CN106834323A - Gene editing method based on streptomyces virginiae IBL14 gene cas7-5-3 - Google Patents

Abstract

The invention discloses a gene editing method based on the Streptomyces virginia IBL14 gene cas 7‑5‑3, which realizes for the first time the gene editing of the prokaryotic genome by the CRISPR‑Cas I system; Gene editing offers new additions and options. The system can be used to conveniently, quickly and effectively edit the genomes of Escherichia coli, Bacillus subtilis and other prokaryotic organisms. The optimized gene editing system is expected to be applied to the gene editing of other organisms.

Description

A Gene Editing Based on Cas7-5-3 of Streptomyces virginia IBL14 Gene method

technical field

The invention relates to gene editing technology in the field of biotechnology, specifically a gene editing method based on the cas 7-5-3 gene of Streptomyces virginia IBL14.

Background technique

Streptomyces virginiae IBL14 (Streptomyces virginiae IBL14) is an actinomycete isolated and screened from the sludge near the steroid drug manufacturer. It can effectively transform and degrade progesterone, flavonoids, cortisol and cholesterol and other steroidal compounds_ENREF_1（Wang, F.-Q.; Zhang, C.-G.; Li, B.; Wei,D.-Z.; Tong, W.-Y., New microbiological transformations of steroids by Streptomyces virginiae IBL-14. Environmental science & technology 2009, 43(15), 5967-5974). CRISPR (clustered regμlarly interspaced short palindromic repeat sequences) is called clustered regularly interspaced short palindromic repeat sequences, which were first discovered in Escherichia coli_ENREF_1 in the 1980s (Ishino, Y.; Shinagawa, H.; Makino, K.; Amemura, M.; Nakata, A., Nucleotide Sequence of the iap Gene, Responsible for AlkalinePhosphatase Isozyme Conversion in Escherichia coli, and Identification of the Gene Product. JOURNAL OF BACTERIOLOGY 1987, 169 (12), 5429-5433). In 2002 R. Jansen et al. compared and analyzed the clustered regularly spaced short palindromic repeat sequence in the prokaryotic genome in detail, and found that the conserved gene sequence _ENREF_1 was often accompanied by the repeat sequence (Jansen, R.; Embden, J.D. ; Gaastra, W.; Schouls, L. M., Identification of genes that are associated with DNA repeats in prokaryotes. Mol Microbiol 2002, 43 (6), 1565-75), they named this unique repeat sequence CRISPR, conserved gene sequence The encoded proteins are called Cas accessory proteins (CRISPR-associated proteins). At present, it has been found that the CRISPR-Cas system is widely distributed in prokaryotes_ENREF_1 (http://crispr.u-psud.fr/), and only the Cas9 protein in the CRISPR-Cas type II system has been commercially applied to gene editing.

So far, although many discoveries have been made about the CRISPR-Cas type I system, there is no report on its application to gene editing. Previously, we used the type I-B-sv14 CRISPR-Cas system in Streptomyces virginia IBL14 combined with an engineered gene editing plasmid to edit the chromosome of Streptomyces virginia IBL14_ENREF_1 (Tong Wangyu; Yong Dexiang; Li Xue; Qiu Caihua A CRISPR-Cas system in Streptomyces virginia IBL14 and its application for gene editing. 2015110028173, 2015), but it has not been able to achieve gene editing in other biological genomes. The present invention connects the gene cas7-5-3 in the Streptomyces virginia IBL14 type I-B-sv14 CRISPR-Cas system with a plasmid (plasmid) to obtain a Cas7-5-3 protein expression plasmid plasmamid-cas7-5-3 , combining the designed gene editing plasmid (plasmid-t/gDNA) or other plasmids for the first time to achieve gene editing in prokaryotes Escherichia coli and Bacillus subtilis. And the Cas7-5-3 protein (containing 1323 amino acids) has a molecular size similar to that of the Cas9 protein (containing 1368 amino acids).

Although the CRISPR-Cas type I system _ENREF_1 has been found in Escherichia coli (Semenova, E.; Savitskaya, E.; Musharova, O.; Strotskaya, A.; Vorontsova, D.; Datsenko, K.A.; Logacheva, M. D.; Severinov, K., Highly efficient primed spaceacquisition from targets destroyed by the Escherichia coli type I-E CRISPR-Cas interfering complex. Proceedings of the National Academy of Sciences of the United States of America 2016, 113 (27), 7626-7631), but so far There is no report on gene editing by the CRISPR-Cas type I system in E. coli; in particular, the CRISPR-Cas system_ENREF_1 has not been found in Bacillus subtilis (http://crispr.u-psud.fr/). The present invention realizes the purpose of verifying the efficacy of the plasmid system by knocking out the E. coli β-galactosidase gene lacZ and the Bacillus subtilis lactate dehydrogenase gene ldh.

Contents of the invention

The technical problem to be solved by the present invention is to provide a CRISPR-Cas type I-B-sv14 system Cas7-5-3 protein expression plasmid system similar to the Cas9 protein in the CRISPR-Cas type II system, combined with gene editing plasmids or other plasmids for prokaryotic Gene editing of organisms.

To achieve this purpose, the technical solution adopted by the present invention to solve its problems is: a gene editing method based on Streptomyces virginia IBL14 gene cas 7-5-3, characterized in that: Streptomyces virginia IBL14 Contains 3 genes cas 7-5-3, the nucleic acid sequence of which is as described in Attached Table 1, after the construction of protein expression plasmid (attached 1), the construction of gene editing plasmid (attached 2) and the steps of obtaining and testing recombinants (Fig. 3) It can effectively edit the genome of prokaryotic organisms.

The gene editing method based on Streptomyces virginia IBL14 gene cas 7-5-3 is characterized in that: the steps of protein expression plasmid construction, gene editing plasmid construction and recombinant acquisition and inspection steps are as follows:

(1) Design primers according to the cas 7-5-3 gene of Streptomyces virginiae IBL14 and related information sequences (Appendix 2), use the genome of Streptomyces virginiae IBL14 as a template, and use DNA polymerase to amplify by PCR reaction Amplify the cas 7-5-3 gene and connect it to the plasmid (plasmid) to obtain the Cas7-5-3 protein expression plasmid plasma- cas 7-5-3;

(2) Design primers (Appendix 2) according to the DNA sequence information of the prokaryotic target gene, and use the prokaryotic genome as a template to amplify through PCR reactions to obtain restriction endonuclease recognition and cleavage sites at the end and overlap PCR The PCR fragments of the upper and lower homology arms of the target gene with complementary sequences, and the upper and lower homology arms are connected by overlap PCR to obtain a gene editing template (template DNA/t-DNA). Targeting gene fragment (guide-DNA/g-DNA) containing the lactose operon promoter and RNA terminator (Supplementary Table 2), and the gene editing template and the targeting gene according to the cohesive ends on the restriction site The fragment is connected to the plasmid to obtain a gene editing plasmid (plasmid-t/gDNA);

(3) Prepare competent prokaryotic cells, and transform the protein expression plasmid plasmid- cas 7-5-3 obtained in step (1) and various targeted gene editing plasmids obtained in step (2) into target bacteria Different gene-edited recombinants were obtained in the competent state; PCR and gene sequencing and/or functional analysis were performed on the recombinant chromosomes to confirm the edited target recombinants.

The gene editing method based on the cas 7-5-3 gene of Streptomyces virginia IBL14 is characterized in that: the prokaryotic organisms refer to Escherichia coli, Bacillus subtilis and other prokaryotic microorganisms.

The described gene editing method based on Streptomyces virginia IBL14 gene cas 7-5-3 is characterized in that: the gene editing refers to the application of the protein expression plasmid in combination with a gene editing plasmid or other plasmids that can modify the prokaryotic Chromosomal gene knockout, insertion, scarless point mutation and any combination of cells.

Beneficial effect

The present invention is a new gene editing system based on the cas gene of the CRISPR-Cas I type system of Streptomyces virginia IBL14, and realizes the gene editing of the prokaryotic genome by the CRISPR-Cas I system for the first time; CRISPR-Cas type II Cas provides new complements and options for gene editing. The system can be used to conveniently, quickly and effectively edit the genomes of prokaryotic organisms such as Escherichia coli and Bacillus subtilis. The optimized gene editing system is expected to be applied to the gene editing of other organisms.

Description of drawings

Figure 1 shows the construction of the protein expression plasmid pHT304-cas7-5-3. Ori / origin: DNA replication initiation site; lac promoter / lactose promoter: the promoter of the lactose operon, combined with RNA polymerase to initiate DNA transcription; CAP binding site / catabolite activator protein binding site: cAMP receptor protein binding site , to promote RNase transcription activity; AmpR / ampicillin resistance: ampicillin resistance; Erm /erythromycin: erythromycin

Figure 2 shows the construction of gene editing plasmids, (A) construction of gene editing plasmid pKC1139- lacZ -t/g-DNA, (B) construction of gene editing plasmid pKC1139- ldh -t/g-DNA. Ori pSG5 / the origin from pSG5: the origin of replication on the pSG5 plasmid vector; oriTRK2 / the origin of conjugal transfer from RK2: the origin of conjugal transfer from RK2 plasmid vector; lac promoter / lactose promoter: the promoter of the lactose operon ;T7 promoter: T7 promoter, which initiates DNA transcription; AmpR / ampicillin resistance: ampicillin resistance; AprR / apramycin resistance: apramycin resistance; Cm / Chloramphenicol: chloramphenicol

Figure 3 shows the results of screening and verification of transformants. (A) Blue-white screening results of Escherichia coli JM109(DE3), the blue indicates the original strain, and the white indicates the recombinant strain; (B) The external PCR electrophoresis image of the gene lacZ , lane M: 5000 bp DNAladder, lane 1: blank control, Swimming lane 2: the lacZ gene PCR of plasmid pHT304- cas 7-5-3, pKC1139- lacZ -t/g-DNA and pKD46 transformants, swimming lane 3: the lacZ gene PCR of wild-type EC JM109 (DE3) genome (in swimming lane 2 The DNA band is smaller than the DNA band in lane 3, indicating successful gene knockout); (C) PCR sequencing results of the lacZ gene of the plasmid pHT304- cas 7-5-3, pKC1139- lacZ -t/g-DNA, and pKD46 transformants (The sequence result is consistent with the designed gene sequence in Table 2, indicating that the recombinant is correct).

detailed description

In order to fully understand the technical content of the present invention, the technical solution of the present invention will be further introduced and illustrated below in conjunction with specific examples, in order to better explain the content of the present invention, and the following examples do not limit the protection scope of the present invention. In addition, the following materials are used in the listed examples unless otherwise specified:

1) Strains and plasmids

Streptomyces virginiae IBL14 / SV IBL14; Escherichia coli DH5α / EC DH5α; Escherichia coli JM109 / EC JM109; Bacillus subtilis 168 / BS 168, plasmid pHT304; pKC1139; pKD46.

2) Medium

LB liquid medium

Add 5 g of yeast powder, 10 g of peptone, and 10 g of NaCl, add an appropriate amount of tap water to dissolve, adjust the volume to 1 L, adjust the pH to 7.0-7.2, subpackage and bandage, and sterilize at 121 °C/20 min.

LB solid medium

Add 5 g of yeast powder, 10 g of peptone, 10 g of NaCl, and 20 g of agar, add an appropriate amount of tap water to dissolve, adjust the volume to 1 L, adjust the pH to 7.0-7.2, subpackage and bandage, and sterilize at 121 °C/20 min.

Spizizen Minimal Medium

10×Spizzen basic salt medium 100 mL/L, 50% (w/v) glucose 10 mL/L, tryptophan stock solution (5 mg/mL) 10 m L/L, agar 1.5% w/v), 121 Sterilize at °C/20 min.

GM1 medium (5mL)

40% glucose 100 μl, 20 mg/mL acid hydrolyzed casein 100 μl, 50 mg/mL yeast extract 100 μl, 20% (w/w) MgSO4.7H2O 5 μl, 10×Spizzen minimal medium 500 μl , water 3195 μl, the above substances need to be sterilized at 121 ℃/20 min before mixing.

GM2 medium (5mL)

100 μl of 40% glucose, 50 μl of 20 mg/mL acid hydrolyzed casein, 40 μl of 20% (w/w) MgSO4.7H2O, 500 μl of 10×Spizzen basic medium, 3310 μl of water, 121 ℃ before mixing /20 min for sterilization.

All reagents used are commercially available.

Example 1 (Three-plasmid one-step co-transformation method to knock out EC JM109 lacZ gene)

(1) Construction of protein expression plasmid pHT304- cas 7-5-3

According to the whole genome sequencing information of SV IBL14 and the sequence information of plasmid pHT-304, the cas 7-5-3 gene-specific primers cas -F and cas -R with the complementary sequence of plasmid pHT-304 were designed (Table 2). Genomic DNA of SV IBL14 was extracted, and cas gene PCR amplification was carried out using TransStart FastPfu DNA Polymerase produced by Quanshijin Biotechnology Co., Ltd. The reaction conditions were: 95°C for 5 min, 94°C for 30 s, 55°C for 30 s, 72°C for 2 min, 2.5 U of Pfu DNA Polymerase produced by Sangon Bioengineering (Shanghai) Co., Ltd. (50 μl reaction system), 30 cycles, 72 ° C for 10 min. The PCR product was detected by 1% agarose electrophoresis, recovered by the kit, and the purified cas full-length gene was obtained. The cas full-length gene sequence was connected to the plasmid pHT-304 by one-step method to obtain the Cas7-5-3 protein expression plasmid pHT304- cas 7-5-3.

(2) Construction of gene editing plasmid pKC1139- lacZ -t-DNA

(A) Gene lacZ primer design and amplification of the full-length lacZ gene

According to the genome sequencing information of EC JM109, gene lacZ -specific primers lacZ -F and lacZ -R were designed (Table 2). Extract EC JM109 genomic DNA, use Pfu DNA Polymerase for lacZ gene PCR amplification, reaction conditions: 95 °C for 5 min, 94 °C for 30 s, 52 °C for 30 s, 72 °C for 1 min, 2.5 U Pfu DNA Polymerase (50 μl reaction system) , 30 cycles, 10 min at 72°C. The PCR product was detected by 1.5% agarose electrophoresis, recovered from the kit, and the purified lacZ full-length gene fragment was obtained for future use.

(B) Preparation of upstream and downstream homology arms

According to the full sequence of the lacZ gene (Table 1), the primers lacZ -UF and lacZ -UR of the upstream homology arm of the lacZ gene, and the primers lacZ -DF and lacZ -DR of the downstream homology arm of the lacZ gene (Table 2) were designed. ), and the upper homology arm upstream primer contains a HindIII restriction endonuclease site, and the lower homology arm downstream primer contains an XbaI restriction endonuclease site. Using the purified lacZ gene DNA as a template, the upper and lower homology arms were first amplified respectively. The reaction conditions were: 95°C for 5 min, 94°C for 30 s, 58°C for 30 s, 72°C for 45 s, 2.5 U Pfu DNA Polymerase ( 50 μl reaction system), 30 cycles, 72 °C for 10 min. The PCR product was detected by 1.5% agarose electrophoresis, recovered by the kit, and the purified DNA fragments of the upper and lower homology arms were obtained for future use.

(C) Construction of gene editing template vector pKC1139- lacZ -t-DNA

Mix 0.5 μl of the purified product of the upper homology arm and the purified product of the lower homology arm as a template, and perform overlap PCR in a 25 μl reaction system. The reaction conditions are: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 1 min, and annealing at 58 °C for 1 min , extend at 72°C for 30 s, add 1 μl each of primers UF and DR after one cycle, and continue PCR. The reaction conditions are: pre-denaturation at 95°C for 5 min, denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 1 min 30 s, 30 cycles, 72 ℃ 10 min. 1.5% agarose gel electrophoresis was used to detect and purify the amplified product to obtain a gene editing template; the obtained gene editing template was cut out of sticky ends by HindIII restriction endonuclease and XbaI restriction endonuclease, and then passed through full gold The T4 ligase produced by Biotechnology Co., Ltd. was used to connect it to the pKC1139 plasmid to obtain the gene editing template vector pKC1139- lacZ -t-DNA.

(D) Construction of gene editing plasmid pKC1139- lacZ -t/g-DNA

The targeted gene fragment containing the lactose operon promoter and the guide DNA- lacZ junction product (Table 2) was directly synthesized by Chuzhou General Biological Company, with BamHI and EcoRI restriction sites added at the beginning and end, respectively, the middle is the promoter, and the repeat sequence (repeat), spacer sequence (spacer), repeat sequence (repeat) and terminator; the synthetic targeted gene fragments were cut out of sticky ends by BamHI and EcoRI restriction endonucleases, and then ligated to the obtained DNA by T4 ligase The gene editing plasmid pKC1139- lacZ -t/g-DNA was obtained from the gene editing template vector pKC1139- lacZ -t-DNA.

(3) Acquisition and inspection of recombinants

(A) Competent one-step preparation of EC JM109

In the ultra-clean bench, use a sterilized toothpick to pick up the single clone on the EC JM109 plate and culture it overnight in 30 ml LB liquid medium at 37 °C and 200 rpm; transfer 100 μl of the overnight cultured bacterial solution to a new Incubate in LB liquid medium at 37°C and 200 rpm for about 2 h until the OD600 value of the bacterial liquid is 0.4-0.6; take 1 ml of the above bacterial liquid into a 1.5 ml EP tube, and centrifuge at 5000 rpm×g at 4°C for 5 min After removing the supernatant completely, take 50 μl of the SSCS solution that has been pre-cooled on ice and pipette the bacterial cell pellet evenly to obtain the competent cells of EC JM109, and store them at -80 ℃ for later use (the whole process is done on ice conduct).

(B) Co-transformation of plasmids pKD46, pHT304- cas 7-5-3 and pKC1139- lacZ -t/g-DNA

The plasmids pKD46, pHT304- cas 7-5-3 and pKC1139- lacZ -t/g-DNA were well mixed and transformed into EC JM109 competent cells, and then coated with 5 μl isopropyl-β-D-thiopyridine Galactopyranoside (IPTG 200 mg/ml), 40 μl 5-bromo-4-chloro-3-indole-β-D-galactopyranoside (X-gal 20 mg/ml ) and 20 μl arabinose (10 mM/L) apramycin and ampicillin-resistant LB solid medium and cultured overnight at 30 °C to obtain transformants.

(C) Recombinant chromosome PCR and gene sequencing analysis

The white monoclonal clone was picked as a template, and the lacZ gene verification primer lacZ -F/ lacZ -R was used for PCR amplification reaction. The reaction conditions were: 95°C for 5 min, 94°C for 30 s, 58°C for 30 s, 72°C for 2 min 40 s, 2.5 U EasyTaq DNA Polymerase (25 μl reaction system) produced by Sangon Bioengineering (Shanghai) Co., Ltd., 30 cycles, 72 ° C for 10 min. The PCR product was detected by 1% agarose electrophoresis, and the amplified band of recombinant chromosomal DNA was observed to decrease, and the lacZ gene was successfully knocked out by General Biosystems (Anhui) Co., Ltd. sequencing (Figure 3).

Example 2 (two-step co-transformation with three plasmids to knock out the EC JM109 lacZ gene)

(1) Construction of protein expression plasmid pHT304- cas 7-5-3

Same as embodiment 1 step (1)

(2) Construction of gene editing plasmid pKC1139- lacZ -t-DNA

Same as embodiment 1 step (2)

(3) Acquisition and inspection of recombinants

(A) Transformation of plasmid pHT304- cas 7-5-3 and competent preparation of EC JM109- pHT304- cas 7-5-3

The plasmid pHT304- cas 7-5-3 was transformed into EC JM109 competent, and the transformants were screened by erythromycin resistance, and then the transformants were used to prepare competent cells to obtain EC containing plasmid pHT304- cas 7-5-3 Competence of JM109- pHT304- cas 7-5-3 strain. The competent preparation method is the same as step (3A) in Example 1.

(B) Co-transformation of plasmids pKD46 and pKC1139- lacZ -t/g-DNA

Mix the plasmids pKD46 and pKC1139- lacZ -t/g-DNA well and transform them into EC JM109-pHT304- cas 7-5-3 competent medium, in which 5 μl IPTG, 40 μl X-gal and 20 μl Arabidopsis The erythromycin, apramycin and ampicillin LB solid medium with glycosides were cultivated overnight at 30°C to obtain transformants.

(C) Recombinant chromosome PCR and gene sequencing analysis

The white monoclonal clone was picked as a template, and the lacZ gene verification primer lacZ -F/ lacZ -R was used for PCR amplification reaction. The reaction conditions were: 95°C for 5 min, 94°C for 30 s, 58°C for 30 s, 72°C for 2 min 40 s, 2.5 UEasyTaq DNA Polymerase (25 μl reaction system), 30 cycles, 72 ° C for 10 min. The PCR product was detected by 1% agarose electrophoresis, and the size change of the recombinant chromosomal DNA amplification band was observed, and the lacZ gene was successfully knocked out by gene sequencing.

Example 3 (Two-plasmid one-step co-transformation method to knock out EC JM109 lacZ gene)

(1) Construction of protein expression plasmid pHT304- cas 7-5-3

Same as embodiment 1 step (1)

(2) Construction of gene editing plasmid pKD46- lacZ -t/g-DNA

Except that the homology arm and the guide DNA- lacZ target gene fragment were sequentially connected to the plasmid pKD46 to form the gene editing plasmid pKD46- lacZ -t/g-DNA, the rest of the steps were the same as step (2) in Example 1.

(3) Acquisition and inspection of recombinants

Same as embodiment 1 step (3)

Embodiment 4 (knockout of BS 168 gene ldh and insertion of chloramphenicol resistance gene)

(1) Construction of protein expression plasmid pHT304- cas 7-5-3

Same as embodiment 1 step (1)

(2) Construction of gene editing plasmid pKC1139- ldh -t/g-DNA

(A) Gene ldh primer design and amplification of ldh full-length gene

Design gene primers as ldh -F and ldh -R (Table 2). Using BS 168 genomic DNA as a template, the ldh gene fragment was amplified. Reaction conditions: 95°C for 5 min, 94°C for 30 s, 55°C for 30 s, 72°C for 2 min, 2.5 U Pfu DNA Polymerase (50 μl reaction system), 30 cycles, 72°C for 10 min. The PCR product was detected by 1.5% agarose electrophoresis, recovered from the kit, and the purified ldh full-length gene fragment was obtained for future use.

(B) Preparation of upstream and downstream homology arms

The upper and lower homology arm primers ldh -UF, ldh-UCmR, ldh -UCmF, ldh -CmDR, ldh - CmDF , ldh -DR were designed respectively (Table 2). Using the purified ldh gene and Cm gene DNA as templates, the upper, Cm , and downstream homology arms were first amplified respectively. The reaction conditions were: 95°C for 5 min, 94°C for 30 s, 55°C for 30 s, 72°C for 1 min 20 s, 2.5 U Pfu DNA Polymerase (50 μl reaction system), 30 cycles, 72 ° C for 10 min. The PCR product was detected by 1.5% agarose electrophoresis, recovered by the kit, and purified upper, Cm , and downstream homology arm DNA fragments were obtained for future use.

(C) Construction of gene editing template vector pKC1139- ldh -t-DNA

Take 1.0 μl of the purified product of the upper homology arm, the purified product of the cm and the purified product of the lower homology arm and mix it as a template, and perform overlap PCR in a 25 μl reaction system. Anneal for 1 min at °C, extend for 30 s at 72°C, add 1 μl each of primers UF and DR after one cycle, and continue PCR. Extend for 2 min at ℃, and perform 30 cycles, 10 min at 72 ℃. 1.5% agarose gel electrophoresis to detect the amplified product and purify it to obtain the gene editing template; cut out the cohesive ends of the obtained gene editing template with HindIII and XbaI restriction endonucleases, and then connect it to the pKC1139 plasmid with T4 ligase On the above, the gene editing template vector pKC1139- ldh -t-DNA was obtained.

(D) Construction of gene editing plasmid pKC1139- ldh -t/g-DNA

The targeted gene fragment containing the lactose operon promoter and guide DNA- ldh ligation product (Table 2) was directly synthesized by Chuzhou General Biology Co., Ltd., with BamHI and EcoRI restriction sites added at the beginning and end, respectively, the middle is the promoter, and the repeat sequence (repeat), spacer sequence (spacer), repeat sequence (repeat) and terminator; the synthetic targeted gene fragments were cut out of sticky ends by BamHI and EcoRI restriction endonucleases, and then ligated to the obtained DNA by T4 ligase The gene editing plasmid pKC1139- ldh -t/g-DNA was obtained from the gene editing template vector pKC1139- ldh -t-DNA.

(3) Acquisition and inspection of recombinants

(A) Preparation of BS 168 competent cells

Pick a wild-type BS 168 monoclonal and culture it overnight in 5 ml GM1 medium at 37 °C and 130 rpm. The next day, 1 ml was transferred to 9 ml GM1 medium, and cultured at 37 °C, 200 rpm for 3.5 h. Transfer 5 ml of GM1 culture medium to 45 ml of GM2 medium, and culture at 37 °C, 130 rpm for 1.5 h. Centrifuge at 4000 rpm for 10 min at 4 °C. Retain an appropriate amount of supernatant to resuspend the cells, and divide into 2 ml EP tubes to obtain BS 168 competence for future use.

(B) Co-transformation of plasmids pHT304- cas 7-5-3 and pKC1139- ldh -t/g-DNA

Mix the plasmids pHT304- cas 7-5-3 and pKC1139- ldh -t/g-DNA well and add them to the prepared competent cells, let stand at 37°C for 1 hour, and incubate at 37°C at 220 rpm for 3-4 hours . Pipette an appropriate amount of bacterial liquid and spread it on the LB medium containing Cm resistance, and cultivate it in a 37 °C incubator.

(C) Recombinant chromosome PCR and gene sequencing analysis

Single clones were picked on LB plates containing Cm antibiotics, cultured overnight at 37°C, 200 rpm. The next day, the genome was extracted for PCR, and the product was detected by 1% agarose electrophoresis to observe the change in the size of the amplified band of recombinant chromosomal DNA, and gene sequencing proved that the BS 168- ldh gene knockout and the chloramphenicol resistance gene insertion were successful .

Embodiment 5 (single plasmid knocks out BS 168 ldh gene)

(1) Construction of protein expression plasmid pHT304- cas 7-5-3

Same as embodiment 1 step (1)

(2) Construction of gene editing plasmid pHT304- cas 7-5-3- ldh -t/g-DNA

Except that the temple-DNA- ldh target gene fragment does not contain the Cm gene, other steps are the same as step (2) in Example 4.

(3) Acquisition and inspection of recombinants

Same as embodiment 4 step (3)

The above description only uses examples to further illustrate the technical content of the present invention, so that readers can understand more easily, but it does not mean that the implementation of the present invention is limited to this, and any technical extension or re-creation according to the present invention is subject to protection of the invention.

Table 1 Full-length sequences of Streptomyces virginia IBL14 gene cas7-5-3, Escherichia coli JM109 gene lacZ and Bacillus subtilis BS168 gene ldh

gene sequence

A) cas7

(999bp) gtggtcgccggtgccccgaacaacggggagggcgaggacaacacggggcgtgtgaagaagctgagggtcgggcgggaggagttcccgtacgtgtccgcgcaggcgttccgtcggtggttgcgtgactcgctgccggcgcaggagccgcgttcggtggtcactcgctcgggcagcggtgccaagcagcaggcacacaccgcgggccggccggacctgcacctggacgatgatctgttcggctacatggtcgcggtgaaggggaaggggggaagctaccagcgggacaccgtgctggctaccgggactttagtttcagtggtgccgcagcgtccgacgttggacttcggcacgatgagccgggacttcccggctggtgagcacccggtgattcactcgcacgagctgtacagcgcgaccctggccggcgatgttctgctggatctgccgcgggctggggtcttcgagacggacggcaacgggttgcgcgtggcgatcagccctgccgtcgctgaggaagcggcgaagaacggggcggaggtcaccacgctgcggggcagtgcggccattcggttgccgcttactgagcggcaccggcggatcggcacgctgttgcggacgctggcgtcggtgcgtggtggggccaagcaggctctgcactacggggaccgggccccttcattggtcttgttggctcctctcaagggtggcgtcaatccgttcacccgtgttctgggcgcccgcgacggtaagcctgtgttcctgagcgatgtcctgcgcgaggagctcgaggcgtgggcggatgagctggacgggccggtgctgctgggctgggccccggggtttctcggcgatcagcgtgagcaggtccgccgcgagctcaaggatctgattgacgagggccgtgtcgtcctgagccatcctcgtgtgctgctgacccagctggccgaccggatcgagcagggtgatcatgacgcgtggttcgaggactccgc ggcgtga

B) cas5

(663bp)

gtgacgggtacggaggtcacggccctgcagatcacggtgacggcgccggttgtctccttccgtaatccgctgtatgccggggtgcaggtgacgctgccgtgtccgccgccggccaccgtcggcggcctcctcgccgcagcggctggggggtgggagcaggtcaatccggagctgcgtttcgcgatggcgttccacgctggcggcaaggcggtcgatctcgagacgtaccacccgctggacgcgtctgggaagaaggcgtcgcctgccccgcgtaaccgggagttccttacggcggccgagctcaccgtgtggctggtcgacgaccctgaagggtggcagcgccgcctgcgtcggccggtgtggccgctgcggctgggccgcagccaggacctggtcggtatccgcaccggcctggttccgttgcgcgcggagcccggcgagcagcggtccgccgtggtgccggagacggcggggaggatgggaaccctactgcggctgccgactgcggtctctgggggccgggaccgtacccggtgggacagctaccggttcgacagctcgggccgcagtgaccatgtggtcgtaggcggctggtcgactgccgggggacaggcagtcattctgctgccctcggcccatcccgataccgtcgcgcgttcctga

C) cas3

(2316bp)

gtgggccgtctggacgcggtggaggacgtcttcggcggcaggttctggcccgtcgtggaactcgctggcctcacccacgacgccggcaagattcccgaaggcttccagcggatgctggcgggatacagccgtgcctggggtgagcgtcacgaagtcgcctcgttgggcttcctgcccgcgctcatcggcgacccggacgtgctgttgtgggtggcgaccgcggtcgccacccaccatcgtccgctgaccggccagaacggacgcgacctgcagactctctacagcggtgtcaccatcaccgagctcgcgcaccgtttcgggccttttgacccacgcgctgtccccgccttggaggcctggcttcgtgcgagcgccatccgggtcggcctccccgcggccgctgttccagacgacggcacgctcaccgacaccggagtggtcgctggcgcccaccagctgctggaggagattttggaccgttgggcagaccgtgtgaggcctgaggtgggcttggccgctgtactgctgcagggggcggtcaccctggccgaccacttgtcctccgcccatcaggctctgcccaccgtccagccgttgggggccgggttccggtcccggttggagaaggagttcgctgaacgcggcaggaccctgcgtgcccaccagctggaggccgccaccgttaccggacatcttctgctgcgcgggccgaccggcagtgggaagaccgaggctgccctgctgtgggctgccagccaggtcgaggccctgaaggcggaaggccggggcgtgccgcgtgtgtttttcactctcccctacctggcctccatcaacgccatggcaacacggctgggtgacactctcggcgatggtgaggctgtcggcgttgcccactcccgcgccgcctcctaccaccttgcccaggccatcgccccgcaggacggcgacgaggaggacgaacacggagccccctgccgtgttgacgcggccgccaaggcct tgtcccgggccgctgccaccaagctgttccgcgagagtgtccgcgtcgccaccccctaccagcttctgcgggccgccctggccgggccggcccactccggcatcctcatcgacgccgcgaactcggtgttcatcctggacgaactccacgcctacgacgcccgcaggctcggctacatcctggccagtgcccggctgtgggaacgcctcggtggacggatcacagtcctgtccgcgaccctgcccagggccctggccgacctgttcgagagcaccctcaccgcccccatcaccttcctcgacacccccgacctcgggctgccggcgcgccacctcctgcacacccgaggccaccatctcaccgacccggccacactggaggagatccgtctgcggctgtcccgggacgagtcggtcctggtgatcgccaacaacgtgtcccaggccatcgccctgtacgaacagctcgcacccgacgtgtgtgaacgcttcggtcaggacgccgcgctactgctgcactcccggtttcgacggatggaccggtcccggattgagcagaagatcgccgaccggttcgccactgtggcacctgatgcccagaacagccgtaagccgggcctggtcgttgccacgcaggtggtcgaggtcagtctcgacgtcgacttcgatgtgctgttcactggagcggctccgctcgaggccctcctgcagcgcttcggccggaccaaccgcgtcggggcccgcccgccggccgacgtcatcgtccaccatcccgcctggaccacacgccgccgacagcccggcgagtacgccgacggcatctacccacgggagccggtcgagtccgcgtggcacatcctcacccgcaatcacgggcgagtcatcgacgaagcggacgccaccgcgtggctggacgaggtctacgccacggactggggcaggcaatggcaccgcgaggtgctggagcggcgagaaagattcgaccgtgcgttcctgcagtt ccgctaccccttcgaagaccgcactgacctggccgataccttcgacgaactcttcgacggctccgaagccatcctcgccgaagaccaggacgcctactcagccgcactggccgcaccagacggcgaccaccccggagctggccggctcctcgcagaggaatacctcatccccgttccccactgggccagccccctcagccgctacgagaagcagctcaaagtccgcgtcatcaacggcgactaccaccccgaccacggcctcatggcggtccgggggctgccccagcccgcctaccgcgccggggaggtcttgtga

D) lacZ (3075bp)

atgaccatgattacggattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgctttgcctggtttccggcaccagaagcggtgccggaaagctggctggagtgcgatcttcctgaggccgatactgtcgtcgtcccctcaaactggcagatgcacggttacgatgcgcccatctacaccaacgtgacctatcccattacggtcaatccgccgtttgttcccacggagaatccgacgggttgttactcgctcacatttaatgttgatgaaagctggctacaggaaggccagacgcgaattatttttgatggcgttaactcggcgtttcatctgtggtgcaacgggcgctgggtcggttacggccaggacagtcgtttgccgtctgaatttgacctgagcgcatttttacgcgccggagaaaaccgcctcgcggtgatggtgctgcgctggagtgacggcagttatctggaagatcaggatatgtggcggatgagcggcattttccgtgacgtctcgttgctgcataaaccgactacacaaatcagcgatttccatgttgccactcgctttaatgatgatttcagccgcgctgtactggaggctgaagttcagatgtgcggcgagttgcgtgactacctacgggtaacagtttctttatggcagggtgaaacgcaggtcgccagcggcaccgcgcctttcggcggtgaaattatcgatgagcgtggtggttatgccgatcgcgtcacactacgtctgaacgtcgaaaacccgaaactgtggagcgccgaaatcccgaatctctatcgtgcggtggttgaactgcacaccgccgacggcacgctgattgaagcagaagcctgcgatgtcggtttcc gcgaggtgcggattgaaaatggtctgctgctgctgaacggcaagccgttgctgattcgaggcgttaaccgtcacgagcatcatcctctgcatggtcaggtcatggatgagcagacgatggtgcaggatatcctgctgatgaagcagaacaactttaacgccgtgcgctgttcgcattatccgaaccatccgctgtggtacacgctgtgcgaccgctacggcctgtatgtggtggatgaagccaatattgaaacccacggcatggtgccaatgaatcgtctgaccgatgatccgcgctggctaccggcgatgagcgaacgcgtaacgcgaatggtgcagcgcgatcgtaatcacccgagtgtgatcatctggtcgctggggaatgaatcaggccacggcgctaatcacgacgcgctgtatcgctggatcaaatctgtcgatccttcccgcccggtgcagtatgaaggcggcggagccgacaccacggccaccgatattatttgcccgatgtacgcgcgcgtggatgaagaccagcccttcccggctgtgccgaaatggtccatcaaaaaatggctttcgctacctggagagacgcgcccgctgatcctttgcgaatacgcccacgcgatgggtaacagtcttggcggtttcgctaaatactggcaggcgtttcgtcagtatccccgtttacagggcggcttcgtctgggactgggtggatcagtcgctgattaaatatgatgaaaacggcaacccgtggtcggcttacggcggtgattttggcgatacgccgaacgatcgccagttctgtatgaacggtctggtctttgccgaccgcacgccgcatccagcgctgacggaagcaaaacaccagcagcagtttttccagttccgtttatccgggcaaaccatcgaagtgaccagcgaatacctgttccgtcatagcgataacgagctcctgcactggatggtggcgctggatggtaagccgctggcaagcgg tgaagtgcctctggatgtcgctccacaaggtaaacagttgattgaactgcctgaactaccgcagccggagagcgccgggcaactctggctcacagtacgcgtagtgcaaccgaacgcgaccgcatggtcagaagccgggcacatcagcgcctggcagcagtggcgtctggcggaaaacctcagtgtgacgctccccgccgcgtcccacgccatcccgcatctgaccaccagcgaaatggatttttgcatcgagctgggtaataagcgttggcaatttaaccgccagtcaggctttctttcacagatgtggattggcgataaaaaacaactgctgacgccgctgcgcgatcagttcacccgtgcaccgctggataacgacattggcgtaagtgaagcgacccgcattgaccctaacgcctgggtcgaacgctggaaggcggcgggccattaccaggccgaagcagcgttgttgcagtgcacggcagatacacttgctgatgcggtgctgattacgaccgctcacgcgtggcagcatcaggggaaaaccttatttatcagccggaaaacctaccggattgatggtagtggtcaaatggcgattaccgttgatgttgaagtggcgagcgatacaccgcatccggcgcggattggcctgaactgccagctggcgcaggtagcagagcgggtaaactggctcggattagggccgcaagaaaactatcccgaccgccttactgccgcctgttttgaccgctgggatctgccattgtcagacatgtataccccgtacgtcttcccgagcgaaaacggtctgcgctgcgggacgcgcgaattgaattatggcccacaccagtggcgcggcgacttccagttcaacatcagccgctacagtcaacagcaactgatggaaaccagccatcgccatctgctgcacgcggaagaaggcacatggctgaatatcgacggtttccatatggggattggtggcgacgactcctgg agcccgtcagtatcggcggaattccagctgagcgccggtcgctaccattaccagttggtctggtgtcaaaaataa

E) ldh

(966bp)

atgatgaacaaacatgtaaataaagtagctttaatcggagcgggttttgttggaagcagttatgcatttgcgttaattaaccaaggaatcacagatgagcttgtggtcattgatgtaaataaagaaaaagcaatgggcgatgtgatggatttaaaccacggaaaggcgtttgcgccacaaccggtcaaaacatcttacggaacatatgaagactgcaaggatgctgatattgtctgcatttgcgccggagcaaaccaaaaacctggtgagacacgccttgaattagtagaaaagaacttgaagattttcaaaggcatcgttagtgaagtcatggcgagcggatttgacggcattttcttagtcgcgacaaatccggttgatatcctgacttacgcaacatggaaattcagcggcctgccaaaagagcgggtgattggaagcggcacaacacttgattctgcgagattccgtttcatgctgagcgaatactttggcgcagcgcctcaaaacgtacacgcgcatattatcggagagcacggcgacacagagcttcctgtttggagccacgcgaatgtcggcggtgtgccggtcagtgaactcgttgagaaaaacgatgcgtacaaacaagaggagctggaccaaattgtagatgatgtgaaaaacgcagcttaccatatcattgagaaaaaaggcgcgacttattatggggttgcgatgagtcttgctcgcattacaaaagccattcttcataatgaaaacagcatattaactgtcagcacatatttggacgggcaatacggtgcagatgacgtgtacatcggtgtgccggctgtcgtgaatcgcggagggatcgcaggtatcactgagctgaacttaaatgagaaagaaaaagaacagttccttcacagcgccggcgtccttaaaaacattttaaaacctcattttgcagaacaaaaagtcaactaa

Table 2 Primers and edited original sequence list

Primer name Sequence (5'→3')

304cas-F taacaatttcacacaggaaacagctgtggtcgccggtgccccgaac

304cas-R ttggcgggtgtcggggctggcttaatcacaagacctccccggcgc

lacZ-F tacccaacttaatcgccttgcagcaca

lacZ-R ccgtcgatattcagccatgtgccttctt

lacZ-UF

lacZ-UR

lacZ-DF

lacZ-DR

guide DNA-lacZ

ldh-F

ldh-R

ldh-UF

ldh-UCmR

ldh-UCmF

ldh-CmDR

ldh-CmDF

ldh-DR

ldh-UR

ldh-DF

guide DNA-ldh

cccAAGCTTatgagcgtggtggttatgcc

acgaagccgccctgtaaacccatgccgtgggtttcaata

tattgaaacccacggcatgggtttacagggcggcttcgt

gcgtTCTAGAatgcgggtcgcttcacttac

cgGGATCCtaatacgactcactatagggaatattgtcctcatcgccccttcgaggggtcgcaacccgcccggtgcagtatgaaggcggcggagccgacaccacggtcctcatcgccccttcgaggggtcgcaacctagcataaccccttggggcctctaaacgggtcttgaggggttttttgGAATTCcg

atgatgaacaaacatgtaa

ttagttgactttttgttc

ccAAGCTTcgatgagatggatttaaacca

cacaggtaggcgcgccatgttgcgtaagtcaggata

tatcctgacttacgcaacatggcgcgcctacctgtg

tttttctcaacgagttcactccttacgccccgcc

ggcggggcgtaaggagtgaactcgttgagaaaaa

gcgtTCTAGAgcgattcacgacagcc

tttttctcaacgagttcactatgttgcgtaagtcaggata

tatcctgacttacgcaacatagtgaactcgttgagaaaaa

cgGGATCCtaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaagtcctcatcgccccttcgaggggtcgcaacagcggcctgccaaaagagcgggtgattggaagcggcacaagtcctcatcgccccttcgaggggtcgcaacctagcataaccccttggggcctctaaacgggtcttgaggggttttttgGAATTCcg

The capital letter is the enzyme cutting site, the wavy line is the protective base, the bold bold of the primer is the complementary region, the single underline is the promoter promoter, the italic is the spacer, the bold bold is the repeat, and the double underline is the terminator

Claims (4)

1. A gene editing method based on Streptomyces virginia IBL14 gene cas 7-5-3, characterized in that: Streptomyces virginia IBL14 contains 3 genes cas 7-5-3, expressed by protein The steps of plasmid construction, gene editing plasmid construction and recombinant acquisition and verification can edit the prokaryotic genome. 2. a kind of gene editing method based on Streptomyces virginia IBL14 gene cas 7-5-3 according to claim 1, is characterized in that: the steps are as follows: (1) Design primers based on the sequence of cas 7-5-3 of the Streptomyces virginia IBL14 gene and related information, use the genome of Streptomyces virginia IBL14 as a template, and use DNA polymerase to amplify the cas 7- 5-3 gene, and connected to the plasmid plasmid, to obtain the Cas7-5-3 protein expression plasmid plasmid- cas 7-5-3; (2) Design primers according to the DNA sequence information of the prokaryotic target gene, use the prokaryotic genome as a template, and amplify the target target with restriction endonuclease recognition and cleavage sites at the end and overlapping PCR complementary sequences through PCR reactions respectively PCR fragments of the upper and lower homology arms of the gene, and use overlap PCR to connect the upper and lower homology arms to obtain the gene editing template DNA/t-DNA, and design and directly synthesize the lactose operon promoter and RNA at the beginning and end according to the sequence information of the target gene of prokaryotes The target gene fragment guide-DNA/g-DNA of the terminator, and connect the gene editing template and the target gene fragment to the plasmid according to the sticky end on the restriction enzyme site to obtain the gene editing plasmid plasmamid-t/gDNA; (3) Prepare competent prokaryotic cells, and transform the protein expression plasmid plasmid- cas 7-5-3 obtained in step (1) and various targeted gene editing plasmids obtained in step (2) into target bacteria Different gene-edited recombinants were obtained in the competent state; PCR and gene sequencing and/or functional analysis were performed on the recombinant chromosomes to confirm the edited target recombinants. 3. A gene editing method based on the cas 7-5-3 gene of Streptomyces virginia IBL14 according to claim 1, characterized in that: said prokaryotic organisms refer to Escherichia coli, Bacillus subtilis and other prokaryotic microorganisms. 4. A kind of gene editing method based on Streptomyces virginia IBL14 gene cas 7-5-3 according to claim 1, characterized in that: said gene editing refers to the use of the protein expression plasmid in combination with the gene editing plasmid Or other plasmids can carry out knockout, insertion, scarless point mutation and any combination on the chromosomal genes of prokaryotic cells.