forward sequence, and add "AGCAC" to the 3' end G", add "5'-AAAAC-3'" to the 5' end of the reverse sequence, and "G" to the 3' end. The sequence of Spacer DNA is as follows:

Escherichia coli spacer DNA:

Zhengda spacer DNA1: AGCACaaattgctccccattcttttG (SEQ ID.18)

Anti-large spacer DNA1: AAAACATAAGAATGGGGAGCAATTTG (SEQ ID.19)

Zhengda spacer DNA2: AGCACgcaagcacgccttagtaaccG (SEQ ID.20)

Anti-large spacer DNA2: AAAACGGTTACTAAGGCGTGCTTGCG (SEQ ID.21)

Pseudomonas aeruginosa spacer DNA:

Ortho-copper spacer DNA1: AGCACAACGCCAGCCATGCGTGTACG (SEQ ID.22)

Anticopper spacer DNA1: AAAACGTACACGCATGGCTGGCGTTG (SEQ ID.23)

Copper-positive spacer DNA2: AGCACCATCCTCAAGCGCCTGTTCAG (SEQ ID.24)

Anticopper spacer DNA2: AAAACTGAACAGGCGCTTGAGGATGG (SEQ ID.25)

Acinetobacter baumannii spacer DNA:

Abalone spacer DNA1: AGCACTTAAGTTCTTAGGTCAACGGG (SEQ ID.26)

Anti-Abalone spacer DNA2: AAAACCCGTTGACCTAAGAACTTAAG (SEQ ID.27)

Abalone spacer DNA2: AGCACTTGCCTGTGGCATTGTCTGGG (SEQ ID.28)

Anti-Abalone spacer DNA2: AAAACCCCAGACAATGCCACAGGCAAG (SEQ ID.29)

3) The following takes large spacer1 and large spacer2 as examples, and the synthesis method of other spacer DNA is the same:

① Synthesize the sequences 5'-AGCACaaattgctccccattcttt-3' and 5'-GATAAGAATGGGGAGCAATTT-3', mix the two in equal proportions, heat denature and anneal to make the two form complementary chains, and obtain double-stranded DNA with prominent bases at both ends - large spacerD1':

5'-AGCACaaattgctccccattcttat-3'

|||||||||||||||||||||

3'-TTTAACGAGGGGTAAGAATAG-5',

The obtained double-stranded DNA can be inserted into the restriction site of BsaI or BsmAI.

② Synthesize the sequences 5'-AGCACgcaagcacgccttagtaacc-3' and 5'-GGGTTACTAAGGCGTGCTTGC-3', mix the two in equal proportions, heat denature and anneal to make the two form complementary chains, and obtain double-stranded DNA with protruding bases at both ends - large spacerD2':

5'-AGCACgcaagcacgccttagtaacc-3'

|||||||||||||||||||||

3'-CGTTCGTGCGGAATCATTGGG-5',

The obtained double-stranded DNA can be inserted into the restriction site of BsaI or BsmAI.

4) The large spacerD1' and large spacerD2' were cloned into pCr-NHEJ successively and passed through the BsaI or BsmAI restriction site to construct the CRISPR-Cas9 vector targeting the target gene.

5) After successful construction, transform the host bacteria by electroporation, screen resistant clones on an antibiotic plate, and perform colony PCR with a primer located in the knockout region and a primer in the non-knockout region to amplify the target gene (electrophoresis The results are shown in Figure 6A). If the target gene cannot be amplified, it proves that the knockout is successful, otherwise it means that the target gene of the colony has not been knocked out successfully. The primer pair sequences for the three genes are as follows:

Escherichia coli gene knockout identification primers:

Big P1 (SEQ ID.30): 5'-CCCATTCTTATCGGCCTGAG-3'

Big P2 (SEQ ID.31): 5'-CTTGGCGTTGTACAACGTGG-3'

Pseudomonas aeruginosa gene knockout identification primers:

Bao P1 (SEQ ID32.): 5'-AAGTTCTTAGGTCAACGGTG-3'

Bao P2 (SEQ ID.33): 5'-TGTATGTACAGGCGAACTTC-3'

Acinetobacter baumannii gene knockout identification primers:

Copper P1 (SEQ ID.34): 5'-AGCCATGCGTGTACTGGTTC-3'

Copper P2 (SEQ ID.35): 5'-GCTGTCGGTTTTCGCCGGAG-3'

The methods for competent preparation and electroporation transformation of Pseudomonas aeruginosa and Acinetobacter baumannii are exactly the same as the above-mentioned gene knockout method for Escherichia coli. The identification results of knockout genes in Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii are shown in Figure 6A, B and C.

Example 5.

1. Amplification of cas9, ku, and ligD genes and connection of three gene fragments.

1) Amplification of cas9 gene: primers PPLtetO-1-1-cas9U (SEQ ID.4;) and cas9D (SEQ ID.5;) were used to amplify the cas9 gene, and the template was pCas9 plasmid (Addgene, GI:669193757). PCR reaction system: 1 μL of pfu high-fidelity DNA polymerase (Beijing Tiangen), 5 μL of 10×PCR buffer, 1 μL of upstream primer (5 μM), 1 μL of downstream primer (5 μM), 10 ng of template DNA, supplemented with sterilized ultrapure water to 50 μL.

2) ku gene amplification: the ku gene was amplified using primers PPLtetO-1-1-KuU (SEQ ID.6;) and PkuD (SEQ ID.7;), and the template was the pET16b-MtKu plasmid (Science. 2002, 6; 297(5587):1686-9). PCR reaction system: 1 μL of pfu high-fidelity DNA polymerase, 5 μL of 10×PCR buffer, 1 μL of upstream primer (5 μM), 1 μL of downstream primer (5 μM), 10 ng of template DNA, supplemented with sterilized ultrapure water to 50 μL. PCR cycle conditions: Denaturation at 98°C for 10 s, extension at 68°C for 2 min, a total of 30 cycles, and final extension at 72°C for 5 min.

3) ligD gene amplification: the ku gene was amplified using primers PPLtetO-1-1- PligDU (SEQ ID.8;) and PligDD (SEQ ID.9;), and the template was the pET16b-MtligD plasmid [Science. 2002, 6; 297(5587):1686-9]. PCR reaction system: 1 μL of pfu high-fidelity DNA polymerase, 5 μL of 10×PCR buffer, 1 μL of upstream primer (5 μM), 1 μL of downstream primer (5 μM), 10 ng of template DNA, supplemented with sterilized ultrapure water to 50 μL. PCR cycle conditions: Denaturation at 98°C for 10 s, extension at 68°C for 2 min, a total of 30 cycles, and final extension at 72°C for 5 min.

4) PCR product recovery: The above three PCR products were recovered using a PCR product recovery kit from QIAGEN.

5) In-fusion PCR ligation and amplification of cas9, ku, and ligD genes: take the same amount of PCR products of the three genes, and use TBiotech's In-fusion PCR kit for ligation. Reaction system: The PCR products of cas9 , ku and ligD were mixed according to the molar ratio of 1:1:1 (the total amount of DNA was 1 μg), 5×In-Fusion HD Enzyme Premix (In-Fusion? HD Cloning Plus, Clontech, TAKARA ) 2 μL, supplemented with sterilized ultrapure water to 10 μL, and incubated at 37°C for 1 h to connect the above three PCR products. The above ligation products were amplified with primers PPLtetO-1-cas9U and RPligDD. PCR reaction system: 1 μL of pfu high-fidelity DNA polymerase, 5 μL of 10×PCR buffer, 1 μL of upstream primer (5 μM), 1 μL of downstream primer (5 μM), and 1 μL of template. Take 3 μL of PCR product for electrophoresis.

2. Plasmid transformation, extraction and digestion of the broad host plasmid PBBR1MCS-2.

1) Calcium chloride method to prepare E. coli DH5α competent: use an inoculation loop to pick DH5α colonies of monoclonal E. coli into 2ml LB liquid medium, shake and culture overnight at 37°C. The above-mentioned overnight cultured bacteria were transferred to 5ml LB liquid medium at a ratio of 1:100 and cultured with shaking for 2-3 hours (OD ₆₀₀ about 0.2<0.4). Put the above-mentioned bacterial solution in the logarithmic phase of growth on ice for 10 minutes, centrifuge at 8000 rpm at 4°C for 30 seconds, and discard the supernatant. Suspend the cells in 400 μL of 100 mmol/L calcium chloride solution, bathe in ice for 10 minutes, centrifuge at 4°C at 4000 rpm for 5 minutes, and discard the supernatant. Then suspend the bacteria in 400 μL of 100 mmol/L calcium chloride solution pre-cooled on ice. This bacterial solution is the competent bacteria. Pack 100 μL in sterilized Eppendorf tubes, and put them in a refrigerator at 4 °C for transformation.

2) Plasmid transformation: Add 1 μL of plasmid DNA (100ng/μL) to 100 μL of competent bacteria (in a 1.5mL centrifuge tube), mix well, and place on ice for 30 minutes. Immediately place it in a water bath at 42°C for 90 seconds for heat shock, and immediately place it in an ice-water bath to cool for 2 minutes after taking it out. Add 900 μL of SOC liquid medium (without kanamycin) to the bacteria, shake gently on a shaker at 37°C at 200 rpm for 1 hour. Take 10 μL of coating and 1.5% LB agar solid medium (containing kanamycin 50ug/ml) on a plate, culture at 37°C for 12 hours, and grow a single colony.

3) Plasmid expansion culture and mass extraction: pick a single colony, inoculate in 3ml LB liquid medium (containing kanamycin 50ug/ml), shake gently on a shaker at 37°C at 200 rpm, and incubate for 12 hours. Plasmids were extracted using a plasmid extraction kit (high-purity plasmid mini-extraction kit, Beijing Tiangen).

4) Plasmid digestion: Reaction system: 1 μg plasmid, 1 μL each of KpnI (NEB) and HindIII (NEB), 1 μL of 10× buffer Cutsmart, supplemented with water to 20 μL, digested at 37°C for 2 hours. Digested products were recovered using a DNA gel recovery kit (QIAGEN).

3. Ligation of cas9, ku, and ligD genes to the vector: Take the three gene ligation fragments and the enzyme-digested vector pBBR1MCS-2 and mix them at a molar ratio of 1:1 (the total amount of DNA is 1 μg), add 5×In-Fusion HD Enzyme Premix (In-Fusion? HD Cloning Plus, Clontech, TAKARA) 2μL, supplemented with sterilized ultrapure water to 10μL, incubated at 37°C for 1h, to connect the above two DNA fragments, pNHEJ was obtained when the connection was successful.

4. Design of sgRNA: first obtain the gene sequences of tolC (GI:224015931), RND family drug transporter (locus_tag="A1S_1773") and RND-type multi-drug resistance efflux pump gene mexA (locus_tag="OU9_00435") on PUBMED , and then select the first 250nt sequence of the open reading frame as the target site, and use the online sgRNA design software (http://crispr.dbcls.jp/) to find the spacer sequence on the target gene. The sequence pattern is 3'-N ₂₀ NGG -5', and the spacer is the sequence complementary to N ₂₀ . Then, BLAST software was used on pubmed to compare with the whole genome of Pseudomonas aeruginosa PAO1, and the 12-base repeat spacer sequence at the 3' end was removed to avoid off-target effects.

5. Cloning of Spacer DNA:

Since the construction of the vectors for the three genes is completely the same, only the construction of the knockout vector of the Escherichia coli tolC gene is described here as an example, and the construction steps and experimental conditions of the other two vectors are completely the same.

Preparation of double-stranded Spacer DNA:

Take the synthesized Zhengda spacer DNA1 (SEQ ID.18) and anti-big spacer DNA1 (SEQ ID.19) fragments, mix them at equimolar concentrations, and then denature and anneal.

The denaturation and annealing system is:,

2.5μl forward spacer DNA (100μM)

2.5μl reverse spacer DNA (100μM)

1μl NEB buffer2

4 μl sterile water

Run in the PCR instrument according to the following program: 95°C, 5min; 95–85°C at-2°C/s; 85–25°C at-0.1°C/s. After annealing, the two DNA single strands complement each other to form a double strand. The denaturation and annealing conditions of Zhengda spacer DNA2 (SEQ ID.20) and anti-big spacer DNA1 (SEQ ID.21) are the same as above.

Cloning of double-stranded Spacer DNA

6. First carry out single enzyme digestion on the plasmid, reaction system: 1 μg of plasmid pNHEJ, 1 μL of BsaI (NEB) or BsmAI (NEB), 1 μL of 10× buffer Cutsmart, add water to 20 μL, digest at 37°C for 2 hours, and then digest with the enzyme The cut products were recovered using a DNA gel recovery kit (QIAGEN). Mix double-stranded Spacer DNA and vector recovery fragments at a ratio of 1:1, add 5×In-Fusion HD Enzyme Premix (In-Fusion? HD Cloning Plus, Clontech, TAKARA) 2 μL, add sterilized ultrapure water to 10 μL , and incubated at 37°C for 1h to ligate the above two DNA fragments. The ligation product was transformed into Escherichia coli DH5α and the plasmid was extracted (competent preparation by calcium chloride method, plasmid transformation, and extraction were as described above). Then the plasmid was digested with BsmAI (NEB) or BsaI (NEB). Reaction system: 1 μL, 1 μL of 10× buffer Cutsmart, supplemented with water to 20 μL, digested at 37°C for 2 hours, and then recovered the digested product with a DNA gel recovery kit (QIAGEN). Mix double-stranded Spacer DNA and vector recovery fragments at a ratio of 1:1 (the total amount of DNA is 1 μg), add 5×in-Fusion HD Enzyme Premix (In-Fusion? HD Cloning Plus, Clontech, TAKARA) 2 μL, supplement Sterilize ultrapure water to 10 μL and incubate at 37°C for 1 h to ligate the above two DNA fragments. After the connection is successful, the plasmid vector for gene site-directed knockout is obtained. The ligated product was transformed into Escherichia coli DH5α, and the plasmid was extracted by the method described above.

Note: Since the host strain DH5α carries the TolC gene, after accepting the plasmid, the TolC gene has already been knocked out, and the corresponding site of the target gene has been cut. connect. If DH5α receives a gene knockout plasmid that knocks out Pseudomonas aeruginosa or Acinetobacter baumannii, the above situation will not happen theoretically.

gene knockout experiment

Preparation of competent bacteria for electroshock transformation (the preparation methods of Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii are exactly the same, and are described here):

1) Preparation of electroporation competent cells

① Pick up monoclonal colonies with a pipette tip and put them into a glass test tube filled with 2ml LB liquid medium. 37°C, 220rpm, incubate for 14-16 hours.

② On the second day, inoculate in 100ml LB liquid medium at a volume ratio of 1:100, shake at 37°C and 220rpm for 2-3 hours, measure OD every half hour, when the OD value reaches 0.3-0.4 , stop culturing.

③ Pre-cool the bacterial culture solution on ice for 30 minutes, then divide the bacterial solution into 50ml pre-cooled centrifuge tubes, and centrifuge at 4000g for 10 minutes at 4°C.

④ Discard the supernatant, add a small amount of sterilized ultrapure water to the centrifuge tube to suspend the precipitate, then add an appropriate amount of sterilized ultrapure water, and centrifuge at 4000rpm for 10 minutes at 4°C.

⑤ Repeat step 4.

⑥ After discarding the supernatant, add a small amount of sterilized and pre-cooled 10% glycerol on ice to resuspend the bacteria, then fill up with 10% glycerol, centrifuge at 4°C, 4000rpm for 10min.

⑦ Discard the supernatant, add 2ml of 10% glycerol to each centrifuge tube, and resuspend the bacteria, which is used for electrotransformation.

2) Electroconversion

① Take 2 μl of CRISPR-Cas9 vectors targeting different genes in a 1.5ml centrifuge tube, and place it together with a 0.1CM electrode cup on ice for pre-cooling.

② Transfer 40μL of thawed competent cells to this 1.5ml centrifuge tube, mix carefully, and place on ice for 10min.

③ Transfer the mixture to the pre-cooled electrode cup, tap the electrode cup gently to make the mixture evenly enter the bottom of the electrode cup;

④ Push the electrode cup into the electrotransformer for electroporation transformation (1.8kV, 6mS). Immediately after the electroporation, add 1000 μl of SOC liquid medium to the electroporation cup, resuspend the cells, and transfer to a 1.5ml centrifuge tube middle.

⑤ Resuscitate at 37°C, 220-250rpm for 1 hour.

⑥ Take 10 μL of the transformation product plus 90 μL SOC and spread it on an LB plate containing kanamycin (50 μg/ml), put it in a 37°C greenhouse, and culture overnight.

⑦The colony was selected the next day, and the positive rate of gene knockout was identified by colony PCR.

3) Knockout rate identification

PCR was performed using identifying primers as previously described. Mix the PCR reaction system in the PCR tube according to the following conditions:

pfu high-fidelity DNA polymerase 1 μL, 10×PCR buffer 5 μL, upstream primer (5 μM ) 1 μL, downstream primer (5 μM ) 1 μL, add sterilized ultrapure water to 50 μL, then randomly select 10 colonies from the plate, and inoculate A very small amount of bacteria was picked by the needle tip and inoculated into the above PCR reaction system. Then perform PCR amplification: PCR cycle conditions: denaturation at 98°C for 10 s, extension at 52°C for 2 min, a total of 30 cycles, and final extension at 72°C for 5 min.

The identification results showed that the gene knockout efficiency of this method could reach 87.5%-100%.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than limiting the protection scope of the present invention, although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand , the technical solution of the present invention may be modified or equivalently replaced without departing from the spirit and scope of the technical solution of the present invention.

gene sequence list

<110> Guangdong Medical College

<120> A pCr-NHEJ vector and its construction method and its application for bacterial gene knockout

<160> 15

<210> SEQ ID NO: 1

<211> 12621

<212> DNA

<213> pCr-NHEJ

<400> SEQ ID NO: 1

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gttctttgcggacggtccaatacgcagtcaaggacttggtggccgagcgctggatctccgtcgtgaagctcaacggccccggcaccgtgtcggcctacgtggtcaatgaccgcgtggcgtggggccagccccgcgaccagttgcgcctgtcggtgttcagtgccgccgtggtggttgatcacgacgaccaggacgaatcgctgttggggcatggcgacctgcgccgcatcccgaccctgtatccgggcgagcagcaactaccgaccggccccggcgaggagccgcccagccagcccggcattccgggcatggaaccagacctgccagccttgaccgaaacggaggaatgggaacggcgcgggcagcagcgcctgccgatgcccgatgagccgtgttttctggacgatggcgagccgttggagccgccgacacgggtcacgctgccgcgccggtagcacttgggttgcgcagcaacccgtaagtgcgctgttccagactatcggctgtagccgcctcgccgccctataccttgtctgcctccccgcgttgcgtcgcggtgcatggagccgggccacctcgacctgaatggaagccggcggcacctcgctaacggattcaccgtttttatcaggctctgggaggcagaataaatgatcatatcgtcaattattacctccacggggagagcctgagcaaactggcctcaggcatttgagaagcacacggtcacactgcttccggtagtcaataaaccggtaaaccagcaatagacataagcggctatttaacgaccctgccctgaaccgacgaccgggtcgaatttgctttcgaatttctgccattcatccgcttattatcacttattcaggcgtagcaccaggcgtttaagggcaccaataactgccttaaaaaaattacgccccgccctgccactcatcgcagtcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaaca aaatattaacgcttacaatttccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgagcgcgcgtaatacgactcactatagggcgaattggagctccaccgcggtggcGAGATTGACATCCCTATCAGTGATAGAGATACTGAGCACGAGACCaaaGGTCTCgttttagagctaGAAAtagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcTTTTTTTGACGACGACGACGACGAGATTGACATCCCTATCAGTGATAGAGATACTGAGCACGAGACaaaGTCTCgttttagagctaGAAAtagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcTTTTTTTgatcccccgggctgcaggaattcgatatcaGAGATTGACATCCCTATCAGTGATAGAGATACTGAGCACAAGGAGTATACCATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGATCACTGATGATTATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTGACAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGTTATCTACAGGAGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGTCATCCTATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAA CTATCTATCATCTGCGAAAAAAATTGGCAGATTCTACTGATAAAGTGGATTTGCGCTTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCATTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTTATCCAGTTGGTACAAACCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGTAGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAAAAATGGATTGTTTGGGAATCTCATTGCTTTGTCATTGGGATTGACCCCTAATTTTAAATCAAATTTTGATTTGGCAGAAGATACTAAATTACAGCTTTCAAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTGTTTTTGGCAGCTAAGAATTTATCAGATGCTATTTTACTTTCAGATATCCTAAGAGTAAATAGTGAAATAACTAAGGCTCCCCTATCAGCTTCAATGATTAAACGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGACAACAACTTCCAGAAAAGTATAAAGAAATCTTTTTTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATAAATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGCGAAACTAAATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTTGACAACGGCTCTATTCCCTATCAAATTCACTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATCCATTTTTAAAAGACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCG GAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAAGTACTACCAAAACATAGTTTGCTTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACTGAGGGAATGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGATTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGCGTTGAAGATCGGTTTAATACGTCATTAGGTACCTACCATGATTTGCTAAAAATTATCAAAGATAAAGATTTTTTGGATAATGAAGAAAATGAAGATATTTTAGAGGATATTGTTTTAACATTGACCTTATTTGAAGATAAGGAAATGATTGAGGAACGACTTAAAAAGTATGCTAACCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCATTATACTGGCTGGGGGCGTTTGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTTGAAATCAGATGGTTTTGCCAATCGCAACTTTATGCAGCTTATCAATGATGATAGTTTAACATTCAAGGAAGCTATTCAAAAAGCACAGGTGTCTGGACAAGGCCATAGTTTACATGAACAGATTGCTAACTTAGCTGGCAGTCCTGCTATTAAAAAAGGTATTTTACAGACTGTAAAAATTGTTGATGAACTGGTCAAAGTAATGGGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAAAATCAGACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAACGAATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTT GAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATTATCTCCAAAATGGAAGAGACATGTATGTGGACCAAGAATTAGATATTAATCGTTTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCCTTAAAGACGATTCAATAGACAATAAGGTGCTAACACGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAACTATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTAACGAAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTTTGGATAGTCGCATGAATACTAAATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATCTAAATTAGTTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCCATGATGCGTATCTTAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTTGAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGTTCGTAAAATGATTGCTAAGTCTGAGCAGGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCTAATATCATGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGAAACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCCCCAAGTCAATATTGTCAAGAAAACAGAAGTACAGACAGGTGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGACAAGCTTATTGCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTG ATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTTGAAAAAAATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAGAAAAGACTTAATCATTAAACTACCTAAATATAGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAAAAGGAAATGAGCTGGCTTTGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGTCATTATGAAAAATTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTGGAGCAGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATTTTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCAATACGTGAACAAGCAAAAAATATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATATTTTGATACAACAATTGAACGAAACCGTTACAAATCAATTAAGGAAGTCTTAGATGCAACCCTCATCCATCAATCCATCACTGGTCTTTATGAAACACGCATTGATTTGAGTCAGCTAGGAGGTGACTGAGAGATTGACATCCCTATCAGTGATAGAGATACTGAGCACAAGGAGTATACCATGCGAGCCATTTGGACGGGTTCGATCGCCTTCGGGCTGGTGAACGTGCCGGTCAAGGTGTACAGCGCTACCGCAGACCACGACATCAGGTTCCACCAGGTGCACGCCAAGGACAACGGACGCATCCGGTACAAGCGCGTCTGCGAGGCGTGTGGCGAGGTGGTCGACTACCGCGATCTTGCCCGGGCCTACGAGTCCGGCGACGGCCAAATGGTGGCGATCACCGACGACGACATCGCCAGCTT GCCTGAAGAACGCAGCCGGGAGATCGAGGTGTTGGAGTTCGTCCCCGCCGCCGACGTGGACCCGATGATGTTCGACCGCAGCTACTTTTTGGAGCCTGATTCGAAGTCGTCGAAATCGTATGTGCTGCTGGCTAAGACACTCGCCGAGACCGACCGGATGGCGATCGTGCATTTCACGCTGCGCAACAAGACCAGGCTGGCGGCGTTGCGCGTCAAGGATTTCGGCAAGCGAGAGGTGATGATGGTGCACACGTTGCTGTGGCCCGATGAGATCCGCGACCCCGACTTCCCGGTGCTGGACCAGAAGGTGGAGATCAAACCCGCGGAACTCAAGATGGCCGGCCAGGTGGTGGACTCGATGGCCGACGACTTCAATCCGGACCGCTACCACGACACCTACCAGGAGCAGTTACAGGAGCTGATCGACACCAAACTCGAAGGTGGGCAGGCATAAGGTGGGCAGGCATGAGATTGACATCCCTATCAGTGATAGAGATACTGAGCACAAGGAGTATACCATGGGTTCGGCGTCGGAGCAACGGGTGACGCTGACCAACGCCGACAAGGTGCTCTATCCCGCCACCGGGACCACAAAGTCCGATATCTTCGACTACTACGCCGGTGTTGCCGAAGTCATGCTCGGCCACATCGCGGGACGGCCGGCGACGCGCAAGCGCTGGCCTAACGGCGTCGACCAACCCGCGTTCTTCGAAAAGCAGTTGGCGTTGTCGGCGCCGCCTTGGCTGTCACGTGCAACGGTGGCGCACCGGTCCGGGACGACGACCTATCCGATCATCGATAGCGCAACCGGGCTGGCCTGGATCGCCCAACAGGCGGCGCTGGAGGTGCACGTGCCGCAGTGGCGGTTTGTCGCCGAGCCCGGATCAGGTGAGTTAAATCCGGGCCCGGCAACGCGTTTGGTGTTCGACCTGGACCCGGGCGAAGGCGTGATGATGGCCCAGCTGGCCGAGGTGGCGCGCGCGGTTCGTGATCTTCTCGC CGATATCGGGTTGGTCACCTTCCCGGTCACCAGCGGCAGCAAGGGATTGCATCTGTACACACCGCTGGATGAGCCGGTGAGCAGCAGGGGAGCCACGGTGTTGGCCAAGCGCGTCGCGCAGCGATTGGAGCAGGCGATGCCCGCGTTGGTCACCTCGACCATGACCAAAAGCCTGCGGGCCGGGAAGGTGTTTGTGGACTGGAGCCAGAACAGCGGCTCGAAGACCACCATCGCGCCGTACTCACTACGTGGCCGGACGCATCCGACCGTCGCGGCGCCACGCACCTGGGCGGAGCTCGACGACCCCGCACTGCGTCAGCTCTCCTACGACGAGGTGCTGACCCGGATTGCCCGCGACGGCGATCTGCTCGAGCGGCTGGATGCCGACGCTCCGGTAGCGGACCGGTTGACCCGATACCGCCGCATGCGCGACGCATCGAAAACTCCCGAGCCGATTCCCACGGCGAAACCCGTTACCGGAGACGGCAATACGTTCGTCATCCAGGAGCATCACGCGCGTCGGCCGCACTACGATTTCCGGCTGGAATGCGACGGCGTGCTGGTCTCGTGGGCGGTACCGAAAAACCTGCCCGACAACACATCGGTTAACCATCTAGCGATACACACCGAGGACCACCCGCTGGAATACGCCACGTTCGAGGGCGCGATTCCCAGCGGGGAGTACGGCGCCGGCAAGGTGATCATCTGGGACTCCGGCACTTACGACACCGAGAAGTTCCACGATGACCCGCACACGGGGGAGGTCATCGTGAATCTGCACGGCGGCCGGATCTCTGGGCGTTATGCGCTGATTCGGACCAACGGCGATCGGTGGCTGGCGCACCGCCTAAAGAATCAGAAAGACCAGAAGGTGTTCGAGTTCGACAATCTGGCCCCAATGCTTGCCACGCACGGCACGGTGGCCGGTCTAAAGGCCAGCCAGTGGGCGTTCGAAGGCAAGTGGGACGGCTACCGGTTGCTGGTTGAGGCTGACCACGGC GCCGTGCGGCTGCGGTCCCGCAGCGGGCGCGATGTCACCGCCGAGTATCCGCAATTGCGGGCATTGGCGGAGGATCTCGCCGATCACCACGTGGTGCTGGACGGCGAGGCCGTCGTACTTGACTCCTCTGGTGTGCCCAGCTTCAGCCAGATGCAGAATCGGGGCCGCGACACCCGTGTCGAGTTCTGGGCGTTCGACCTGCTCTACCTCGACGGCCGCGCGCTGCTAGGCACCCGCTACCAAGACCGGCGTAAGCTGCTCGAAACCCTAGCTAACGCAACCAGTCTCACCGTTCCCGAGCTGCTGCCCGGTGACGGCGCCCAAGCGTTTGCGTGCTCGCGCAAGCACGGCTGGGAGGGCGTGATCGCCAAGAGGCGTGACTCGCGCTATCAGCCGGGCCGGCGCTGCGCGTCGTGGGTCAAGGACAAGCACTGGAACACCCAGGAAGTCGTCATTGGTGGCTGGCGCGCCGGGGAAGGCGGGCGCAGCAGTGGCGTCGGGTCGCTGCTCATGGGCATCCCCGGTCCAGGTGGGCTGCAGTTCGCCGGGCGGGTCGGTACCGGCCTCAGCGAACGCGAACTGGCCAACCTCAAGGAGATGCTGGCGCCGCTGCATACCGACGAGTCCCCCTTCGACGTACCACTGCCCGCGCGTGACGCCAAGGGCATCACATATGTCAAGCCGGCGCTGGTTGCAGAGGTGCGCTACAGCGAGTGGACTCCGGAGGGCCGGCTGCGTCAATCAAGCTGGCGTGGGCTGCGGCCGGACAAGAAACCCAGTGAGGTGGTGCGCGAATGAccagcttttgttccctttagtgagggttaattgcgcgcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcggg aaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgcatgcataaaaactgttgtaattcattaagcattctgccgacatggaagccatcacaaacggcatgatgaacctgaatcgccagcggcatcagcaccttgtcgccttgcgtataatatttgcccatgggggtgggcgaagaactccagcatgagatccccgcgctggaggatcatccagccggcgtcccggaaaacgattccgaagcccaacctttcatagaaggcggcggtggaatcgaaatctcgtgatggcaggttgggcgtcgcttggtcggtcatttcgaaccccagagtcccgctcagaagaactcgtcaagaaggcgatagaaggcgatgcgctgcgaatcgggagcggcgataccgtaaagcacgaggaagcggtcagcccattcgccgccaagctcttcagcaatatcacgggtagccaacgctatgtcctgatagcggtccgccacacccagccggccacagtcgatgaatccagaaaagcggccattttccaccatgatattcggcaagcaggcatcgccatgggtcacgacgagatcctcgccgtcgggcatgcgcgccttgagcctggcgaacagttcggctggcgcgagcccctgatgctcttcgtccagatcatcctgatcgacaagaccggcttccatccgagtacgtgctcgctcgatgcgatgtttcgcttggtggtcgaatgggcaggtagccggatcaagcgtatgcagccgccgcattgcatcagccatgatggatactttctcggcaggagcaaggtgagatgacaggagatcctgccccggcacttcgcccaatagcagccagtcccttcccgcttcagtgacaacgtcgagcacagctgcgcaaggaacgcccgtcgtggccagccacgatagccgcgctgcctcgtcctgcagttcattcagggca ccggacaggtcggtcttgacaaaaagaaccgggcgcccctgcgctgacagccggaacacggcggcatcagagcagccgattgtctgttgtgcccagtcatagccgaatagcctctccacccaagcggccggagaacctgcgtgcaatccatcttgttcaatcatgcgaaacgatcctcatcctgtctcttgatcagatcttgatcccctgcgccatcagatccttggcggcaagaaagccatccagtttactttgcagggcttcccaaccttaccagagggcgccccagctggcaattccggttcgcttgctgtccataaaaccgcccagtctagctatcgccatgtaagcccactgcaagctacctgctttctctttgcgcttgcgttttcccttgtccagatagcccagtagctgacattcatcccaggtggcacttttcggggaaatgtgcgcgcccgcgttcctgctggcgctgggcctgtttctggcgctggacttcccgctgttccgtcagcagcttttcgcccacggccttgatgatcgcggcggccttggcctgcatatcccgattcaacggccccagggcgtccagaacgggcttcaggcgctcccgaaggt

the

<210> SEQ ID NO: 2

<211> 89

<212>DNA

<213> PPLtetO-1-cas9U

<400> SEQ ID NO: 2

gctctagaactagtg GA GATTGA CATCCCTATCAGTGATAGA GATACT GAGCACAAGGAGTATACC ATGGATAAGAAATACTCAATAGG

the

<210> SEQ ID NO: 3

<211> 37

<212>DNA

<213> cas9D

<400> SEQ ID NO: 3

agggatgtcaatctc TCAGTCACCTCCTAGCTGACTC

the

<210> SEQ ID NO: 4

<211> 88

<212> DNA

<213> PPLtetO-1-KuU

<400> SEQ ID NO: 4

gctctagaactagtg GA GATTGA CATCCCTATCAGTGATAGA GATACT GAGCACAAGGAGTATACC ATGCGAGCCATTTGGACGGGTT

the

<210> SEQ ID NO: 5

<211> 23

<212> DNA

<213> PkuD

<400> SEQ ID NO: 5

ATGCCTGCCCACCTTCGAGTTTG

the

<210> SEQ ID NO: 6

<211> 84

<212>DNA

<213> PLtetO-1-ligDU

<400> SEQ ID NO: 6

gctctagaactagtg GA GATTGA CATCCCTATCAGTGATAGA GATACT GAGCACAAGGAGTATACC ATGGGTTCGGCGTCGGAG

the

<210> SEQ ID NO: 7

<211> 83

<212>DNA

<213> PligDD

<400> SEQ ID NO: 7

GGGAACAAAGCTGG CAAAAAACCCCTCAAGACCCGTTTAGAGGCCCCAAGGGGTTATGCTAGTCATTCGCGCACCACCTCACT

the

<210> SEQ ID NO: 8

<211> 81

<212> DNA

<213> BsaIU

<400> SEQ ID NO: 8

GA GATTGA CATCCCTATCAGTGATAGA GATACT GAGCAC gagacc aaa GGTCTC gttttagagctaGAAAtagcaagttaa

the

<210> SEQ ID NO: 9

<211> 72

<212> DNA

<213> BsaID

<400> SEQ ID NO: 9

GAAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTC

the

<210> SEQ ID NO: 10

<211> 33

<212>DNA

<213> PBSaIU

<400> SEQ ID NO: 10

ctccaccgcggtggcGAGATTGACATCCCTATC

the

<210> SEQ ID NO: 11

<211> 33

<212> DNA

<213> PBsaID

<400> SEQ ID NO: 11

GTCGTCGTCGTCGTCAAAAAAAGCACCGACTCG

the

<210> SEQ ID NO: 12

<211> 79

<212>DNA

<213> BsmAIU

<400> SEQ ID NO: 12

GA GATTGA CATCCCTATCAGTGATAGA GATACT GAGCAC gagac aaa gtctcg ttttagagctaGAAAtagcaagttaa

the

<210> SEQ ID NO: 13

<211> 71

<212>DNA

<213> BsmAID

<400> SEQ ID NO: 13

GAAAtagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcTTTTTTT

the

<210> SEQ ID NO: 14

<211> 33

<212> DNA

<213> PBsmAIU

<400> SEQ ID NO: 14

GACGACGACGACGAC GA GATTGA CATCCCTATC

the

<210> SEQ ID NO: 15

<211> 33

<212>DNA

<213> PBsmAID

<400> SEQ ID NO: 15

GCCACCGCGGTGGAGAAAAAAAGCACCGACTCG

Claims (7)

1. A pCr-NHEJ vector, characterized in that: the sequence of the pCr-NHEJ vector has a sequence shown in SEQ ID NO. 1.

2. Use of the pCr-NHEJ vector of claim 1 for site-directed bacterial gene knock-out.

3. The method of claim 1 for constructing pCr-NHEJ vector, wherein: the method comprises the following steps:

(1) broad host plasmids using gram-negative bacteria_PBBR1MCS-2 (GI: 773412) as basic vector;

(2) the following three protein genes were amplified separately using upstream and downstream primers with prokaryotic promoter PLtetO-1 for mRNA transcription:cas9（GI:674296984）、ku(GI: 444893469) andligD(GI: 444893469) to obtaincas9The amplification product,kuAmplification products andligD(ii) amplification products; wherein,cas9、kuandligDthe open reading frames of the three protein genes are all located downstream of the independent PLtetO-1 so thatcas9、kuAndligDthe expression of the three protein genes is regulated and controlled by the promoter; the above-mentionedcas9Amplification product, thekuAmplification product and the sameligDThe amplified products have 15nt base overlaps;

(3) the above-mentionedcas93' end of amplification product and the sameku5' end of the amplification product, theku3' end of amplification product and the sameligDThe 3' ends of the amplified products are overlapped by 15nt bases, and the amplified products are connected by in-fusion PCRcas9Amplification product, thekuAmplification product and the sameligDAmplifying the product to obtain a first adaptor;

(4) double digestion of the basic vector with the restriction endonucleases KpnI and HindIII_PBBR1MCS-2 to obtain the enzyme digestion product of the basic vector; performing gel electrophoresis and gel cutting on the enzyme digestion product, and purifying the enzyme digestion product by adopting a DNA recovery kit; then adopting in-fusion PCR to connect the purified enzyme digestion product and the first connector to obtain a vector pNHEJ;

(5) carrying out double enzyme digestion on the vector pNHEJ by using NotI and SpeI to obtain an enzyme digestion product of the vector pNHEJ;

(6) simultaneously introducing two adjacent inverted BsaI enzyme cutting sites and two adjacent inverted BsmAI enzyme cutting sites into the vector pNHEJ for transcription to form sgRNA capable of simultaneously combining two positions of a target gene; wherein, the DNA segment with two adjacent inverted BsaI enzyme cutting sites is DBsaI, and the DNA segment with two adjacent inverted BsmAI enzyme cutting sites is DBsmaI;

(7) linking DBsai and DBsmaI through in-fusion PCR to obtain a second ligation product;

(8) and (3) connecting the second connection product with the enzyme digestion product of the vector pNHEJ by in-fusion PCR to obtain a CRISPR-Cas9 system for knocking out the target gene, namely a pCr-NHEJ vector.

4. The method of claim 3, wherein the vector comprises the following steps: in the step (2), the primer PPLtetO-1-cas9U with the sequence shown in SEQ ID NO. 2 and the primer PCas9D with the sequence shown in SEQ ID NO. 3 are adopted to amplify the primercas9Protein gene, obtainingcas9(ii) amplification products;

the amplification of the DNA fragment was carried out using the primer PPLtetO-1-KuU having the sequence shown in SEQ ID NO. 4 and the primer PkuD having the sequence shown in SEQ ID NO. 5kuProtein gene, obtainingku(ii) amplification products;

the amplification of the said sequence was carried out using the primer PLtetO-1-ligDU with the sequence indicated by SEQ ID NO. 6 and the primer PIigDD with the sequence indicated by SEQ ID NO. 7ligDProtein gene, obtainingligDAnd (4) amplifying the product.

5. The method of claim 3, wherein the vector comprises the following steps: in the step (6), the DNA fragment DBsai with two adjacent inverted BsaI cleavage sites is constructed as follows:

firstly, directly synthesizing a DNA sequence BsaIU with a sequence shown in SEQ ID NO. 8 and a DNA sequence BsaID with a sequence shown in SEQ ID NO. 9, wherein the 3 'end of the BsaIU and the 5' end of the BsaID have 15nt overlapping sequences; wherein, BsaID contains tracrRNA sequence;

BsaIU and BsaID were ligated by in-fusion PCR; then, a primer PBsaiU with a sequence shown by SEQ ID NO. 10 and a primer PBsaiD with a sequence shown by SEQ ID NO. 11 are adopted for PCR amplification, and a DNA fragment DBsai with two adjacent inverted BsaI enzyme cutting sites is obtained.

6. The method of claim 3, wherein the vector comprises the following steps: in the step (6), the DNA fragment DBSMAI with two adjacent inverted BsmAI enzyme cutting sites is constructed as follows:

firstly, directly synthesizing a DNA sequence BsmIU with a sequence shown in SEQ ID NO. 12 and a DNA sequence BsmAID with a sequence shown in SEQ ID NO. 13; wherein BsmAID contains tracrRNA sequence;

ligating said DNA sequence BsmAIU and said DNA sequence BsmAID by in-fusion PCR; then, a primer PBsmaU with a sequence shown in SEQ ID NO. 14 and a primer PBsmaD with a sequence shown in SEQ ID NO. 15 are adopted for PCR amplification, and a DNA fragment DBsmaI with two adjacent inverted BsmAI enzyme cutting sites is obtained.

7. Use of the pCr-NHEJ vector constructed by the method of any one of claims 3 to 6 for site-directed bacterial gene knock-out.