CN116254284A

CN116254284A - pCasRA plasmid and preparation method and application thereof

Info

Publication number: CN116254284A
Application number: CN202310081742.0A
Authority: CN
Inventors: 熊文广; 常依; 曾振灵; 万鹏; 谢龙飞; 张旭财
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2023-01-17
Filing date: 2023-01-17
Publication date: 2023-06-13

Abstract

The invention discloses a pCasRA plasmid, the nucleotide information of which is shown as SEQ ID NO: 1. The pCasRA plasmid takes the pCas9 plasmid as a vector skeleton, a replicon of the oriT and pRA0726 plasmids and a cefoxitin Ding Kang gene of a conjugation transfer site are connected to the vector skeleton, and the crRNA part in the pCas9 plasmid is replaced by a connecting product of a Cas9 protein gene (SEQ ID NO: 20) and an integrated sgRNA (SEQ ID NO: 7). The pCasRA plasmid can be transferred into Richbacter anatipestifer in escherichia coli through conjugation, and can stably express Cas9 protein genes in Richbacter anatipestifer to edit genome DNA so as to realize gene knockout and/or single-base mutation.

Description

pCasRA plasmid and preparation method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a pCasRA plasmid and a preparation method and application thereof.

Background

The Riemerella anatipestifer mainly infects duckling, turkeys, geese and other birds, is widely popular nationwide, and is horizontally transmitted mainly through respiratory tract, digestive tract, mosquito bite and other approaches, such as spray, polluted physical objects, water source, excrement, damaged lower limb skin and the like. The disease mainly presents with typical "three week inflammation" symptoms, namely ballooning inflammation, perihepatic inflammation and cellulosic pericarditis. The affected young birds mostly have multiple clinical symptoms such as listlessness, diarrhea, head and neck tremor, and stark strain, and clinically the disease is also called duck infectious serositis, duck septicemia or duck plague syndrome, and the death rate can reach 80 percent.

The serotypes of the Riemerella anatipestifer are complex, the cross protection effect among the serotypes is not obvious, and in addition, the situation that multiple serotypes are popular at the same time in the same farm in production is possible, so that the effective prevention and control of the serotypes are difficult to carry out by vaccine means. In addition, the Riemerella anatipestifer has a wide drug resistance spectrum, and researches report that the Riemerella anatipestifer has drug resistance to various antibiotics such as chloramphenicol, tetracyclines, quinolones, macrolides, lincomamides, aminoglycosides, cephalosporins and the like. Therefore, it is also difficult to control infection of Riemerella anatipestifer by antibiotic therapy in production. In order to better understand virulence factors and pathogenic mechanisms of the riemerella anatipestifer, a new way for preventing and controlling the riemerella anatipestifer is searched, and an efficient gene editing method is established. At present, the gene research of the Riemerella anatipestifer adopts the traditional homologous recombination technology, but the development of the Riemerella anatipestifer in the experimental research is limited due to the limitation of the homologous recombination technology.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides a pCasRA plasmid, a preparation method and application thereof.

A first object of the present invention is to provide a pCasRA plasmid.

The second object of the invention is to provide the application of the pCasRA plasmid in rapid gene editing of Riemerella anatipestifer.

A third object of the present invention is to provide a method for producing the above pCasRA plasmid.

The fourth object of the invention is to provide a kit for rapid gene editing of Riemerella anatipestifer.

CRISPR/Cas9 is a novel gene editing technology developed in recent years. It has great potential to innovate genome modification and mutation technology in living body. This technology was originally derived from the innate immune system of microorganisms and is a very effective method for microorganisms against phage invasion. It is mainly composed of an endonuclease Cas9 and an sgRNA that mediates RNA. Cas9 endonuclease is able to specifically bind to sgrnas, target recognition of the genome and achieve double strand breaks of genomic DNA by base complementary pairing of the sgrnas with genomic DNA. By combining gene recombinase, the technology can rapidly, conveniently and accurately target any part of modified and mutated genome. With the breakthrough development of large-scale DNA microarray synthesis technology, the technology can realize large-scale transcription inhibition and activation at the genome level or specific gene group, and greatly improves the efficiency of functional gene screening.

The CRISPR/Cas9 genome editing technology has realized efficient genome editing in mammalian cells and some model organisms, such as escherichia coli, yeast and the like, but has not realized genome editing in Riemerella anatipestifer, so that the genetic operation of Riemerella anatipestifer is greatly simplified, the screening efficiency of functional genes is improved, and the method lays a foundation for subsequent gene biological function research, drug target discovery, novel drug design and gene therapy.

In order to achieve the above object, the present invention is realized by the following means:

a pCasRA plasmid having nucleotide sequence information as set forth in SEQ ID NO: 1.

The invention also claims the application of the pCasRA plasmid in the rapid gene editing of Riemerella anatipestifer.

Preferably, the gene editing is a gene knockout and/or a single base mutation.

More preferably, the competent E.coli in step S4 is competent E.coli DH 5. Alpha.

More preferably, the gene knockout is a knockout of the dprA gene (nc_ 017045.1) with gene number nc_ 017045.1.

More preferably, the single base mutation is a mutation of cytosine C at position 271 in the dprA gene to adenine a.

The gene knockout method comprises the following steps:

s1, selecting a DNA fragment of 20bp before an NGG sequence on a gene to be knocked out as interval DNA, obtaining a reverse complementary sequence of the interval DNA, adding GAAA at the 5 'end of the interval DNA, adding AAAC at the 5' end of the reverse complementary sequence, annealing and connecting the interval DNA and the reverse complementary sequence, and then inserting the interval DNA and the reverse complementary sequence into a BsaI cleavage site of the pCasRA plasmid of claim 1 to obtain the pCasRA-spacer plasmid; n of the NGG sequence is any base;

s2, amplifying a DNA fragment of 600-1000 bp before the 5 '-end of the target gene to obtain an upstream DNA fragment of the target gene, amplifying a DNA fragment of 600-1000 bp after the 3' -end of the target gene to obtain a downstream DNA fragment of the target gene, and connecting the upstream DNA fragment of the target gene with the downstream DNA fragment of the target gene to obtain a connecting product of the upstream DNA fragment and the downstream DNA fragment of the target gene;

s3, connecting the connection product of the upstream and downstream DNA fragments of the target gene obtained in the step S2 to the SaII enzyme cutting site of the pCasRA-spacer plasmid obtained in the step S1 to obtain a connection product;

s4, converting the connection product obtained in the step S3 into competent escherichia coli, culturing, and extracting plasmids to obtain pCasRA-target gene plasmids;

s5, transferring the pCasRA-target gene plasmid obtained in the step S4 into Riemerella anatipestifer, and thus achieving gene knockout.

The invention also claims a preparation method of the pCasRA plasmid, which comprises the following steps:

s1, taking a pCas9 plasmid as a carrier framework, taking a pEX18Gm plasmid as a template to clone an oriT conjugation transfer site, synthesizing a replicon of a pRA0726 plasmid and a cefoxitin resistance gene by using published data on NCBI, and connecting the replicon and the cefoxitin resistance gene to the carrier framework to construct an escherichia coli-riemerella anatipestifer shuttle carrier plasmid;

the nucleotide sequence of the oriT joint transfer site is shown in SEQ ID NO:2 is shown in the figure; the nucleotide sequence of replicon of pRA0726 plasmid is shown as SEQ ID NO:3 is shown in the figure;

s2, replacing a promoter of the Cas9 gene in the escherichia coli-Riemerella anatipestifer shuttle vector plasmid obtained in the step S1 by using a promoter of the Riemerella anatipestifer RA-CH-1 genome;

the nucleotide sequence of the promoter of the Riemerella anatipestifer RA-CH-1 genome is shown in SEQ ID NO:4 is shown in the figure; the nucleotide sequence of the promoter of the Cas9 gene is shown in SEQ ID NO:5 is shown in the figure;

s3, cloning an sgRNA sequence by taking the pCasSA plasmid as a template; cloning rpsL gene promoter by taking a Riemerella anatipestifer standard strain 11845 genome as a template, and connecting the rpsL gene promoter with an sgRNA sequence to obtain integrated sgRNA; amplifying by taking the pCas9 plasmid as a template to obtain a Cas9 protein gene, and connecting the Cas9 protein gene with the 5' end of the integrated sgRNA to obtain a connecting product;

the nucleotide information of the sgRNA sequence is shown as SEQ ID NO: shown at 17; the nucleotide sequence of the rpsL gene promoter is shown in SEQ ID NO:6 is shown in the figure; the nucleotide sequence of the integrated sgRNA is shown as SEQ ID NO: shown in figure 7; the nucleotide sequence of the Cas9 protein gene is shown as SEQ ID NO: shown at 20;

s4, simultaneously carrying out enzyme digestion on the connection product obtained in the step S3 and the escherichia coli-riemerella anatipestifer shuttle vector plasmid obtained in the step S1 by using SaII enzyme and SacI enzyme, and then connecting the two.

The invention also claims a kit for rapid gene editing of Riemerella anatipestifer, which comprises the pCasRA plasmid; the kit further contains BsaI enzyme and/or SaII enzyme.

Preferably, the gene editing is a gene knockout and/or a single base mutation.

More preferably, the gene knockout is a knockout of the dprA gene with gene number nc_ 017045.1.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a pCasRA plasmid, the nucleotide information of which is shown as SEQ ID NO: 1. The pCasRA plasmid takes the pCas9 plasmid as a vector skeleton, a replicon of the oriT and pRA0726 plasmids and a cefoxitin Ding Kang gene of a conjugation transfer site are connected to the vector skeleton, and the crRNA part in the pCas9 plasmid is replaced by a connecting product of a Cas9 protein gene (SEQ ID NO: 20) and an integrated sgRNA (SEQ ID NO: 7). The pCasRA plasmid is a shuttle plasmid which can be replicated and passaged in escherichia coli and riemerella anatipestifer, contains a conjugation transfer site oriT and can be transferred to the riemerella anatipestifer in the escherichia coli through conjugation; the plasmid shows chloramphenicol resistance in escherichia coli and cefazei Ding Kang in Riemerella anatipestifer, and can be used for strain screening; the plasmid can stably express Cas9 protein genes in Riemerella anatipestifer, and can edit genome DNA, so that the functions of gene knockout, single base mutation and the like are realized.

Drawings

FIG. 1 is a schematic diagram of the pCasRA plasmid;

FIG. 2 is a graph showing the results of gel electrophoresis of pCasRA plasmid for gene knockout experiments;

FIG. 3 is a diagram of the sequencing results of pCasRA plasmid for gene knockout experiments;

FIG. 4 is a diagram of the sequencing results of pCasRA plasmid for single base mutation experiments.

Detailed Description

The invention will be further described in detail with reference to the drawings and specific examples, which are given solely for the purpose of illustration and are not intended to limit the scope of the invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.

EXAMPLE 1 construction of pCasRA plasmid

1. Experimental method

(1) Construction of recombinant plasmid 1

PCR amplification is carried out from pEX18GM plasmid to obtain a conjugation transfer site oriT, and the nucleotide sequence of the conjugation transfer site oriT is shown as SEQ ID NO. 2; wherein the 5' primer sequence for amplifying oriT is shown in SEQ ID NO:8, the 3' primer sequence for amplifying oriT is shown as SEQ ID NO: shown at 9.

5' primer for amplifying oriT (SEQ ID NO: 8): 5'-ccgTCTAGActagagtcgatcttcgccagc-3';

3' primer (SEQ ID NO: 9) for amplifying oriT: 5'-cggTCTAGAcgcctgatgcggtattttctcc-3'.

Wherein the uppercase is XbaI cleavage site.

The PCR amplification reaction system is as follows: 25. Mu.L of PrimerStar Max Premix, 1. Mu.L of 5 'primer (10. Mu.M) for amplifying oriT (SEQ ID NO: 8), 1. Mu.L of 3' primer (10. Mu.M) for amplifying oriT (SEQ ID NO: 9), 2. Mu.L of template DNA (100 ng/. Mu.L of pEX18GM plasmid (AAC 27024.1)), 21. Mu.L of ddH ₂ O。

The PCR amplification reaction procedure was: 98 ℃ for 1min;98 ℃,15s,55 ℃,15s,72 ℃,30s,30 cycles; 72℃for 10min.

And after the PCR reaction is finished, the PCR product is recovered and purified by using a PCR product purification recovery kit (Beijing An Nuolun biotechnology Co., ltd.), and the recovery and purification steps are carried out according to a kit use operation manual, so as to obtain the amplified oriT fragment.

The pCas9 plasmid was digested with XbaI, wherein the digestion reaction system was: mu.L of 10 XCutSmart Buffer (NEB), 1. Mu.L of XbaI enzyme (NEB), 1. Mu.g of pCas9 plasmid, and ddH was added ₂ O was replenished to 50 μl. Linearized pCas9 plasmid was obtained.

The amplified oriT fragment is subjected to enzyme digestion by using XbaI enzyme, wherein the enzyme digestion reaction system is as follows: mu.L of 10 XCutSmart Buffer (NEB), 1. Mu.L of XbaI enzyme (NEB), 1. Mu.g of amplified oriT fragment and ddH were added ₂ O was replenished to 50 μl. Obtaining the oriT fragment after enzyme digestion.

pC to be linearizedThe as9 plasmid and the oriT fragment after enzyme digestion are connected by T4DNA ligase, and the connection reaction system is as follows: 1. Mu.L of 10 XT 4DNA ligase Buffer (NEB), 1. Mu.L of T4DNA library (NEB), 30 to 50ng of linearized pCas9 plasmid, linearized pCas9 plasmid and digested oriT fragment in a molar ratio of 1:3 adding ddH ₂ O was replenished to 10 μl.

The pCas9 plasmid with the oriT fragment ligated was obtained.

10 mu L of pCas9 plasmid connected with oriT fragment is transformed into competent escherichia coli Dh5α strain, the transformed Dh5α strain is spread on LB solid medium containing 50 mu g/mL chloramphenicol, after DH5 α strain is completely absorbed by LB solid medium, the medium is placed in a 37 ℃ incubator for inversion and overnight culture.

The transformed strains grown on the medium were subjected to PCR verification after every other day and sent to biological company (Optimago Corp.) for sequencing, the strain with correct sequence was transferred and stored and plasmid was extracted with a plasmid miniprep kit (Beijing Tiangen Biochemical technologies Co., ltd.) to obtain recombinant plasmid 1.

(2) Construction of recombinant plasmid 2

The promoter of the genome of the RA-CH-1 strain, the replicon of the pRA0726 plasmid (KU 997673.1, SEQ ID NO: 3) and the cefoxitin Ding Kang gene fragment (NG_ 047633.1) published on NCBI were synthesized by the Biotechnology company and were introduced into the pUC57 plasmid to obtain a recombinant pUC57 plasmid, the nucleotide sequence of which is shown in SEQ ID NO: shown at 12.

The nucleotide sequence of the promoter of the Riemerella anatipestifer RA-CH-1 genome is shown in SEQ ID NO: 4.

PCR amplification is carried out by taking the recombinant pUC57 plasmid as a template to obtain a promoter of the Riemerella anatipestifer RA-CH-1 genome, a replicon of pRA0726 plasmid and a cefazeirox Ding Kang gene. Wherein the 5' primer sequences of the promoter for amplifying the genome of the Riemerella anatipestifer RA-CH-1, the replicon of the pRA0726 plasmid and the cefoxitin Ding Kang gene are shown as SEQ ID NO:10 is shown in the figure; the 3' primer sequences of the promoter for amplifying the genome of the Riemerella anatipestifer RA-CH-1, the replicon of the pRA0726 plasmid and the cefoxitin Ding Kang gene are shown as SEQ ID NO: 11.

5' primer (SEQ ID NO: 10): 5'-cggGCTGAGGaattttaaataattttttta-3';

3' primer (SEQ ID NO: 11): 5'-cggCCTCAGCaaaatcagttctttagcga-3';

wherein the uppercase is the cleavage site of BbvCI enzyme.

The PCR amplification reaction system is as follows: 25. Mu.L of PrimerStar Max Premix, 1. Mu.L of 5 'primer (10. Mu.M) (SEQ ID NO: 10), 1. Mu.L of 3' primer (10. Mu.M) (SEQ ID NO: 11), 2. Mu.L of template DNA (100 ng/. Mu.L of pUC57 plasmid), 21. Mu.L of ddH ₂ O。

The PCR amplification reaction procedure was: 98 ℃ for 1min;98 ℃,15s,51 ℃,15s,72 ℃,90s,30 cycles; 72℃for 10min.

And after the PCR reaction is finished, the PCR product is recovered and purified by using a PCR product purification and recovery kit (Beijing An Nuolun biotechnology Co., ltd.), and the step of recovering and purifying is carried out according to a kit using an operation manual, so that a PCR product 1, namely the amplified promoter of the Riemerella anatipestifer RA-CH-1 genome, the replicon of the pRA0726 plasmid and the cefazene Ding Kang gene fragment is obtained.

Removing the promoter (SEQ ID NO: 5) of the Cas9 protein gene of the recombinant plasmid 1 obtained in the step (1) through inverse PCR amplification, wherein the sequence of the 5' primer amplified through inverse PCR is shown as SEQ ID NO: 13; the 3' primer sequence of inverse PCR amplification is shown as SEQ ID NO: 14.

5' primer (SEQ ID NO: 13): 5'-ggcGCTGAGGcccaatacttaataataaatagattataacac-3';

3' primer (SEQ ID NO: 14): 5'-ccgCCTCAGCatggataagaaatactcaataggc-3';

wherein the uppercase is the cleavage site of BbvCI enzyme.

The PCR amplification reaction system is as follows: 25. Mu.L of PrimerStar Max Premix, 1. Mu.L of 5 'primer (10. Mu.M) (SEQ ID NO: 13), 1. Mu.L of 3' primer (10. Mu.M) (SEQ ID NO: 14), 2. Mu.L of template DNA (100 ng/. Mu.L of pCas9 plasmid), 21. Mu.L of ddH ₂ O。

The PCR amplification reaction procedure was: 98 ℃ for 3min;98 ℃,15s,57 ℃,15s,72 ℃,3min,30 cycles; 72℃for 10min.

And after the PCR reaction is finished, the PCR product is recovered and purified by using a PCR product purification recovery kit (Beijing An Nuolun biotechnology Co., ltd.), and the step of recovering and purifying is carried out according to the kit using an operation manual, so as to obtain a PCR product 2, namely the recombinant plasmid 1 amplified by inverse PCR.

And respectively carrying out enzyme digestion on the PCR product 1 and the PCR product 2 by BbvCI enzyme to obtain a linearized PCR product 2 and an digested PCR product 1. And (3) connecting the linearized PCR product 2 and the digested PCR product 1 by using T4DNA ligase to obtain a connecting product, namely a recombinant plasmid 1 which is connected with a promoter of a Riemerella anatipestifer RA-CH-1 genome, a replicon of pRA0726 plasmid and inverse PCR amplification of cefazene Ding Kang gene.

10 mu L of the ligation product is transformed into competent escherichia coli Dh5α strain, the transformed Dh5α strain is spread on LB solid medium containing 50 mu g/mL chloramphenicol, and after DH5 α strain is completely absorbed by LB solid medium, the medium is placed in a 37 ℃ incubator for inversion and overnight culture.

The transformed strains grown on the medium were subjected to PCR verification after every other day and sent to biological company (Optimago Corp.) for sequencing, the strain with correct sequence was transferred and stored and plasmid was extracted with a plasmid miniprep kit (Beijing Tiangen Biochemical technologies Co., ltd.) to obtain recombinant plasmid 2.

(3) Construction of pCasRA plasmid

PCR amplification of rpsL gene promoter was performed using the genome of Riemerella anatipestifer 11845 strain (NC-017045.1) as a template. The nucleotide sequence of the rpsL gene promoter is shown in SEQ ID NO:6 is shown in the figure; the 5' primer sequence for amplifying the rpsL gene promoter is shown as SEQ ID NO: 15; the 3' primer sequence for amplifying the rpsL gene promoter is shown as SEQ ID NO: shown at 16.

5' primer (SEQ ID NO: 15): 5'-aggtgactgaagtatattttcagacaactttatccatt-3';

3' primer (SEQ ID NO: 16): 5'-gtctcgtttccattatatatagtaggcatttaattgct-3'.

The PCR amplification reaction system is as follows: 25 μL of PrimerStar Max Premix,1 μL of 5' primer1. Mu.L of 3' -primer (10. Mu.M) (SEQ ID NO: 15), 2. Mu.L of template DNA (100 ng/. Mu.L of genome of Riemerella anatipestifer 11845 strain (NC-017045.1)), 21. Mu.L of ddH ₂ O。

The PCR amplification reaction procedure was: 98 ℃ for 1min;98 ℃,15s,51 ℃,15s,72 ℃,15s,30 cycles; 72℃for 10min.

After the PCR reaction, the PCR product was recovered and purified using a PCR product recovery kit (Beijing An Nuolun Biotechnology Co., ltd.) according to the procedure of the kit using an operation manual to obtain the rpsL gene promoter.

PCR amplification is carried out by taking pCasSA plasmid as a template to obtain a sgRNA sequence, and the nucleotide sequence of the sgRNA is shown as SEQ ID NO:17, wherein the 5' primer sequence for amplifying the sgRNA sequence is set forth in SEQ ID NO:18, the 3' primer sequence for amplifying sgRNA sequence is shown as SEQ ID NO: 19.

5' primer (SEQ ID NO: 18): 5'-agcaattaaatgcctactatatataatggaaacgagac-3';

3' primer (SEQ ID NO: 19): 5'-aggGTCGACattctagaccatgggtat-3';

wherein the uppercase is the cleavage site of the saII enzyme.

The PCR amplification reaction system is as follows: 25. Mu.L of PrimerStar Max Premix, 1. Mu.L of 5 'primer (10. Mu.M) (SEQ ID NO: 18), 1. Mu.L of 3' primer (10. Mu.M) (SEQ ID NO: 19), 2. Mu.L of template DNA (100 ng/. Mu.L of pCasSA plasmid), 21. Mu.L of ddH ₂ O。

After the PCR reaction, the PCR product was recovered and purified using a PCR product recovery kit (Beijing An Nuolun Biotechnology Co., ltd.) according to the manual of the kit to obtain the sgRNA sequence.

PCR amplification is carried out by taking pCas9 plasmid as a template to obtain a Cas9 protein gene, wherein the nucleotide sequence of the Cas9 protein gene is shown as SEQ ID NO:20, the 5' primer sequence for amplifying Cas9 protein gene is shown as SEQ ID NO:21, the 3' primer sequence for amplifying the Cas9 protein gene is as shown in SEQ ID NO: shown at 22.

5' primer (SEQ ID NO: 21): 5' -ggcgagctcccgctgcttttaaatat-3’；

3' primer (SEQ ID NO: 22): 5'-aatggataaagttgtctgaaaatatacttcagtcacct-3';

wherein the underlined part is the cleavage site of SacI enzyme.

The PCR amplification reaction system is as follows: 25. Mu.L of PrimerStar Max Premix, 1. Mu.L of 5 'primer (10. Mu.M) (SEQ ID NO: 21), 1. Mu.L of 3' primer (10. Mu.M) (SEQ ID NO: 22), 2. Mu.L of template DNA (100 ng/. Mu.L of pCas9 plasmid), 21. Mu.L of ddH ₂ O。

And after the PCR reaction is finished, the PCR product is recovered and purified by using a PCR product purification recovery kit (Beijing An Nuolun biotechnology Co., ltd.), and the recovery and purification steps are carried out according to a kit use operation manual to obtain the Cas9 protein gene.

The rpsL gene promoter and the sgRNA sequence are connected, and a connection reaction system is as follows: 25. Mu.L of PrimerStar Max Premix, 0.5. Mu.L of rpsL gene promoter, 0.5. Mu.L of sgRNA sequence, 22. Mu.L of ddH ₂ O。

After the connection reaction system is prepared, carrying out PCR reaction, wherein the PCR reaction procedure is as follows: 98 ℃ for 30s;98 ℃,15s,51 ℃,15s,72 ℃,15s,15 cycles; 72℃for 5min.

After completion of the PCR reaction, 1. Mu.L of 5 'primer (10. Mu.M) (SEQ ID NO: 15) for amplifying rpsL gene promoter and 1. Mu.L of 3' primer (10. Mu.M) (SEQ ID NO: 19) for amplifying sgRNA sequence were added thereto, and the same PCR reaction procedure was maintained, and amplification was continued for 30 cycles.

The PCR product was then recovered and purified using a PCR product purification recovery kit (Beijing An Nuolun Biotechnology Co., ltd.) and the recovery and purification steps were performed according to the kit using an operation manual to obtain a ligation product 1, i.e., a product in which the rpsL gene promoter was ligated with the sgRNA sequence (integrated sgRNA (SEQ ID NO: 7)).

Connecting the connection product 1 with the Cas9 protein gene, wherein a connection reaction system is as follows: 25. Mu.L of PrimerStar Max Premix, 0.5. Mu.L of ligation product 1, 0.5. Mu.L of Cas9 protein gene, 22. Mu.L of ddH ₂ O。

After the completion of the PCR reaction, 1. Mu.L of 5 'primer (10. Mu.M) (SEQ ID NO: 15) for amplifying rpsL gene promoter and 1. Mu.L of 3' primer (10. Mu.M) (SEQ ID NO: 22) for amplifying Cas9 protein gene were added thereto, and the same PCR reaction procedure was maintained and amplification was continued for 30 cycles.

And then, the PCR product is recovered and purified by using a PCR product purification and recovery kit (Beijing An Nuolun Biotechnology Co., ltd.), and the recovery and purification steps are carried out according to the kit using an operation manual, so as to obtain a ligation product 2, namely a ligated fragment.

Simultaneously carrying out enzyme digestion on the recombinant plasmid 2 obtained in the step (2) by using SaII enzyme and SacI enzyme to obtain a linearized recombinant plasmid 2; and simultaneously carrying out enzyme digestion on the ligation product 2 by using SaII enzyme and SacI enzyme to obtain the digested ligation product 2.

The linearized recombinant plasmid 2 and the digested ligation product 2 are ligated by using T4DNA ligase, and the ligation reaction system is as follows: 1. Mu.L of 10 XT 4DNA ligase Buffer (NEB), 1. Mu.L of T4DNA library (NEB), 30 to 50ng of linearized recombinant plasmid 2, the linearized recombinant plasmid 2 and the ligation product 2 after cleavage were in a molar ratio of 1:3 adding ddH ₂ O was replenished to 10 μl.

After the preparation of the ligation reaction system, the ligation reaction was carried out overnight at 16℃to give ligation product 3. 10 mu L of the ligation product 3 was transformed into competent E.coli Dh5α strain, the transformed Dh5α strain was plated on LB solid medium containing 50. Mu.g/mL chloramphenicol, and after the DH5 α strain was completely absorbed by LB solid medium, the medium was placed in an incubator at 37℃and cultured upside down overnight.

The transformed strains grown on the medium were subjected to PCR verification after every other day and sent to biological company (Optimago Corp.) for sequencing, the strain with correct sequence was transferred and stored and plasmid was extracted with a plasmid miniprep kit (Beijing Tiangen Biochemical technology Co., ltd.) to obtain pCasRA plasmid.

2. Experimental results

The pattern diagram of the pCasRA plasmid is shown in figure 1, and the nucleotide sequence of the pCasRA plasmid is shown in SEQ ID NO: 1.

Example 2pCasRA plasmid for Gene knockout in Riemerella anatipestifer

Taking the dprA gene (NC_ 017045.1) as an example, the invention performs gene knockout in the Riemerella anatipestifer 11845 strain (NC_ 017045.1) (but not limited to the dprA gene, and all genes meeting the conditions of the embodiment can be knocked out).

1. Experimental method

(1) Construction of pCasRA-dprA-spacer plasmid

A DNA fragment of 20 bases before a certain NGG (N is any base) sequence is selected on the dprA gene as a spacer fragment (SEQ ID NO: 23), and the reverse complement of the spacer fragment is synthesized. Adding GAAA to the 5' end of the spacer fragment to obtain a modified spacer fragment (SEQ ID NO: 24); AAAC was added to the 5' -end of the reverse complement to give a modified reverse complement (SEQ ID NO: 25).

The spacer fragment (SEQ ID NO: 23): 5'-taacattcacctaaaagacg-3';

modified spacer fragment (SEQ ID NO: 24): 5'-GAAAtaacattcacctaaaagacg-3';

modified reverse complement (SEQ ID NO: 25): 3'-attgtaagtggattttctgcCAAA-5'.

And carrying out annealing connection on the modified spacer fragment and the modified reverse complementary sequence.

The reaction system is as follows: 1. Mu.L of modified spacer fragment (100. Mu.M) (SEQ ID NO: 24), 1. Mu.L of modified reverse complement sequence (100. Mu.M) (SEQ ID NO: 25), 1. Mu.L of T4 PNK Buffer, 0.5. Mu.L of T4 PNK enzyme, 6.5. Mu.L of ddH ₂ O。

Adding at 95deg.C for 30min, naturally cooling to room temperature to obtain annealed ligation product (i.e. space double-stranded DNA), and adding ddH ₂ O dilutes the annealed ligation product 200-fold to obtain diluted space double-stranded DNA.

The pCasRA plasmid prepared in the example 1 is subjected to enzyme digestion by BsaI enzyme to obtain a linearized pCasRA plasmid, and the enzyme digestion reaction system is as follows: mu.L of 10 XCutSmart Buffer (NEB), 1. Mu.L of BsaI (NEB), 1. Mu.g of pCasRA plasmid, and ddH was added ₂ O was replenished to 50 μl. After preparing the enzyme digestion reaction system, the reaction is carried out for 3 to 5 hours at 37 ℃. After the PCR reaction, the PCR product was recovered and purified using a PCR product purification recovery kit (Beijing An Nuolun Biotechnology Co., ltd.) according to the kit usage instruction manual to obtain a linearized pCasRA plasmid.

The diluted space double-stranded DNA and the linearized pCasRA plasmid are connected by using T4DNA ligase, and a connection reaction system is as follows: 1. Mu.L of 10 XT 4DNA ligase Buffer (NEB), 1. Mu.L of T4DNA library (NEB), 30 to 50ng of linearized pCasRA plasmid, linearized pCasRA plasmid and diluted spacer double-stranded DNA in a molar ratio of 1:3 adding ddH ₂ O was replenished to 10 μl.

The ligation reaction system was prepared, mixed and reacted overnight at 16℃to ligate the spacer double-stranded DNA between BsaI cleavage sites of the pCasRA plasmid, thereby obtaining the pCasRA plasmid with the spacer double-stranded DNA ligated thereto, and the ligase was inactivated at 65℃for 10min.

The pCasRA plasmid connected with the space double-stranded DNA is transformed into competent escherichia coli Dh5α strain, the transformed Dh5α strain is paved on LB solid medium containing 50 mug/mL chloramphenicol, after DH5 α strain is completely absorbed by LB solid medium, the medium is placed in a 37 ℃ incubator for inversion and overnight culture.

The transformed strains grown on the medium were subjected to PCR verification after every other day and sent to biological company (Optimago Corp.) for sequencing, the strain with correct sequence was transferred and stored and plasmid was extracted with a plasmid miniprep kit (Beijing Tiangen Biochemical technology Co., ltd.) to obtain pCasRA-dprA-space plasmid.

(2) Amplified ligation of upstream and downstream fragments of dprA Gene

The DNA fragments of 600bp on the upstream and downstream of the dprA gene are amplified, and the specific amplification primers are as follows:

5' primer sequence (SEQ ID NO: 26) for amplifying the DNA fragment upstream of the dprA gene:

5’-aggGTCGACctcataatttccatatccga-3’；

3' primer sequence (SEQ ID NO: 27) for amplifying the DNA fragment upstream of the dprA gene:

5’-atggcaaggcgctagttagttgaaaaattacttttttaaaa-3’；

5' primer sequence (SEQ ID NO: 28) for amplifying a DNA fragment downstream of the dprA gene:

5’-ttttaaaaaagtaatttttcaactaactagcgccttgccat-3’；

3' primer sequence (SEQ ID NO: 29) for amplifying a DNA fragment downstream of the dprA gene:

5’-aacGTCGACggctatggcggcaagtc-3’；

wherein the uppercase is the cleavage site of the SaII enzyme.

Genomic DNA was extracted from Riemerella anatipestifer 11845 strain (NC-017045.1) using a bacterial genomic DNA extraction kit as template DNA for PCR amplification.

Amplifying a DNA fragment of 600bp at the upstream of the dprA gene, wherein the reaction system is as follows: 25. Mu.L of PrimerStar Max Premix, 1. Mu.L of 5 '-primer sequence (10. Mu.M) (SEQ ID NO: 26) for amplifying the DNA fragment upstream of the dprA gene, 1. Mu.L of 3' -primer sequence (10. Mu.M) (SEQ ID NO: 27) for amplifying the DNA fragment upstream of the dprA gene, 2. Mu.L of template DNA (100 ng/. Mu.L of genome of Riemerella anatipestifer 11845 strain (NC-017045.1)), 21. Mu.L of ddH ₂ O。

The PCR reaction procedure was: 98 ℃ for 1min;98 ℃,15s,54 ℃,15s,72 ℃,30s,30 cycles; 72℃for 5min. And obtaining the upstream fragment of the dprA gene after the reaction is finished.

Amplifying a DNA fragment of 600bp at the downstream of the dprA gene, wherein the reaction system is as follows: 25. Mu.L of PrimerStar Max Premix, 1. Mu.L of 5 '-primer sequence (10. Mu.M) (SEQ ID NO: 28) for amplifying a DNA fragment downstream of the dprA gene, 1. Mu.L of 3' -primer sequence (10. Mu.M) (SEQ ID NO: 29) for amplifying a DNA fragment downstream of the dprA gene, 2. Mu.L of template DNA (100 ng/. Mu.L of genome of Riemerella anatipestifer 11845 strain (NC-017045.1)),21 mu L ddH ₂ O。

The PCR reaction program is consistent with that of the upstream fragment of the dprA gene, and the downstream fragment of the dprA gene is obtained.

Connecting an upstream fragment of the dprA gene with a downstream fragment of the dprA gene, wherein a connection reaction system is as follows: 25. Mu.L of PrimerStar Max Premix, 0.5. Mu.L of dprA upstream fragment, 0.5. Mu.L of dprA downstream fragment, 22. Mu.L of ddH ₂ O。

After the connection reaction system is prepared, carrying out PCR reaction, wherein the PCR reaction procedure is as follows: 98 ℃ for 30s;98 ℃,15s,54 ℃,15s,72 ℃,30s,15 cycles; 72℃for 5min.

After completion of the PCR reaction, 1. Mu.L of 5 '-primer sequence (10. Mu.M) (SEQ ID NO: 26) for amplifying the DNA fragment upstream of the dprA gene and 1. Mu.L of 3' -primer sequence (10. Mu.M) (SEQ ID NO: 29) for amplifying the DNA fragment downstream of the dprA gene were added thereto, and the same PCR reaction procedure was maintained, and amplification was continued for 30 cycles.

And then, the PCR product is recovered and purified by using a PCR product purification and recovery kit (Beijing An Nuolun Biotechnology Co., ltd.) and the recovery and purification steps are carried out according to a kit using an operation manual to obtain a dprA upstream and downstream fragment ligation product.

(3) Construction of pCasRA-dprA plasmid

Carrying out enzyme digestion on the pCasRA-dprA-spacer plasmid obtained in the step (1) by using SaII enzyme to obtain a linearized pCasRA-dprA-spacer plasmid; and (3) carrying out enzyme digestion on the dprA upstream and downstream fragment connection product obtained in the step (2) by using SaII enzyme to obtain the digested dprA upstream and downstream fragment connection product.

The linearized pCasRA-dprA-space plasmid and the digested dprA upstream and downstream fragment connection products are connected by T4DNA ligase, and the connection reaction system is as follows: 1. Mu.L of 10 XT 4DNA ligase Buffer (NEB), 1. Mu.L of T4DNA ligase (NEB), 30-50 ng of linearized pCasRA-dprA-space plasmid, linearized pCasRA-dprA-space plasmid and digested dprA upstream and downstream fragment ligation products in a molar ratio of 1:3 adding ddH ₂ O was replenished to 10 μl.

After the ligation reaction system was prepared, the ligation product 4 (pCasRA-dprA-spacer plasmid to which the ligation product of the upstream and downstream fragments of dprA was ligated) was obtained by overnight reaction at 16 ℃. 10 mu L of the ligation product 4 was transformed into competent E.coli Dh5α strain, the transformed Dh5α strain was plated on LB solid medium containing 50. Mu.g/mL chloramphenicol, and after the DH5 α strain was completely absorbed by LB solid medium, the medium was placed in an incubator at 37℃and cultured upside down overnight.

The transformed strains grown on the medium were subjected to PCR verification after every other day and sent to biological company (Optimago Corp.) for sequencing, the strain with correct sequence was transferred and stored and plasmid was extracted with a plasmid miniprep kit (Beijing Tiangen Biochemical technology Co., ltd.) to obtain pCasRA-dprA plasmid.

(4) Knockout of dprA Gene in Riemerella anatipestifer

Transforming the pCasRA-dprA plasmid obtained in the step (3) into a competent escherichia coli S17-1 strain, and spreading the plasmid on an LB solid medium containing 50 mug/mL chloramphenicol; meanwhile, the Riemerella anatipestifer ATCC 11845 strain is streaked and cultured on a TSA solid plate.

Escherichia coli S17-1 and Riemerella anatipestifer ATCC 11845 strain containing pCasRA-dprA plasmid were cultured to OD with LB and TSB, respectively ₆₀₀ About 0.6.

Taking E.coli S17-1 containing pCasRA-dprA plasmid as an example: taking 1mL of escherichia coli S17-1 bacterial liquid containing pCasRA-dprA plasmid, centrifuging in a high-speed table type low-temperature centrifuge at 4 ℃ and 5000rpm for 5min in a 2mL EP tube, removing the supernatant, and collecting thalli; 10mmol/L MgSO after ice bath with 1mL ₄ Washing the solution, re-suspending the thallus, centrifuging at 4deg.C and 5000rpm for 5min, removing supernatant, collecting thallus, and repeating for 3 times to obtain Escherichia coli S17-1 thallus.

The same treatment was performed on the Riemerella anatipestifer ATCC 11845 strain to obtain ATCC 11845 strain.

With 100. Mu.L of MgSO ₄ Re-suspending coliform bacteria S17-1 and ATCC 11845 bacteria with solution (10 mmol/L) and mixing to obtain mixed bacterial liquid, attaching sterilizing filter membrane with aperture of 0.22 μm to TSA solid culture medium, dripping the mixed bacterial liquid onto the filter membrane, culturing in a 37 deg.C incubator for 24 hr, and collecting the bacterial liquid with clean forcepsThe filters were placed in sterile petri dishes with 5mL MgSO ₄ And eluting the thalli by using the solution (10 mmol/L) to obtain an eluted bacterial solution. 100 mu L of the eluted bacterial liquid is coated on a TSA agar plate containing 50 mu g/mL kanamycin and 1 mu g/mL cefoxitin, the TSA agar plate is placed in a 37 ℃ incubator for inversion culture, and 12 monoclonal colonies are obtained after collection, namely the infected Riemerella anatipestifer ATCC 11845.

(5) Verification of Gene knockout

12 monoclonal colonies were inoculated into 12 50. Mu.L of TSB (tryptone soy broth) broth, respectively, to give corresponding ATCC bacterial solutions.

Selecting one part of ATCC bacterial liquid, and carrying out PCR amplification by taking the ATCC bacterial liquid as a template to obtain a PCR amplification product. The reaction system is as follows: 25. Mu.L of PrimeStar Max Premix, 1. Mu.L of 5 '-primer (10. Mu.M) (SEQ ID NO: 30), 1. Mu.L of 3' -primer (10. Mu.M) (SEQ ID NO: 31), 2. Mu.L of template DNA (ATCC bacterial liquid), 21. Mu.L of ddH ₂ O。

5' primer (SEQ ID NO: 30): 5'-gctaatcctttaccagtaagtac-3';

3' primer (SEQ ID NO: 31): 5'-gagagaactctatcgcttataga-3'.

The amplified 8 PCR amplified products (8 single colonies) were subjected to gel electrophoresis on a 1% agarose gel, the electrophoresis bands were observed, and the PCR amplified products with correct bands were sent to biological company (Optimago) for sequencing verification.

The remaining 11 ATCC bacterial solutions were subjected to the same treatment and detection verification.

2. Experimental results

The results of gel electrophoresis are shown in fig. 2, and the results show that: of the 96 single colonies obtained by amplifying 12 ATCC bacterial liquid, 14 single colonies showed gene deletion, which indicates successful gene knockout.

The sequencing verification result is shown in fig. 3, wherein the red box part in the figure indicates that the gene is deleted, and only the 5 'end part sequence and the 3' end part sequence of the deleted gene are given in order to be displayed on one figure because the actually deleted gene is overlong. It is demonstrated that the present embodiment can effectively achieve gene knockout.

EXAMPLE 3 use of pCasRA plasmid for Single base mutation in Riemerella anatipestifer

The present invention targets cytosine C at position 271 in the dprA gene (NC_ 017045.1) and mutates it to adenine A. (not limited to the 271 th amino acid of the mutant dprA gene)

1. Experimental method

(1) Construction of pCasRA-dprAm plasmid

The pCasRA-dprA-spacer plasmid obtained in the step (1) of example 2 was digested with SaII enzyme to obtain a linearized pCasRA-dprA-spacer plasmid.

The DNA fragments of 600bp respectively at the upstream and downstream of the single base mutation site (271 position in the dprA gene) of the dprA gene are amplified, and the specific amplification primers are as follows:

amplifying a 5' primer sequence (SEQ ID NO: 32) of a DNA fragment upstream of a single base mutation site of the dprA gene:

5’-ggaGTCGACagctccaaaactttagaag-3’；

amplifying 3' primer sequence of DNA fragment upstream of single base mutation site of dprA gene (SEQ ID NO:):

5’-tacgacatcttaatcagcttactcgtcttttaggtgaa-3’；

amplifying 5' primer sequence of DNA fragment downstream of single base mutation site of dprA gene (SEQ ID NO: 34):

5’-ttcacctaaaagacgagtaagctgattaagatgtcgta-3’；

3' primer sequence (SEQ ID NO: 35) of DNA fragment downstream of single base mutation site of amplified dprA gene:

5’-aggGTCGACgacactattgtttagcaaa-3’；

wherein the uppercase is the cleavage site of the SaII enzyme, and the underlined is the point mutated gene.

According to the method of the step (2) in the example, the genomic DNA of the Riemerella anatipestifer 11845 strain in the step (2) in the example 2 is used as a template, and the DNA fragments of 600bp at the upstream and downstream of the single base mutation site of the dprA gene are amplified respectively to obtain the single base mutation upstream fragment of the dprA gene and the single base mutation downstream fragment of the dprA gene.

And connecting the single-base mutation upstream fragment of the dprA gene with the single-base mutation downstream fragment of the dprA gene to obtain a single-base mutation connecting fragment of the dprA gene.

And (3) carrying out enzyme digestion on the single-base mutant connecting fragment of the dprA gene by using SaII enzyme to obtain the single-base mutant connecting fragment of the dprA gene after enzyme digestion.

The linearized pCasRA-dprA-space plasmid and the digested dprA gene single base mutant ligation fragment were ligated as described in step (3) of example 2 to give pCasRA-dprA plasmid.

(2) Single base mutation in Riemerella anatipestifer and verification

The pCasRA-dprAm plasmid was transferred into the Riemerella anatipestifer ATCC 11845 strain and cultured to obtain 12 monoclonal colonies, which were the modified Riemerella anatipestifer, according to the method described in step (4) of example 2.

The 12 monoclonal colonies were inoculated into 12 50. Mu.L of TSB (tryptone soy broth) broth, respectively, to obtain the corresponding bacterial solutions.

And selecting one part of bacterial liquid, and carrying out PCR amplification by taking the bacterial liquid as a template to obtain a PCR amplification product 1. The reaction system is as follows: 25. Mu.L of PrimeStar Max Premix, 1. Mu.L of 5 'primer (10. Mu.M) (SEQ ID NO: 36), 1. Mu.L of 3' primer (10. Mu.M) (SEQ ID NO: 37), 2. Mu.L of template DNA, 21. Mu.L of ddH ₂ O。

5' primer (SEQ ID NO: 36): 5'-ctaaatgatagaatatctcctccca-3';

3' primer (SEQ ID NO: 37): 5'-cgacatcttaatcagcttactc-3'.

The PCR amplified product 1 was subjected to gel electrophoresis on a 1% agarose gel, the electrophoresis band was observed, and the PCR amplified product with the correct band was sent to biological company (Optimus Praeparata) for sequencing verification.

Wherein the primers for sequencing verification are:

5' primer (SEQ ID NO: 38): 5'-ctaaatgatagaatatctcctccca-3';

3' primer (SEQ ID NO: 39): 5'-atggtaaatgcggaagaaatt-3'.

The remaining 11 parts of bacterial liquid are subjected to the same treatment detection verification.

2. Experimental results

The results of sequencing verification are shown in FIG. 4, wherein the red boxes represent single base mutation success, and the results show that: of the 12 monoclonal colonies, 4 were successful in single base mutation, indicating that the method described in this example can effectively achieve single base mutation.

Construction and verification of pCasRA1 plasmid with non-substituted sgRNA of comparative example 1

1. Experimental method

Comparative example 1 differs from example 1 in that: comparative example 1 preparation of pCasRA1 plasmid the sgRNA sequence was not amplified and ligated with the rpsL gene promoter using the pCasSA plasmid as a template in step (3) of example 1, and the remaining steps were the same.

The nucleotide sequence of the prepared pCasRA1 plasmid is shown as SEQ ID NO: 40.

Connecting the prepared pCasRA1 plasmid with a target through BsaI enzyme digestion vectors, wherein the nucleotide sequence information of the target F is shown as SEQ ID NO:41, the nucleotide information of the target R is shown as SEQ ID NO: shown at 42.

Target F (SEQ ID NO: 41): 5' -AAACGTGGTTGTAGAAGCAATCAGG-3’；

Target R (SEQ ID NO: 42): 5' -AAAACCTGATTGCTTCTACAACCAC-3’。

Wherein the underlined part is a complementary base added due to the cleavage specificity of BsaI, and the rest is a target sequence.

The pCasRA1 plasmid with the attached target was transferred into the Riemerella anatipestifer 11845 (NC-017045.1) strain as described in example 2, and the colony growth was observed by plating the bacterial solution, which was designated as Experimental group 1.

Two sets of experiments were repeated and colony growth was recorded as set 2 and set 3.

The control group was set as a strain of Riemerella anatipestifer 11845 (NC_ 017045.1) which was not transformed with the pCasRA1 plasmid with the target, and 3 control groups were set in total, control group 1, control group 2 and control group 3, respectively.

The pCasRA plasmid prepared in the example 1 is connected with a target through BsaI digestion vector, wherein the nucleotide sequence information of the target F is shown as SEQ ID NO:43, the nucleotide information of the target R is shown as SEQ ID NO: shown at 44.

Target F (SEQ ID NO: 43): 5' -GAAAGTGGTTGTAGAAGCAATCAG-3’；

Target R (SEQ ID NO: 44): 5' -AAACCTGATTGCTTCTACAACCAC-3’。

The pCasRA plasmid with the attached target was transferred into the Riemerella anatipestifer 11845 (NC-017045.1) strain as described in example 2, and the bacterial solution was subjected to plate drop observation and designated as Experimental group 4.

Two sets of experiments were repeated and colony growth was recorded as experiment set 5 and experiment set 6.

The control group was set as a strain of Riemerella anatipestifer 11845 (NC-017045.1) which was not transformed with the pCasRA plasmid with the target, and 3 control groups were set in total, control group 4, control group 5 and control group 6, respectively.

2. Experimental results

The colony growth conditions of the test groups 1 to 3 and the control groups 1 to 3 are shown in Table 1.

TABLE 1 colony growth

The results showed that the pCasRA1 plasmid without replacement of sgRNA was not effective against Riemerella anatipestifer 11845 strain.

The colony growth conditions of the test groups 4 to 6 and the control groups 4 to 6 are shown in Table 1.

TABLE 1 colony growth

The results show that the pCasRA plasmid prepared in example 1 has bactericidal effect on Riemerella anatipestifer 11845 strain.

It should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and that other various changes and modifications can be made by one skilled in the art based on the above description and the idea, and it is not necessary or exhaustive to all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.

Claims

1. A pCasRA plasmid, characterized in that the nucleotide sequence of the pCasRA plasmid is shown as SEQ ID NO: 1.

2. Use of the pCasRA plasmid of claim 1 in rapid gene editing of riemerella anatipestifer.

3. The use according to claim 2, wherein the gene editing is a gene knockout and/or a single base mutation.

4. The use according to claim 3, wherein the gene knockout is a knockout of the dprA gene with the knockout gene number nc_ 017045.1.

5. The use according to claim 3, wherein the single base mutation is a mutation of cytosine C at position 271 in the dprA gene to adenine a.

6. A method of gene knockout as claimed in claim 3, comprising the steps of:

s2, amplifying a DNA fragment 600-1000 bp before the 5 '-end of the gene to be knocked out to obtain an upstream DNA fragment of the target gene, amplifying a DNA fragment 600-1000 bp after the 3' -end of the gene to be knocked out to obtain a downstream DNA fragment of the target gene, and connecting the upstream DNA fragment of the target gene with the downstream DNA fragment of the target gene to obtain a connecting product of the upstream DNA fragment and the downstream DNA fragment of the target gene;

7. A kit for rapid gene editing of riemerella anatipestifer, characterized in that the kit comprises the pCasRA plasmid of claim 1; the kit further contains BsaI enzyme and/or SaII enzyme.

8. The kit of claim 7, wherein the gene editing is a gene knockout and/or a single base mutation.

9. The kit of claim 8, wherein the gene knockout is a dprA gene with a knockout gene number nc_ 017045.1.

10. The kit according to claim 8, wherein the single base mutation is a mutation of cytosine C at position 271 in the dprA gene to adenine a.