CN110452922B

CN110452922B - Method for gene editing of pediococcus acidilactici by using endogenous CRISPR system

Info

Publication number: CN110452922B
Application number: CN201910728089.6A
Authority: CN
Inventors: 彭楠; 刘玲; 杨丹露; 张志雨; 梁运祥
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2019-08-08
Filing date: 2019-08-08
Publication date: 2021-03-26
Anticipated expiration: 2039-08-08
Also published as: CN110452922A

Abstract

The invention discloses a pediococcus acidilactici gene editing plasmid, and a construction method thereof comprises the following steps: the mini-CRISPR sequence containing two DNA repetitive sequences and two three-type enzyme cutting sites Bbs1 is cloned to shuttle plasmid pMG36E and transformed into escherichia coli to obtain a transformant, and the pediococcus acidilactici gene editing plasmid pMG36E-C is obtained through activation and extraction. The method for performing gene knockout, insertion and point mutation on pediococcus acidilactici by using the endogenous CRISPR system is also disclosed, an exogenous CRISPR nuclease gene does not need to be cloned, genome editing can be performed on the pediococcus acidilactici efficiently and quickly by using a gene editing plasmid, the editing efficiency can reach 90%, and the method is beneficial to application and popularization; the strain which is edited can complete elimination of the knockout plasmid with resistance only by 2-3 passages, is beneficial to continuous editing of a plurality of genes, changes a nutrient metabolic pathway or inserts a new metabolic pathway, and achieves directed modification.

Description

Method for gene editing of pediococcus acidilactici by using endogenous CRISPR system

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a method for carrying out gene editing on pediococcus acidilactici by utilizing an endogenous CRISPR system, specifically an application method for constructing an editing plasmid, electrically converting the editing plasmid into the pediococcus acidilactici and carrying out gene editing on the pediococcus acidilactici by utilizing the editing plasmid, and is particularly suitable for genetic modification of lactic acid bacteria and biosynthesis of lactic acid bacteria metabolites.

Background

Pediococcus acidilactici is an important group of lactic acid bacteria that can convert carbohydrates into lactic acid through metabolic activity of the bacteria. Lactic acid is an important chemical product, has important application in a plurality of fields such as polymeric materials, foods, cosmetics, medicines and the like, for example, has strong preservative and fresh-keeping effects in the aspect of foods, can be used for storing beverages, meat and fruits, and has the effects of regulating pH value, inhibiting bacteria, prolonging shelf life, seasoning, keeping food color and luster and the like; in the aspect of polymerization materials, lactic acid monomers are polymerized into polylactic acid with good degradability so as to be used for manufacturing environment-friendly packaging materials.

Pediococcus acidilactici is widely used in the feed industry as a probiotic. The pediococcus acidilactici is added into the feed, the lactic acid generated by fermentation can effectively increase the flavor of the feed, improve the feed intake of animals to promote the growth of the animals, reduce the pH value to maintain the acidic environment of intestinal tracts and effectively inhibit the growth and the propagation of pathogenic microorganisms; the bacteriocin metabolized and synthesized by the method shows a wider antibacterial spectrum and has obvious inhibition effect on a plurality of pathogenic bacteria in intestinal tracts; the pediococcus acidilactici is beneficial to maintaining the intestinal health and the micro-ecological balance of animals and enhancing the immune function of animal organisms.

At present, the pediococcus acidilactici is mostly directly utilized by thalli and metabolites thereof, and the research on the pediococcus acidilactici genetic system is less, so that the pediococcus acidilactici cannot be modified according to the production purpose, for example, the unfavorable metabolic pathway of the pediococcus acidilactici is blocked, or the beneficial metabolic pathway of the pediococcus acidilactici is amplified, so that the application of the pediococcus acidilactici is greatly limited.

Disclosure of Invention

An object of the present invention is to solve at least the above problems and to provide at least the advantages described later.

The invention also aims to provide a pediococcus acidilactici gene editing plasmid, which is constructed on the basis of shuttle plasmids of escherichia coli and pediococcus acidilactici, does not need to clone an exogenous CRISPR nuclease gene, and can edit a genome by utilizing an endogenous II-A type CRISPR system.

The invention also aims to provide a method for knocking out genes, a gene inserting method and a gene point mutation method of pediococcus acidilactici by utilizing an endogenous CRISPR system, gene knocking out, inserting or point mutation and the like are carried out on the pediococcus acidilactici by utilizing gene editing plasmids, so that the pediococcus acidilactici is genetically modified, the metabolic pathway of the pediococcus acidilactici is directionally changed, and the purposes of over-expression of beneficial products and no expression of unfavorable products are achieved.

The invention also aims to provide a method for reversely screening pediococcus acidilactici by utilizing pediococcus acidilactici gene editing plasmids to complete gene editing, wherein pediococcus acidilactici subjected to single gene editing is reversely screened to obtain a mutant strain without plasmids, and secondary or more gene editing is carried out according to the preparation competence of the mutant strain, so that continuous gene editing of pediococcus acidilactici is realized, and strains with better expression performance are screened and obtained.

To achieve these objects and other advantages of the present invention, there is provided a pediococcus acidilactici gene-editing plasmid constructed by a method comprising the steps of:

respectively carrying out enzyme digestion on a mini-CRISPR sequence containing two DNA repetitive sequences and two three-type enzyme digestion sites Bbs1 and a shuttle plasmid pMG36E of pediococcus acidilactici, carrying out enzyme ligation on the enzyme digestion products, transforming the enzyme digestion products into escherichia coli, and screening to obtain a transformant transformed into pMG 36E-C;

activating the transformant transformed into pMG36E-C, and extracting plasmids to obtain pediococcus acidilactici gene editing plasmid pMG 36E-C;

wherein the DNA sequence is shown as SEQ ID NO: 1 is shown.

Preferably, the mini-CRISPR sequence and pMG36E have Xba1 and Pst1 enzyme cutting sites.

Preferably, the screening conditions are: and E.coli containing a mini-CRISPR sequence and pMG36E is coated on an LB solid medium plate, cultured at 37 ℃, and a transformant is picked and subjected to PCR verification.

The invention also provides a method for gene knockout of pediococcus acidilactici by using an endogenous CRISPR system, which comprises the following steps:

step 1: designing a spacer for 3' NGG according to PAM of a lactic acid piece coccus intracellular II-A type CRISPR system, respectively designing homologous arms according to upstream and downstream sequences of a gene to be knocked out, cloning the two homologous arms to pMG36E-C of any one of claims 1-3, and constructing a knock-out plasmid pMG 36E-S-LR;

step 2: and (3) electrically transferring the pMG36E-S-LR to sensitive pediococcus acidilactici, screening by using an MRS plate containing erythromycin, picking a single colony and carrying out PCR verification to obtain a gene knockout mutant strain.

The invention also provides a method for gene insertion of pediococcus acidilactici by using an endogenous CRISPR system, which comprises the following steps:

step 1: designing a spacer according to the PAM of the II-A type CRISPR system in the lactic acid coccus cell as a 3' end NGG and a pseudo-insertion site, designing homologous arms according to the upstream and downstream sequences of the pseudo-insertion site, cloning the two homologous arms and a pseudo-insertion gene to the pMG36E-C of any one of claims 1-3, and constructing an insertion plasmid pMG 36E-S-LGR;

step 2: electrically transferring the pMG36E-S-LGR to sensitive pediococcus acidilactici, screening by using an MRS plate containing erythromycin, picking a single colony and carrying out PCR verification to obtain a gene insertion mutant strain.

The invention also provides a method for carrying out gene point mutation on pediococcus acidilactici by using an endogenous CRISPR system, which comprises the following steps:

step 1: designing a spacer according to the PAM of the lactic acid coccus intracellular II-A type CRISPR system as a 3' end NGG and a mutation-simulating site, designing homologous arms according to the upstream and downstream sequences of the mutation-simulating site, cloning the two homologous arms and a target gene to pMG36E-C of any one of claims 1-3, and constructing a mutant plasmid pMG 36E-S-LMR;

and 2, electrically transferring the pMG36E-S-LMR to sensitive pediococcus acidilactici, screening by using an MRS plate containing erythromycin, selecting a single colony and carrying out PCR verification to obtain a point mutation strain.

Preferably, the preparation method of the competent pediococcus acidilactici comprises: the method comprises the following steps: streaking pediococcus acidilactici in the frozen tube to an MRS solid culture medium, and activating overnight by using an MRS liquid culture medium after selecting single pediococcus acidilactici to obtain a seed solution;

transferring the seed solution to an MRS liquid culture medium containing threonine to culture to a logarithmic phase, washing the bacterial solution with an electrotransformation buffer solution containing sucrose for 3-4 times, treating with lysozyme for 20-30min, and suspending cells with the electrotransformation buffer solution containing sucrose and glycerol to obtain the competent pediococcus acidilactici.

Preferably, the conditions for electrotransformation into the sensitive pediococcus acidilactici are: the electrotransfer parameters were 2500V, 25uF, 200. omega. and the plasmid concentration was 500 ng.

The invention also provides a reverse screening method of pediococcus acidilactici for completing gene editing by utilizing the pediococcus acidilactici gene editing plasmid, which is characterized in that a gene editing mutant strain completed according to the method is subjected to streak culture on an MRS solid culture medium without antibiotics, is subjected to 2-3 generations of subculture, and a single colony is selected for PCR verification and is screened to obtain a target strain with lost plasmid.

The invention at least comprises the following beneficial effects:

based on escherichia coli and a shuttle plasmid pMG36E, the mini-CRISPR sequence containing two repetitive sequences and two three-type enzyme cutting sites Bbs1 is cloned to the shuttle plasmid pMG36E, and a pediococcus acidilactici gene editing plasmid pMG36E-C is constructed. The gene editing is carried out on the pediococcus acidilactici based on the endogenous CRISPR, an exogenous Cas gene does not need to be cloned, and the operation is simpler.

The method is characterized in that gene editing is carried out on the pediococcus acidilactici based on endogenous CRISPR, and the nutritional metabolic pathway or the new metabolic pathway of the pediococcus acidilactici is changed through point mutation, deletion mutation or insertion mutation, so that the aim of directionally modifying the pediococcus acidilactici is achieved, a target product or an over-expression target product is obtained through the microbial fermentation effect of a modified strain, and the adverse metabolic pathway of the strain can be blocked, so that the synthesis and secretion of the adverse metabolic product are eliminated.

Compared with the traditional homologous recombination, the efficiency of gene editing by using the technology is higher, so that the gene editing work can be completed in a short time, the time cost is saved, and the application and popularization of the editing technology are facilitated.

The edited strain can complete reverse screening only by two to three passages to obtain the target strain monoclonal with lost plasmid, so that the second gene editing is performed, the continuous editing of a plurality of genes can be completed in a short time, and the target strain with better production and metabolism performance can be obtained.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.

Drawings

FIG. 1 is a map of plasmid pMG36E of the present invention;

FIG. 2 is a map of plasmid pMG36E-C of the present invention:

FIG. 3 is an electrophoresis diagram of the mini-CRISPR fragment cloned into plasmid pMG 36E;

FIG. 4 is an electrophoretogram of the plasmid pMG36E-C of the present invention transformed into Pediococcus acidilactici;

FIG. 5 shows a pkt-deleted pure genotype knockout strain obtained by the method of knocking out the gene of Pediococcus acidilactici using the editing plasmid pMG36E-C of the present invention;

FIG. 6 is a map of the insertion plasmid pMG36E-St-LR of the present invention;

FIG. 7 shows a pure genotype mutant strain with deletion of pkt gene and insertion of tkt gene obtained by the method of gene insertion of Pediococcus acidilactici using the editing plasmid pMG36E-C of the present invention;

FIG. 8 shows the pure genotype mutant strain with inserted tkt gene and with lost Emr gene obtained by reverse screening of edited pediococcus acidilactici genetically engineered bacteria.

Detailed Description

The present invention is described in further detail below to enable those skilled in the art to practice the invention with reference to the description.

It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.

Example 1

The construction method of pediococcus acidilactici editing plasmid specifically comprises the following steps:

A. the inventors of Ongjingki (Wuhan) synthesized a mini-CRISPR fragment (shown in SEQ ID NO: 2) containing two DNA sequences of GTTTCAGAAGGATGTTAAATCAATAAGGTTAAGATC and two type III enzyme cleavage sites Bbs 1;

B. performing enzyme digestion (37 ℃ and 3h) on the mini-CRISPR fragment and pMG36E (the laboratory preservation and the map are shown in figure 1), wherein the enzyme digestion sites are Xba1 and Pst 1;

enzyme-linking transforming the enzyme digestion product to escherichia coli, coating an LB solid culture medium plate containing 400ug/ml of erythromycin, culturing at 37 ℃, selecting a transformant and carrying out PCR verification; designing a pair of universal primers 36F/36R (36F: GGCAATCGTTTCAGCAGAAAAATTC 36R: CGTTTTCAGACTTTGCAAGCTTGC) of pMG36E, carrying out PCR amplification by using the primers to detect whether a band of 300bp is contained or not so as to verify whether the mini-CRISPR fragment is cloned on a plasmid or not, wherein the PCR amplification result is shown in figure 3, and 3# (namely a fourth lane from left to right) is a transformant successfully transferred into pMG36E-C (the map is shown in figure 2);

C. the correct transformant was activated in LB liquid medium containing 400ug/ml erythromycin at 37 ℃ and 180rpm, and the plasmid was extracted to obtain Pediococcus acidilactici gene-editing plasmid pMG 36E-C.

Example 2

The method for preparing the competent pediococcus acidilactici specifically comprises the following steps:

A. streaking the pediococcus acidilactici in the frozen tube to an MRS plate, performing static culture in an incubator at 37 ℃ for 24 hours, picking the pediococcus acidilactici into 20mL of MRS liquid culture solution after single bacteria grow out, and performing culture at 37 ℃ and 180rpm for 12 hours;

B. transfer 400. mu.L of the culture medium to 20mL of fresh MRS culture medium (600. mu.L of D/L-threonine 40mM added), incubate at 37 ℃ and 180rpm for about 3h to OD₆₀₀Is 1.5;

C. 1.5mL of the bacterial solution was centrifuged at 4 ℃ and 10000rpm for 5min, the supernatant was discarded, 1mL of an electrotransfer buffer I (sucrose 205.37g/L, potassium phosphate trihydrate 1.86g/L, magnesium chloride hexahydrate 0.20g/L, pH7.5) was added to suspend the cells, the cells were centrifuged at 4 ℃ and 10000rpm for 5min, the supernatant was discarded, and 1mL of the electrotransfer buffer I was added to repeat the centrifugation once, and the supernatant was discarded. The cells were suspended in 100. mu.L of the electrotransfer buffer I, 10. mu.L of 1mg/mL lysozyme was added, and the mixture was treated in a 37 ℃ water bath for 30 min. Then centrifuging for 5min at 4 ℃ and 10000rpm, removing the supernatant, adding 1mL of electrotransfer buffer I for suspension and centrifugation twice, finally adding 500 microliter of electrotransfer buffer II (171.15 g/L of sucrose, 10% of glycerol) for suspension cells, and subpackaging every 80 microliter to obtain the pediococcus acidilactici electrotransfer competence for preservation at-80 ℃.

Example 3

The method for editing the transformation of the plasmid pMG36E-C into pediococcus acidilactici specifically comprises the following steps:

adding 500ng of pMG36E-C to 80ul of pediococcus acidilactici, performing electric shock transformation with electrotransfer parameters of 2500V, 25uF and 200 omega, and immediately adding 900ul of recovery medium, wherein the recovery medium comprises the following components in mass concentration: 171.15g/L of sucrose, 20g/L of glucose, 10g/L of tryptone, 4g/L of yeast extract, 8g/L of beef extract, 3g/L of sodium acetate, 2g/L of diammonium hydrogen citrate, 2g/L of dipotassium phosphate, 0.2g/L of magnesium sulfate heptahydrate, 0.05g/L of manganese sulfate monohydrate and 80L/L of Tween, then incubating for 3 hours at 37 ℃ and 180rpm, taking 100ul of bacterial liquid, coating MRS solid culture medium plates containing 5ug/mL of erythromycin, and culturing at 37 ℃;

after 2 days, multiple transformants grew on the plates, and the transformation efficiency reached 6 x 10³cfu/ug; after 10 transformants were picked and activated, PCR amplification was performed using a pair of universal primers 36F/36R (36F: GGCAATCGTTTCAGCAGAAAAATTC 36R: CGTTTTCAGACTTTGCAAGCTTGC) on pMG36E to detect whether a 300bp band was contained, and the results of the validation are shown in FIG. 4: the results indicated that 10 transformants had been transformed with the editing plasmid pMG 36E-C.

Example 4

The method for knocking out the gene of pediococcus acidilactici by using the editing plasmid pMG36E-C specifically comprises the following steps:

A. taking orotate ribotransferase pyrE as a gene to be knocked out, designing AACAACAACTAACTCCTGCCCAGCTAGATT as a spacer according to 3' NGG, cloning the gene to pMG36E-C by BbsI single enzyme digestion and enzyme ligation, and constructing a knock-out plasmid pMG 36E-Se;

B. using pediococcus acidilactici genome as a template, designing two pairs of primers, namely:

E-LF：AACTGCAG GGCTGGGGGGCATGGAC

E-LR：AGTTGCGCCCCATTGGTTTGAACCCCTTTATTTGGATTTC

E-RF：TAAAGGGGTTCAAACCAATGGGGCGCAACTACAAC

E-RR：CCCAAGCTTACCCGCAAAGTAGACTTGTGC，

amplifying the upstream and downstream 700bp of pyrE gene by a PCR method to be respectively used as a left arm and a right arm, connecting the upstream and downstream 700bp by an SOE method, cloning to pMG36E-Se by PstI/HindIII double enzyme digestion and enzyme ligation methods, and constructing a knock-out plasmid pMG 36E-Se-LR;

C. the knock-out plasmid pMG36E-Se-LR was transformed into Pediococcus acidilactici by the transformation method described in example 3, screened with MRS plates containing 5ug/ml erythromycin, single colonies were picked and verified by PCR, and a pair of primers pyrE-F/pyrE-R (a sequence including the pyrE gene in the amplified genome) at the two ends of the right arm of the pyrE gene on the genome were designed, namely:

pyrE-F：GGACGGGCACTACTTTACTTGTA

pyrE-R: CGGAGCGGGGTGCATGATAA, performing PCR amplification to obtain two strips with different sizes

Judging to obtain mixed genotype strains;

D. the mixed genotype strains were streaked onto MRS plates without antibiotics, single colonies were picked, streaked again, 17 single colonies were picked for activation and, using primers pyrE-F/pyrE-R and pyrE-F2/pyrE-R2 (pyrE gene on amplified genome),

pyrE-F：GGACGGGCACTACTTTACTTGTA

pyrE-R：CGGAGCGGGGTGCATGATAA

pyrE-F2：ATGACGGAAACACAAACTCAAGC

pyrE-R2：TTATTTAATTGTTGTAGTTGCGCC，

PCR verification was performed, and the PCR verification results are shown in FIG. 5: lanes 2-19 in a are PCR validation results for primers pyrE-F/pyrE-R, lanes 20-26 in a and lanes 2-11 in b are PCR validation results for primers pyrE-F2/pyrE-R2. The resulting single colony of # 11 was shown to be a pyrE-deleted pure knock-out strain.

Example 5

The method for performing gene insertion on pediococcus acidilactici by using the editing plasmid pMG36E-C specifically comprises the following steps:

A. taking bacillus coagulans transketolase gene tkt as a gene to be inserted, designing a section of ACTTGAACTTGCCTGACTTCCGCCAATACG sequence on the pkt gene to be inserted as spacer according to 3' NGG, cloning the gene to pMG36E-C by BbsI single enzyme digestion and enzyme ligation, and constructing an insertion plasmid pMG 36E-St;

B. using Pediococcus acidilactici genome as a template, designing two pairs of primers, amplifying the upstream and downstream 700bp of a non-coding site on the genome by a PCR method to be respectively used as a left arm and a right arm, connecting the left arm, tkt gene and the right arm by an SOE method, cloning to pMG36E-St by PstI/HindIII double enzyme digestion and enzyme connection methods, and constructing an insertion plasmid pMG36E-St-LR (map is shown in FIG. 6);

C. the insertion plasmid pMG36E-St-LR was transformed into Pediococcus acidilactici by the transformation method described in example 3, screened with MRS solid medium plates containing 5ug/ml erythromycin, single colonies were picked, and a pair of primers upstream and downstream of the insertion site pkt on the genome were designed, namely:

pkt knockout-F: GCCGGACGAACGCTCTCTAAC

pkt knockout-R: TGGCTGAGCCAGCTTTTGATG, carrying out PCR amplification, wherein the PCR result is two bands with different sizes, and the mixed genotype strain is judged;

D. streaking the mixed genotype strains to an MRS plate without antibiotics, picking single colonies, streaking again, picking 7 single colonies for activation and using primers tkt-F/tkt-R, namely:

tkt-F：CCTTAATTAA CCCCTTCAGCTTGAGCTC，

tkt-R：TGGCTGAGCCAGCTTTTGATG

and a primer pkt-F2/pkt-R2,

pkt-F2：ATGACAGACTATTCATCTAAAGCTT

pkt-R2: TTATTTAACGTCTTTCCATACCC, PCR verification, results are shown in FIG. 7: 4# Single bacterium

The strain is a pure genotype mutant strain with deletion of the pkt gene and insertion of the tkt gene.

Example 6

The method for carrying out gene mutation on pediococcus acidilactici by using the editing plasmid pMG36E-C specifically comprises the following steps:

A. taking L-type lactate dehydrogenase gene L-LDH as a gene to be mutated, designing AATCATCAACAAGAAGGGTGCTACATTCTA as a spacer according to 3' NGG, cloning the gene to pMG36E-C by BbsI single enzyme digestion and enzyme linkage, and constructing a mutant plasmid pMG 36E-Sldh;

B. using a Pediococcus acidilactici genome as a template, designing two pairs of primers, amplifying 700bp upstream and downstream of an L-LDH gene on the genome by a PCR method to be respectively used as a left arm and a right arm, connecting the left arm, a target gene LDH2 and the right arm by an SOE method, cloning to pMG36E-Sldh by enzyme digestion and enzyme ligation, and constructing a mutant plasmid pMG 36E-Sldh-LR;

C. the mutant plasmid pMG36E-Sldh-LR was transformed into Pediococcus acidilactici by the transformation method described in example 3, screened with MRS plates containing 5ug/ml erythromycin, and single colonies were picked.

D. And selecting the single colony to line on an MRS plate without antibiotics, selecting the single colony, marking again, selecting 10 single colonies for activation, and performing PCR amplification and sequencing on the LDH gene, wherein 1 single colony is a mutant strain in which the gene to be mutated is mutated from L-LDH to LDH 2.

Example 7

The method for carrying out reverse screening on the edited pediococcus acidilactici genetically engineered bacteria specifically comprises the following steps:

streaking mixed genotype pediococcus acidilactici subjected to pkt gene knockout on an antibiotic-free MRS plate for subculture, repeating the process for 2 times, picking 21 single colonies on the plate, and performing gene transfer by using a primer Emr-F: AGTTTATGCATCCCTTAACTTA, Emr-R: TCGACCCATATTTAAAAAGC, the Emr gene on the plasmid was amplified, and the PCR amplification results are shown in FIG. 8: 17# single colony is pkt gene knockout pure genotype mutant strain, and Emr gene is lost, i.e. plasmid loss occurs.

While the embodiments of the invention have been disclosed above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made thereto by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein but is not limited to the particular arrangements shown and described without departing from the general concept defined by the appended claims and their equivalents.

Sequence listing

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Claims

1. A pediococcus acidilactici gene editing plasmid pMG36E-C is characterized in that the construction method of the gene editing plasmid comprises the following steps:

wherein the DNA repetitive sequence is shown as SEQ ID NO: 1 is shown in the specification; the mini-CRISPR sequence is shown as SEQ ID NO: 2, respectively.

2. The pediococcus acidilactici gene editing plasmid pMG36E-C of claim 1, wherein the mini-CRISPR sequence and pMG36E have Xba1, Pst1 enzyme cleavage sites.

3. The pediococcus acidilactici gene-editing plasmid pMG36E-C of claim 1, wherein the screening conditions are: and E.coli containing a mini-CRISPR sequence and pMG36E is coated on an LB solid medium plate, cultured at 37 ℃, and a transformant is picked and subjected to PCR verification.

4. A method for knocking out genes of pediococcus acidilactici by using an endogenous CRISPR system is characterized by comprising the following steps of:

5. A method for gene insertion of pediococcus acidilactici using an endogenous CRISPR system, comprising the steps of:

step 1: designing a spacer according to the PAM of the lactic acid piece coccus intracellular II-A type CRISPR system as a 3' end NGG and a pseudo-insertion site, designing homologous arms according to the upstream and downstream sequences of the pseudo-insertion site, cloning the two homologous arms and a pseudo-insertion gene to the pMG36E-C of any one of claims 1-3, and constructing an insertion plasmid pMG 36E-S-LGR;

6. A method for performing gene point mutation on Pediococcus acidilactici by using an endogenous CRISPR system, which is characterized by comprising the following steps:

7. The method of claim 4, 5 or 6, wherein the competent pediococcus acidilactici is prepared by a method comprising the steps of: marking lactobacillus in the frozen tube to an MRS solid culture medium, and activating overnight by using an MRS liquid culture medium after selecting single lactobacillus;

8. The method of claim 4, 5 or 6, wherein the conditions for electrotransformation into the susceptible pediococcus acidilactici are: the electrotransfer parameters were 2500V, 25uF, 200. omega. and the plasmid concentration was 500 ng/80. mu.l.

9. A method for reverse screening of pediococcus acidilactici by using the pediococcus acidilactici gene-editing plasmid pMG36E-C of claim 1, wherein the gene-editing mutant strain prepared by the method of any one of claims 4-6 is streaked on MRS solid medium without antibiotic, and is subcultured for 2-3 generations, and a single colony is selected for PCR verification and screened to obtain a target strain with lost plasmid.