CN106834323A - A kind of gene editing method based on Virginia streptomycete IBL14 genes cas7 53 - Google Patents
A kind of gene editing method based on Virginia streptomycete IBL14 genes cas7 53 Download PDFInfo
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Abstract
Virginia streptomycete IBL14 genes are based on the invention discloses one kindcas753 gene editing method, realizes gene editing of the CRISPR Cas I systems to prokaryotic gene group first；New supplement and selection is provided for CRISPR Cas II types CAS carries out gene editing.Using the system prokaryotic gene such as Escherichia coli, hay bacillus group can be carried out easily and fast, effectively gene editing.The gene editing system after optimization is expected to be applied in other biological gene editings.
Description
Technical field
It is exactly that one kind is based on Virginia streptomycete the present invention relates to the gene editing technology of biological technical field
IBL14 genescasThe gene editing method of 7-5-3.
Background technology
Virginia streptomycete IBL14 (Streptomyces virginiae IBL14) is that we produce from steroid drugs
One plant of actinomyces that separation screening is arrived in sludge near producer, it can effectively be converted and progesterone of degrading, flavones, cortisol
Steroid compound _ ENREF_1s various with cholesterol etc.（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）Referred to as the short palindrome repetitive sequence of cluster aturegularaintervals, is most found in prior to 20th century the eighties
In Escherichia coli _ ENREF_1（Ishino, Y.; Shinagawa, H.; Makino, K.; Amemura, M.;
Nakata, A., Nucleotide Sequence of the iap Gene, Responsible for Alkaline
Phosphatase Isozyme Conversion in Escherichia coli, and Identification of the
Gene Product. JOURNAL OF BACTERIOLOGY 1987, 169 (12), 5429-5433）.R. in 2002
The short palindrome repetitive sequence of this cluster aturegularaintervals in the comparative analysis prokaryotic gene group in detail such as Jansen, finds
The side of the repetitive sequence is frequently accompanied by conservative gene order _ ENREF_1 occur（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 will
This unique repetitive sequence is named as CRISPR, and the protein of conserved genetic sequences coding is referred to as Cas accessory proteins
(CRISPR-associated proteins).Have now been found that CRISPR-Cas systems be distributed widely in prokaryotes in _
ENREF_1（http://crispr.u-psud.fr/）, business application is in the only CRISPR-Cas II types system of gene editing
Cas9 albumen in system.
So far, though CRISPR-Cas I type systems there are many discoveries, the report that can be applied to gene editing is had no.
Previously, we combined a work using the type I-B-sv14 type CRISPR-Cas systems in Virginia streptomycete IBL14
The gene editing plasmid of journey can carry out gene editing _ ENREF_1 to Virginia streptomycete IBL14 itself chromosomes（Tong Wangyu;
Yong Dexiang;Li Xue;CRISPR-Cas systems in a kind of Virginia streptomycete IBL14 of Qiu Caihua and carry out gene using it
The method 2015110028173,2015 of editor）, but fail to realize carrying out gene editing to other biological genomes.This hair
Gene cas7-5-3 and plasmid in the bright streptomycete IBL14 type I-B-sv14 type CRISPR-Cas systems by Virginia
（plasmid）Connection obtains a Cas7-5-3 protein expressing plasmid plasmid-cas7-5-3, with reference to the gene editing of design
Plasmid（plasmid-t/gDNA）Or other plasmids realize the gene in prokaryotes Escherichia coli and hay bacillus first
Editor.And the Cas7-5-3 albumen（Containing 1323 amino acid）With with Cas9 albumen（Containing 1368 amino acid）Close molecule
Size.
Though CRISPR-Cas I types system _ ENREF_1 is there are in having found Escherichia coli（Semenova, E.;
Savitskaya, E.; Musharova, O.; Strotskaya, A.; Vorontsova, D.; Datsenko, K.
A.; Logacheva, M. D.; Severinov, K., Highly efficient primed spacer
acquisition 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 not yet by big
CRISPR-Cas I types systems in enterobacteria carry out the report of gene editing；Particularly in hay bacillus it is not yet found that
CRISPR-Cas systems _ ENREF_1（http://crispr.u-psud.fr/）.The present invention is by knocking out Escherichia coli β-half
Lactose glycoside enzyme gene lacZ and hay bacillus lactate dehydrogenase gene ldh, thus realize verify the plasmid system effect mesh
's.
The content of the invention
The technical problem to be solved in the present invention is to provide Cas9 albumen in a kind of II type systems similar to CRISPR-Cas
CRISPR-Cas type I-B-sv14 system Cas7-5-3 protein expressing plasmid systems, with reference to gene editing plasmid or other matter
Grain carries out the gene editing of prokaryotes.
It is that, up to this purpose, the technical scheme that its problem of solution of the invention is used is：One kind is based on Virginia streptomycete
IBL14 genescasThe gene editing method of 7-5-3, it is characterised in that：Contain 3 genes in Virginia streptomycete IBL14cas7-5-3, its nucleotide sequence builds as described by subordinate list 1 through protein expressing plasmid（Accompanying drawing 1）, gene editing plasmid construction
（Accompanying drawing 2）Acquisition with recon effectively can enter edlin with checking procedure (accompanying drawing 3) to prokaryotic gene group.
Described one kind is based on Virginia streptomycete IBL14 genescasThe gene editing method of 7-5-3, its feature exists
In：Including protein expressing plasmid build, the acquisition of gene editing plasmid construction and recon and checking procedure it is as follows：
（1）According to Virginia streptomycete IBL14 genescas7-5-3 and relevant information primers（Subordinate list 2）, to tie up
Ji Niya streptomycete IBL14 genomes are masterplate, and reacting amplification by PCR with archaeal dna polymerase obtainscas7-5-3 genes, and even
It is connected to plasmid（plasmid）On, obtain Cas7-5-3 protein expressing plasmids plasmid-cas7-5-3；
（2）According to prokaryotes target gene DNA sequence dna information design primer（Subordinate list 2）, with prokaryotic gene group as masterplate,
By PCR reactions, amplification obtains end with restriction enzyme identification and cleavage site and overlap PCR complementations respectively
The upper and lower homology arm PCR fragment of target gene of sequence, and upper and lower homology arm is connected into obtain gene editing masterplate with overlap PCR
(template DNA/t-DNA), while designed according to prokaryotes target gene sequence information and be directly synthesized head and the tail wrapping respectively
Target gene fragment (guide-DNA/g-DNA) containing lactose operon promoter and RNA terminators（Subordinate list 2）, and according to limit
Be connected to for gene editing masterplate and target gene fragment gene editing obtained on plasmid by the cohesive end on property restriction enzyme site processed
Plasmid (plasmid-t/gDNA)；
（3）Prokaryote competence is prepared, and will be by step（1）The protein expressing plasmid plasmid- for obtainingcas7-5-3
With by step（2）The various target gene editor plasmids for obtaining obtain different genes volumes in being transformed into target bacterium competence respectively
Recon after volume；Performing PCR and gene sequencing and/or functional analysis are entered to restructuring daughter chromosome, to confirm the purpose after editor
Recon.
Described one kind is based on Virginia streptomycete IBL14 genescasThe gene editing method of 7-5-3, its feature exists
In：The prokaryotes refer to Escherichia coli, hay bacillus and other prokaryotic micro-organisms.
Described one kind is based on Virginia streptomycete IBL14 genescasThe gene editing method of 7-5-3, its feature exists
In：Described gene editing refers to can be to prokaryotic using the protein expressing plasmid combination gene editing plasmid or other plasmids
Chromogene is knocked out, inserted, seamless point mutation and any combination.
Beneficial effect
The present invention is a kind of CRISPR-Cas I type systems based on Virginia streptomycete IBL14casThe one of gene foundation
New gene editing system is planted, gene editing of the CRISPR-Cas I systems to prokaryotic gene group is realized first；For
CRISPR-Cas II types CAS carries out gene editing there is provided new supplement and selection.Using the system to Escherichia coli, withered grass
The prokaryotic gene such as bacillus group can easily and fast, effectively carry out gene editing.The gene editing system after optimization is expected
It is applied in other biological gene editings.
Brief description of the drawings
Fig. 1 is the structure of protein expressing plasmid pHT304-cas7-5-3.Ori / origin：DNA replication dna initiation site；
lac promoter / lactose promoter：The promoter of lactose operon, starting DNA is combined with RNA polymerase and is turned
Record；CAP binding site / catabolite activator protein binding site:CAP
Binding site, promotes RNase transcriptional activity；AmpR / ampicillin resistance：Amicillin resistance；Erm /
erythromycin:Erythromycin
Fig. 2 is the structure of gene editing plasmid,（A）Gene editing plasmid pKC1139-lacZThe structure of-t/g-DNA,（B）Gene
Editor's plasmid pKC1139-ldhThe structure of-t/g-DNA.Ori pSG5 / the origin from pSG5:PSG5 plasmids are carried
Replication origin on body；oriTRK2 / the origin of conjugal transfer from RK2:RK2 plasmids
Conjugative tiansfer replication origin on carrier；lac promoter / lactose promoter:The startup of lactose operon
Son；T7 promoter:T7 promoters, initial DNA transcriptions；AmpR / ampicillin resistance:Ammonia benzyl mould
Plain resistance；AprR / apramycin resistance:Apramycin resistance；Cm / Chloramphenicol:Chloramphenicol
Fig. 3 is transformant screening and the result.（A）E. coli jm109 (DE3) indigo plant hickie the selection result, blueness is shown to be
Original strain, white is shown to be recombinant bacterial strain；（B）GenelacZExterior PC R electrophoretograms, swimming lane M：5000 bp DNA
Ladder, swimming lane 1：Blank, swimming lane 2：Plasmid pHT304-cas7-5-3、pKC1139-lacZ- t/g-DNA and pKD46 turns
Beggar'slacZGene PCR, swimming lane 3：Wild typeECJM109 (DE3) genomelacZGene PCR（DNA bands in swimming lane 2
It is small compared with DNA bands in swimming lane 3 to show gene knockout success）；（C）Plasmid pHT304-cas7-5-3、pKC1139-lacZ-t/g-
DNA, pKD46 transformantlacZGene PCR sequencing result figure（Sequencing result is consistent with the gene order designed in table 2 to be shown
Recon is correct）.
Specific embodiment
In order to more fully understand technology contents of the invention, technical scheme is made with reference to specific embodiment
It is further described and illustrates, it is intended to preferably explain present disclosure, following examples is not limited the scope of the invention.
Additionally, using following material unless otherwise instructed in listed embodiment：
1）Bacterial strain and plasmid
Virginia streptomyceteStreptomyces virginiae IBL14 /SVIBL14；Escherichia coliEscherichia coli DH5α / ECDH5α；Escherichia coli JM109 / ECJM109；Hay bacillusBacillus subtilis168 /BS168, plasmid pHT304；pKC1139；pKD46.
2）Culture medium
LB fluid nutrient mediums
The g of dusty yeast 5, peptone 10 g, NaCl 10 g, after adding appropriate originally water dissolves, are settled to 1 L, and pH is adjusted to 7.0 ~
7.2, after packing wrapping, 121 DEG C/20 min sterilizings.
LB solid mediums
The g of dusty yeast 5, peptone 10 g, NaCl 10 g, the g of agar 20, after adding appropriate originally water dissolves, are settled to 1L,
PH is adjusted to 7.0 ~ 7.2, after packing wrapping, 121 DEG C/20 min sterilizings.
Spizizen minimal mediums
The basic mL/L of salt culture medium 100 of 10 × Spizzen, the mL/L of 50% (w/v) glucose 10, tryptophan mother liquor（5 mg/m
L）10 m L/L, agar 1.5%w/v）, 121 DEG C/20 min sterilizings.
GM1 culture mediums（5mL）
40% glucose 100 μ l, 20 mg/mL acid hydrolyzed casein 100 μ l, 50 mg/m L yeast extracts 100 μ l, 20%
(w/w) the μ l of 5 μ l, 10 × Spizzen minimal mediums of MgSO4.7H2O 500, the μ l of water 3195, need before above-mentioned substance mixing
121 DEG C/20 min sterilize.
GM2 culture mediums（5mL）
40% glucose 100 μ l, 20 mg/mL acid hydrolyzed casein 50 μ l, 20% (w/w) MgSO4.7H2O 40 μ l, 10 ×
The μ l of Spizzen minimal mediums 500, the μ l of water 3310, need 121 DEG C/20 min to sterilize before above-mentioned substance mixing.
Agents useful for same is commercially available product.
Embodiment 1（The step co-transformation method of three plasmid one is knocked outEC JM109 lacZGene）
（1）Protein expressing plasmid pHT304-casThe structure of 7-5-3
According toSVIBL14 genome sequencings information and plasmid pHT-304 sequence informations, design are complementary with plasmid pHT-304
Sequencecas7-5-3 gene-specific primerscas- F andcas-R（Table 2）.ExtractSVIBL14 genomic DNAs, use full formula
The TransStart FastPfu DNA Polymerase of golden Bioisystech Co., Ltd's production are carried outcasGene PCR is expanded,
Reaction condition：95 DEG C of 5 min, 94 DEG C of 30 s, 55 DEG C of 30 s, 72 DEG C of 2 min, 2.5 U give birth to work bioengineering（Shanghai）
The Pfu DNA Polymerase (50 μ l reaction systems) of limited company's production, 30 circulations, 72 DEG C of 10 min.
PCR products detect that kit is reclaimed, and is purified through 1% agarose electrophoresiscasFull-length gene.Will by one-step methodcas
Full-length gene order is connected with plasmid pHT-304, obtains Cas7-5-3 protein expressing plasmids pHT304-cas7-5-3。
（2）Gene editing plasmid pKC1139-lacZThe structure of-t-DNA
(A) genelacZDesign of primers withlacZThe amplification of full-length gene
According toECJM109 gene order-checking information, designs genelacZSpecific primerlacZ- F andlacZ-R（Table 2）.ExtractECJM109 genomic DNAs, are carried out using Pfu DNA PolymeraselacZGene PCR is expanded, reaction condition：95 ℃ 5
The U Pfu DNA of min, 94 DEG C of 30 s, 52 DEG C of 30 s, 72 DEG C of 1 min, 2.5 Polymerase (50 μ l reactants
System), 30 circulations, 72 DEG C of 10 min.PCR products detect that kit is reclaimed, and is purified through 1.5% agarose electrophoresis
'slacZFull-length gene fragment is standby.
（B）The preparation of upstream and downstream homology arm
According tolacZGene complete sequence（Table 1）DesignlacZUpstream region of gene homology arm primerlacZ- UF andlacZ- UR, downstream are same
Source arm primerlacZ- DF andlacZ- DR (table 2)（Black matrix overstriking is overlap PCR complementary series）, and upper homology arm upstream
Primer restriction enzyme digestion sites containing HindIII, lower homology arm anti-sense primer restriction enzyme digestion sites containing XbaI.
With what is purifiedlacZGene DNA is template, and upstream and downstream homology arm is first expanded respectively, and reaction condition is：95 DEG C of 5 min, 94
DEG C the U Pfu DNA of 30 s, 58 DEG C of 30 s, 72 DEG C of 45 s, 2.5 Polymerase (50 μ l reaction systems), 30
Circulation, 72 DEG C of 10 min.PCR products are detected through 1.5% agarose electrophoresis, and kit is reclaimed, and obtain after purification is upper and lower
Trip homology arm DNA fragments are standby.
（C）Gene editing masterplate carrier pKC1139-lacZThe structure of-t-DNA
Take homology arm purified product to mix as template with the lower μ l of homology arm purified product 0.5,25 μ l reaction systems are carried out
Overlap PCR, reaction condition is：94 DEG C of predegenerations 5 min, 94 DEG C of denaturation l min, 58 DEG C of 1 min of annealing, 72 DEG C are prolonged
30 s are stretched, people's primer UF and each 1 μ l of DR are added after a circulation, continue PCR, reaction condition is：95 DEG C of predegeneration 5 min, 94
DEG C denaturation 30 s, 58 DEG C annealing 30 s, 72 DEG C extension the s of 1 min 30, carry out 30 circulation, 72 DEG C of 10 min.1.5% fine jade
Sepharose electrophoresis detection amplified production is simultaneously purified, and obtains gene editing masterplate；The gene editing masterplate that will be obtained passes through HindIII
Restriction enzyme enzyme, XbaI restriction enzymes cut out cohesive end, are then given birth to by Quan Shijin Bioisystech Co., Ltd
The T4 ligases of product are connected on pKC1139 plasmids, obtain gene editing masterplate carrier pKC1139-lacZ-t-DNA。
（D）Gene editing plasmid pKC1139-lacZThe structure of-t/g-DNA
Containing lactose operon promoter and guide DNA-lacZThe target gene fragment of connection product（Table 2）By the general life in Chuzhou
Thing company is directly synthesized, and head and the tail add BamHI and EcoRI restriction enzyme sites respectively, and centre is successively promoter, repetitive sequence
（repeat）, intervening sequence（spacer）, repetitive sequence（repeat）And terminator；The target gene fragment of synthesis is passed through
BamHI and EcoRI restriction enzymes cut out cohesive end, are then connected to obtain gene editing masterplate by T4 ligases
Carrier pKC1139-lacZ- t-DNA goes up to obtain gene editing plasmid pKC1139-lacZ-t/g-DNA 。
（3）The acquisition of recon and inspection
（A）ECIt is prepared by JM109 competence one-step method
In super-clean bench, with sterilized toothpick pickingECMonoclonal on JM109 flat boards is in 30 ml LB fluid nutrient mediums
In, 37 DEG C, 200 rpm incubated overnights；The μ l of bacterium solution 100 for taking incubated overnight are forwarded in new LB fluid nutrient mediums, and 37
DEG C, 200 rpm cultivate about 2 h, are 0.4 ~ 0.6 to bacterium solution OD600 values；In taking the above-mentioned bacterium solutions of 1 ml to 1.5 ml EP pipes, 4 DEG C
Lower 5000 rpm × g is centrifuged 5 min, after thoroughly eliminating supernatant, take precooling on ice the μ l of SSCS solution 50 by thalline
Precipitation piping and druming is uniform, obtains final productECThe competent cell of JM109, saves backup at -80 DEG C（This process whole process is carried out on ice）.
（B）Plasmid pKD46, pHT304-cas7-5-3 and pKC1139-lacZThe cotransformation of-t/g-DNA
By plasmid pKD46, pHT304-cas7-5-3 and pKC1139-lacZ- t/g-DNA is transformed into after fully mixingEC JM109
In competence, 5 μ l isopropyl-beta D-thio galactopyranosides are being scribbled（IPTG 200 mg/ml）, the 40 bromo- 4- of μ l 5-
Chloro- 3- indoles-β-D- galactopyranosides（X-gal 20 mg/ml ）Peace with 20 μ l arabinoses (10 mM/L) is general mould
30 DEG C of incubated overnights in the LB solid mediums of element and amicillin resistance, obtain transformant.
（C）Restructuring daughter chromosome PCR and gene sequencing analysis
Picking white monoclonal as template, then withlacZGene verifies primerlacZ-F/lacZ- R carries out pcr amplification reaction,
Reaction condition：95 DEG C of 5 min, 94 DEG C of 30 s, 58 DEG C of 30 s, 72 DEG C of 2 min 40 s, 2.5 U life work bioengineering
（Shanghai）The EasyTaq DNA Polymerase (25 μ l reaction systems) of limited company's production, 30 circulations, 72
℃ 10 min.PCR products detect that observation recon chromosome DNA amplification band reduces through 1% agarose electrophoresis, and through logical
Use biosystem（Anhui）Co., Ltd's sequencing is provedlacZGene knockout success（Accompanying drawing 3）.
Embodiment 2（The step cotransformation of three plasmid two is knocked outEC JM109 lacZGene）
（1）Protein expressing plasmid pHT304-casThe structure of 7-5-3
With the step of embodiment 1（1）
（2）Gene editing plasmid pKC1139-lacZThe structure of-t-DNA
With the step of embodiment 1（2）
（3）The acquisition of recon and inspection
（A）Plasmid pHT304-cas7-5-3 convert andEC JM109- pHT304-casIt is prepared by 7-5-3 competence
By plasmid pHT304-cas7-5-3 is transformed intoECIn JM109 competence, by Erythromycinresistant screening transformant, then with
The transformant prepares competence, obtains and contains plasmid pHT304-cas7-5-3'sEC JM109- pHT304-cas7-5-3 bacterial strains
Competence.Competence preparation method is with step in embodiment 1（3A）.
（B）Plasmid pKD46 and pKC1139-lacZThe cotransformation of-t/g-DNA
By plasmid pKD46 and pKC1139-lacZ- t/g-DNA is transformed into after fully mixingEC JM109- pHT304-cas7-5-
In 3 competence, erythromycin, apramycin and the ammonia benzyl of 5 μ l IPTG, 40 μ l X-gal and 20 μ l arabinoses are being scribbled
30 DEG C of incubated overnights, obtain transformant in the LB solid mediums of penicillin.
（C）Restructuring daughter chromosome PCR and gene sequencing analysis
Picking white monoclonal as template, then withlacZGene verifies primerlacZ-F/lacZ- R carries out pcr amplification reaction,
Reaction condition：95 DEG C of 5 min, 94 DEG C of 30 s, 58 DEG C of 30 s, 72 DEG C of 2 min 40 s, 2.5 UEasyTaq DNA
Polymerase (25 μ l reaction systems), 30 circulations, 72 DEG C of 10 min.PCR products are examined through 1% agarose electrophoresis
Survey, the stripe size change of observation recon chromosome DNA amplification, and proved through gene sequencinglacZGene knockout success.
Embodiment 3（The step co-transformation method of two plasmid one is knocked outEC JM109 lacZGene）
（1）Protein expressing plasmid pHT304-casThe structure of 7-5-3
With the step of embodiment 1（1）
（2）Gene editing plasmid pKD46-lacZThe structure of-t/g-DNA
Except by homology arm and guide DNA-lacZTarget fragment is consecutively connected on plasmid pKD46, forms gene editing matter
Grain pKD46-lacZOutside-t/g-DNA, step in remaining step and embodiment 1（2）It is identical.
（3）The acquisition of recon and inspection
With the step of embodiment 1（3）
Embodiment 4（BS168 genesldhKnockout and chloramphenicol resistance gene insertion）
（1）Protein expressing plasmid pHT304-casThe structure of 7-5-3
With the step of embodiment 1（1）
（2）Gene editing plasmid pKC1139-ldhThe structure of-t/g-DNA
（A）GeneldhDesign of primers withldhThe amplification of full-length gene
Designing gene primer isldh- F andldh-R（Table 2）.WithBS168 genomic DNAs are masterplate, amplificationldhGenetic fragment.Instead
Answer condition：The U Pfu DNA of 95 DEG C of 5 min, 94 DEG C of 30 s, 55 DEG C of 30 s, 72 DEG C of 2 min, 2.5 Polymerase
(50 μ l reaction systems), 30 circulations, 72 DEG C of 10 min.PCR products detect through 1.5% agarose electrophoresis, kit
Reclaim, purifiedldhFull-length gene fragment is standby.
（B）The preparation of upstream and downstream homology arm
Separately design upper and lower homology arm primerldh-UF、ldh-UCmR、ldh-UCmF、ldh-CmDR、ldh-CmDF、ldh-DR
（Table 2）.With what is purifiedldhGene andCm Gene DNA is template, first expand respectively,Cm, downstream homology arm, reaction condition
For：95 DEG C of 5 min, 94 DEG C of 30 s, 55 DEG C of 30 s, 72 DEG C of 1min 20 s, 2.5 U Pfu DNA Polymerase
(50 μ l reaction systems), 30 circulations, 72 DEG C of 10 min.PCR products detect that kit is returned through 1.5% agarose electrophoresis
Receive, obtain after purification it is upper,Cm, downstream homology arm DNA fragments it is standby.
（C）Gene editing masterplate carrier pKC1139-ldhThe structure of-t-DNA
Take homology arm purified product,cmPurified product and the μ l of lower homology arm purified product 1.0 mixing are used as template, and 25 μ l are anti-
Answering system carries out overlap PCR, and reaction condition is：94 DEG C of predegenerations 5 min, 94 DEG C of denaturation l min, 55 DEG C of annealing 1
Min, 72 DEG C of 30 s of extension, people's primer UF and each 1 μ l of DR are added after a circulation, continue PCR, and reaction condition is：95 DEG C pre-
5 min, 94 DEG C of denaturation 30 s, 55 DEG C of annealing 30 s, 72 DEG C of extension 2min are denatured, 30 circulations, 72 DEG C of 10 min are carried out.
1.5% agarose gel electrophoresis detection amplified production is simultaneously purified, and obtains gene editing masterplate；The gene editing masterplate of acquisition is passed through
HindIII and XbaI restriction enzymes cut out cohesive end, are then connected to pKC1139 plasmids by T4 ligases
On, obtain gene editing masterplate carrier pKC1139-ldh-t-DNA。
（D）Gene editing plasmid pKC1139-ldhThe structure of-t/g-DNA
Containing lactose operon promoter and guide DNA-ldhThe target gene fragment of connection product（Table 2）By the general life in Chuzhou
Thing company is directly synthesized, and head and the tail add BamHI and EcoRI restriction enzyme sites respectively, and centre is successively promoter, repetitive sequence
（repeat）, intervening sequence（spacer）, repetitive sequence（repeat）And terminator；The target gene fragment of synthesis is passed through
BamHI and EcoRI restriction enzymes cut out cohesive end, are then connected to obtain gene editing masterplate by T4 ligases
Carrier pKC1139-ldh- t-DNA goes up to obtain gene editing plasmid pKC1139-ldh-t/g-DNA 。
（3）The acquisition of recon and inspection
（A）BSThe preparation of 168 competence
Picking wild typeBS168 monoclonals in 5 ml GM1 culture mediums, 37 DEG C, 130 rpm incubated overnights.Next day switching 1
In 9 mlGM1 culture mediums, 37 DEG C, 200 rpm cultivate 3.5 h to ml.5 ml GM1 nutrient solutions of transferring are cultivated in 45 ml GM2
In base, 37 DEG C, 130 rpm cultivate 1.5 h.4 DEG C, 4000 rpm are centrifuged 10 min.Appropriate supernatant re-suspended cell is stayed, is dispensed
To in 2 ml EP pipesBS168 competence are standby.
（B）Plasmid pHT304-cas7-5-3 and pKC1139-ldhThe cotransformation of-t/g-DNA
By plasmid pHT304-cas7-5-3 and pKC1139-ldh- t/g-DNA is added to the competence for preparing after fully mixing
In, 37 DEG C of 1 h of standing, 37 DEG C, 220 rpm culture 3-4 h.Draw appropriate bacterium solution and be applied to the LB cultures containing Cm resistances
On base, in 37 DEG C of incubator cultures.
（C）Restructuring daughter chromosome PCR and gene sequencing analysis
In picking monoclonal on the LB flat boards containing Cm antibiotic, 37 DEG C, 200 rpm, incubated overnight.Next day extracts genome
Enter performing PCR, product is detected through 1% agarose electrophoresis, observe the change of recon chromosome DNA amplification stripe size, and through base
Because sequencing is provedBS168-ldhGene knockout and chloramphenicol resistance gene are inserted successfully.
Embodiment 5（Simple substance grain is knocked outBS 168 ldhGene）
（1）Protein expressing plasmid pHT304-casThe structure of 7-5-3
With the step of embodiment 1（1）
（2）Gene editing plasmid pHT304-cas7-5-3-ldhThe structure of-t/g-DNA
Except temple-DNA-ldhTarget fragment is free ofCm Outside gene, step in remaining step and embodiment 4（2）It is identical.
（3）The acquisition of recon and inspection
With the step of embodiment 4（3）
The above only further illustrates technology contents of the invention with embodiment, is easier to understand in order to reader, but not
Represent embodiments of the present invention and be only limitted to this, any technology done according to the present invention extends or recreates, by of the invention
Protection.
The Virginia streptomycete IBL14 genes cas7-5-3 of table 1, e. coli jm109 gene lacZ and hay bacillus BS168
Gene ldh full length sequences
Gene order
A） cas7
(999bp) gtggtcgccggtgccccgaacaacggggagggcgaggacaacacggggcgtgtgaagaagctgag
ggtcgggcgggaggagttcccgtacgtgtccgcgcaggcgttccgtcggtggttgcgtgactcgctgccggcgcagg
agccgcgttcggtggtcactcgctcgggcagcggtgccaagcagcaggcacacaccgcgggccggccggacctgcac
ctggacgatgatctgttcggctacatggtcgcggtgaaggggaaggggggaagctaccagcgggacaccgtgctggc
taccgggactttagtttcagtggtgccgcagcgtccgacgttggacttcggcacgatgagccgggacttcccggctg
gtgagcacccggtgattcactcgcacgagctgtacagcgcgaccctggccggcgatgttctgctggatctgccgcgg
gctggggtcttcgagacggacggcaacgggttgcgcgtggcgatcagccctgccgtcgctgaggaagcggcgaagaa
cggggcggaggtcaccacgctgcggggcagtgcggccattcggttgccgcttactgagcggcaccggcggatcggca
cgctgttgcggacgctggcgtcggtgcgtggtggggccaagcaggctctgcactacggggaccgggccccttcattg
gtcttgttggctcctctcaagggtggcgtcaatccgttcacccgtgttctgggcgcccgcgacggtaagcctgtgtt
cctgagcgatgtcctgcgcgaggagctcgaggcgtgggcggatgagctggacgggccggtgctgctgggctgggccc
cggggtttctcggcgatcagcgtgagcaggtccgccgcgagctcaaggatctgattgacgagggccgtgtcgtcctg
agccatcctcgtgtgctgctgacccagctggccgaccggatcgagcagggtgatcatgacgcgtggttcgaggactc
cgcggcgtga
B）cas5
(663bp)
gtgacgggtacggaggtcacggccctgcagatcacggtgacggcgccggttgtctccttccgtaatccgctgt
atgccggggtgcaggtgacgctgccgtgtccgccgccggccaccgtcggcggcctcctcgccgcagcggctgggggg
tgggagcaggtcaatccggagctgcgtttcgcgatggcgttccacgctggcggcaaggcggtcgatctcgagacgta
ccacccgctggacgcgtctgggaagaaggcgtcgcctgccccgcgtaaccgggagttccttacggcggccgagctca
ccgtgtggctggtcgacgaccctgaagggtggcagcgccgcctgcgtcggccggtgtggccgctgcggctgggccgc
agccaggacctggtcggtatccgcaccggcctggttccgttgcgcgcggagcccggcgagcagcggtccgccgtggt
gccggagacggcggggaggatgggaaccctactgcggctgccgactgcggtctctgggggccgggaccgtacccggt
gggacagctaccggttcgacagctcgggccgcagtgaccatgtggtcgtaggcggctggtcgactgccgggggacag
gcagtcattctgctgccctcggcccatcccgataccgtcgcgcgttcctga
C）cas3
(2316bp)
gtgggccgtctggacgcggtggaggacgtcttcggcggcaggttctggcccgtcgtggaactcgctggcctca
cccacgacgccggcaagattcccgaaggcttccagcggatgctggcgggatacagccgtgcctggggtgagcgtcac
gaagtcgcctcgttgggcttcctgcccgcgctcatcggcgacccggacgtgctgttgtgggtggcgaccgcggtcgc
cacccaccatcgtccgctgaccggccagaacggacgcgacctgcagactctctacagcggtgtcaccatcaccgagc
tcgcgcaccgtttcgggccttttgacccacgcgctgtccccgccttggaggcctggcttcgtgcgagcgccatccgg
gtcggcctccccgcggccgctgttccagacgacggcacgctcaccgacaccggagtggtcgctggcgcccaccagct
gctggaggagattttggaccgttgggcagaccgtgtgaggcctgaggtgggcttggccgctgtactgctgcaggggg
cggtcaccctggccgaccacttgtcctccgcccatcaggctctgcccaccgtccagccgttgggggccgggttccgg
tcccggttggagaaggagttcgctgaacgcggcaggaccctgcgtgcccaccagctggaggccgccaccgttaccgg
acatcttctgctgcgcgggccgaccggcagtgggaagaccgaggctgccctgctgtgggctgccagccaggtcgagg
ccctgaaggcggaaggccggggcgtgccgcgtgtgtttttcactctcccctacctggcctccatcaacgccatggca
acacggctgggtgacactctcggcgatggtgaggctgtcggcgttgcccactcccgcgccgcctcctaccaccttgc
ccaggccatcgccccgcaggacggcgacgaggaggacgaacacggagccccctgccgtgttgacgcggccgccaagg
ccttgtcccgggccgctgccaccaagctgttccgcgagagtgtccgcgtcgccaccccctaccagcttctgcgggcc
gccctggccgggccggcccactccggcatcctcatcgacgccgcgaactcggtgttcatcctggacgaactccacgc
ctacgacgcccgcaggctcggctacatcctggccagtgcccggctgtgggaacgcctcggtggacggatcacagtcc
tgtccgcgaccctgcccagggccctggccgacctgttcgagagcaccctcaccgcccccatcaccttcctcgacacc
cccgacctcgggctgccggcgcgccacctcctgcacacccgaggccaccatctcaccgacccggccacactggagga
gatccgtctgcggctgtcccgggacgagtcggtcctggtgatcgccaacaacgtgtcccaggccatcgccctgtacg
aacagctcgcacccgacgtgtgtgaacgcttcggtcaggacgccgcgctactgctgcactcccggtttcgacggatg
gaccggtcccggattgagcagaagatcgccgaccggttcgccactgtggcacctgatgcccagaacagccgtaagcc
gggcctggtcgttgccacgcaggtggtcgaggtcagtctcgacgtcgacttcgatgtgctgttcactggagcggctc
cgctcgaggccctcctgcagcgcttcggccggaccaaccgcgtcggggcccgcccgccggccgacgtcatcgtccac
catcccgcctggaccacacgccgccgacagcccggcgagtacgccgacggcatctacccacgggagccggtcgagtc
cgcgtggcacatcctcacccgcaatcacgggcgagtcatcgacgaagcggacgccaccgcgtggctggacgaggtct
acgccacggactggggcaggcaatggcaccgcgaggtgctggagcggcgagaaagattcgaccgtgcgttcctgcag
ttccgctaccccttcgaagaccgcactgacctggccgataccttcgacgaactcttcgacggctccgaagccatcct
cgccgaagaccaggacgcctactcagccgcactggccgcaccagacggcgaccaccccggagctggccggctcctcg
cagaggaatacctcatccccgttccccactgggccagccccctcagccgctacgagaagcagctcaaagtccgcgtc
atcaacggcgactaccaccccgaccacggcctcatggcggtccgggggctgccccagcccgcctaccgcgccgggga
ggtcttgtga
D）lacZ (3075bp)
atgaccatgattacggattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaac
ttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaa
cagttgcgcagcctgaatggcgaatggcgctttgcctggtttccggcaccagaagcggtgccggaaagctggctgga
gtgcgatcttcctgaggccgatactgtcgtcgtcccctcaaactggcagatgcacggttacgatgcgcccatctaca
ccaacgtgacctatcccattacggtcaatccgccgtttgttcccacggagaatccgacgggttgttactcgctcaca
tttaatgttgatgaaagctggctacaggaaggccagacgcgaattatttttgatggcgttaactcggcgtttcatct
gtggtgcaacgggcgctgggtcggttacggccaggacagtcgtttgccgtctgaatttgacctgagcgcatttttac
gcgccggagaaaaccgcctcgcggtgatggtgctgcgctggagtgacggcagttatctggaagatcaggatatgtgg
cggatgagcggcattttccgtgacgtctcgttgctgcataaaccgactacacaaatcagcgatttccatgttgccac
tcgctttaatgatgatttcagccgcgctgtactggaggctgaagttcagatgtgcggcgagttgcgtgactacctac
gggtaacagtttctttatggcagggtgaaacgcaggtcgccagcggcaccgcgcctttcggcggtgaaattatcgat
gagcgtggtggttatgccgatcgcgtcacactacgtctgaacgtcgaaaacccgaaactgtggagcgccgaaatccc
gaatctctatcgtgcggtggttgaactgcacaccgccgacggcacgctgattgaagcagaagcctgcgatgtcggtt
tccgcgaggtgcggattgaaaatggtctgctgctgctgaacggcaagccgttgctgattcgaggcgttaaccgtcac
gagcatcatcctctgcatggtcaggtcatggatgagcagacgatggtgcaggatatcctgctgatgaagcagaacaa
ctttaacgccgtgcgctgttcgcattatccgaaccatccgctgtggtacacgctgtgcgaccgctacggcctgtatg
tggtggatgaagccaatattgaaacccacggcatggtgccaatgaatcgtctgaccgatgatccgcgctggctaccg
gcgatgagcgaacgcgtaacgcgaatggtgcagcgcgatcgtaatcacccgagtgtgatcatctggtcgctggggaa
tgaatcaggccacggcgctaatcacgacgcgctgtatcgctggatcaaatctgtcgatccttcccgcccggtgcagt
atgaaggcggcggagccgacaccacggccaccgatattatttgcccgatgtacgcgcgcgtggatgaagaccagccc
ttcccggctgtgccgaaatggtccatcaaaaaatggctttcgctacctggagagacgcgcccgctgatcctttgcga
atacgcccacgcgatgggtaacagtcttggcggtttcgctaaatactggcaggcgtttcgtcagtatccccgtttac
agggcggcttcgtctgggactgggtggatcagtcgctgattaaatatgatgaaaacggcaacccgtggtcggcttac
ggcggtgattttggcgatacgccgaacgatcgccagttctgtatgaacggtctggtctttgccgaccgcacgccgca
tccagcgctgacggaagcaaaacaccagcagcagtttttccagttccgtttatccgggcaaaccatcgaagtgacca
gcgaatacctgttccgtcatagcgataacgagctcctgcactggatggtggcgctggatggtaagccgctggcaagc
ggtgaagtgcctctggatgtcgctccacaaggtaaacagttgattgaactgcctgaactaccgcagccggagagcgc
cgggcaactctggctcacagtacgcgtagtgcaaccgaacgcgaccgcatggtcagaagccgggcacatcagcgcct
ggcagcagtggcgtctggcggaaaacctcagtgtgacgctccccgccgcgtcccacgccatcccgcatctgaccacc
agcgaaatggatttttgcatcgagctgggtaataagcgttggcaatttaaccgccagtcaggctttctttcacagat
gtggattggcgataaaaaacaactgctgacgccgctgcgcgatcagttcacccgtgcaccgctggataacgacattg
gcgtaagtgaagcgacccgcattgaccctaacgcctgggtcgaacgctggaaggcggcgggccattaccaggccgaa
gcagcgttgttgcagtgcacggcagatacacttgctgatgcggtgctgattacgaccgctcacgcgtggcagcatca
ggggaaaaccttatttatcagccggaaaacctaccggattgatggtagtggtcaaatggcgattaccgttgatgttg
aagtggcgagcgatacaccgcatccggcgcggattggcctgaactgccagctggcgcaggtagcagagcgggtaaac
tggctcggattagggccgcaagaaaactatcccgaccgccttactgccgcctgttttgaccgctgggatctgccatt
gtcagacatgtataccccgtacgtcttcccgagcgaaaacggtctgcgctgcgggacgcgcgaattgaattatggcc
cacaccagtggcgcggcgacttccagttcaacatcagccgctacagtcaacagcaactgatggaaaccagccatcgc
catctgctgcacgcggaagaaggcacatggctgaatatcgacggtttccatatggggattggtggcgacgactcctg
gagcccgtcagtatcggcggaattccagctgagcgccggtcgctaccattaccagttggtctggtgtcaaaaataa
E）ldh
(966bp)
atgatgaacaaacatgtaaataaagtagctttaatcggagcgggttttgttggaagcagttatgcatttgcgt
taattaaccaaggaatcacagatgagcttgtggtcattgatgtaaataaagaaaaagcaatgggcgatgtgatggat
ttaaaccacggaaaggcgtttgcgccacaaccggtcaaaacatcttacggaacatatgaagactgcaaggatgctga
tattgtctgcatttgcgccggagcaaaccaaaaacctggtgagacacgccttgaattagtagaaaagaacttgaaga
ttttcaaaggcatcgttagtgaagtcatggcgagcggatttgacggcattttcttagtcgcgacaaatccggttgat
atcctgacttacgcaacatggaaattcagcggcctgccaaaagagcgggtgattggaagcggcacaacacttgattc
tgcgagattccgtttcatgctgagcgaatactttggcgcagcgcctcaaaacgtacacgcgcatattatcggagagc
acggcgacacagagcttcctgtttggagccacgcgaatgtcggcggtgtgccggtcagtgaactcgttgagaaaaac
gatgcgtacaaacaagaggagctggaccaaattgtagatgatgtgaaaaacgcagcttaccatatcattgagaaaaa
aggcgcgacttattatggggttgcgatgagtcttgctcgcattacaaaagccattcttcataatgaaaacagcatat
taactgtcagcacatatttggacgggcaatacggtgcagatgacgtgtacatcggtgtgccggctgtcgtgaatcgc
ggagggatcgcaggtatcactgagctgaacttaaatgagaaagaaaaagaacagttccttcacagcgccggcgtcct
taaaaacattttaaaacctcattttgcagaacaaaaagtcaactaa
The primer of table 2 and editor's original paper sequence table
Primer 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
cgGGATCCtaatacgactcactatagggaatattgtcctcatcgccccttcgaggggtcgcaacccgcccggt
gcagtatgaaggcggcggagccgacaccacggtcctcatcgccccttcgaggggtcgcaacctagcataaccccttg
gggcctctaaacgggtcttgaggggttttttgGAATTCcg
atgatgaacaaacatgtaa
ttagttgactttttgttc
ccAAGCTTcgatgagatggatttaaacca
cacaggtaggcgcgccatgttgcgtaagtcaggata
tatcctgacttacgcaacatggcgcgcctacctgtg
tttttctcaacgagttcactccttacgccccgcc
ggcggggcgtaaggagtgaactcgttgagaaaaa
gcgtTCTAGAgcgattcacgacagcc
tttttctcaacgagttcactatgttgcgtaagtcaggata
tatcctgacttacgcaacatagtgaactcgttgagaaaaa
cgGGATCCtaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgt
tgtgtggaattgtgagcggataacaagtcctcatcgccccttcgaggggtcgcaacagcggcctgccaaaagagcgg
gtgattggaagcggcacaagtcctcatcgccccttcgaggggtcgcaacctagcataaccccttggggcctctaaac
gggtcttgaggggttttttgGAATTCcg
Capitalization is restriction enzyme site, and wave is protection base, and the overstriking of primer black matrix is complementary region, and single underscore is promoter
Promoter, italic is spacer, and black matrix overstriking is repeat, and double underline is terminator terminator
Claims (4)
1. it is a kind of to be based on Virginia streptomycete IBL14 genescasThe gene editing method of 7-5-3, it is characterised in that：Wei Jini
Contain 3 genes in sub- streptomycete IBL14cas7-5-3, through protein expressing plasmid structure, gene editing plasmid construction and restructuring
The acquisition of son can enter edlin with checking procedure to prokaryotic gene group.
2. according to claim 1 a kind of based on Virginia streptomycete IBL14 genescasThe gene editing side of 7-5-3
Method, it is characterised in that：Step is as follows：
（1）According to Virginia streptomycete IBL14 genescas7-5-3 and relevant information primers, with Virginia chain
Mould IBL14 genomes are masterplate, and reacting amplification by PCR with archaeal dna polymerase obtainscas7-5-3 genes, and it is connected to plasmid
On plasmid, Cas7-5-3 protein expressing plasmids plasmid- is obtainedcas7-5-3；
（2）According to prokaryotes target gene DNA sequence dna information design primer, with prokaryotic gene group as masterplate, by PCR
Amplification obtains target of the end with restriction enzyme identification and cleavage site and overlap PCR complementary series respectively for reaction
To the upper and lower homology arm PCR fragment of gene, and upper and lower homology arm is connected into obtain gene editing masterplate template with overlap PCR
DNA/t-DNA, while designed according to prokaryotes target gene sequence information and be directly synthesized head and the tail being manipulated comprising lactose respectively
The target gene fragment guide-DNA/g-DNA of sub- promoter and RNA terminators, and according to the viscosity on restriction enzyme site
Be connected on plasmid for gene editing masterplate and target gene fragment and obtain gene editing plasmid plasmid-t/gDNA by end；
（3）Prokaryote competence is prepared, and will be by step（1）The protein expressing plasmid plasmid- for obtainingcas7-5-3
With by step（2）The various target gene editor plasmids for obtaining obtain different genes volumes in being transformed into target bacterium competence respectively
Recon after volume；Performing PCR and gene sequencing and/or functional analysis are entered to restructuring daughter chromosome, to confirm the purpose after editor
Recon.
3. according to claim 1 a kind of based on Virginia streptomycete IBL14 genescasThe gene editing side of 7-5-3
Method, it is characterised in that：The prokaryotes refer to Escherichia coli, hay bacillus and other prokaryotic micro-organisms.
4. according to claim 1 a kind of based on Virginia streptomycete IBL14 genescasThe gene editing side of 7-5-3
Method, it is characterised in that：Described gene editing refers to can using the protein expressing plasmid combination gene editing plasmid or other plasmids
The chromogene of prokaryotic is knocked out, is inserted, seamless point mutation and any combination.
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