CN106755037A - A kind of Virginia streptomycete IBL14 type I B sv14 type CAS gene editing systems - Google Patents
A kind of Virginia streptomycete IBL14 type I B sv14 type CAS gene editing systems Download PDFInfo
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Abstract
Present invention firstly discloses a kind of Virginia streptomycete IBL14 type I B sv14 type CAS gene editing systems, including gene editing and immunologic process in the structure and Escherichia coli and Bacillus subtilis of protein expressing plasmid and gene editing plasmid.The system can easily and fast, effectively carry out gene editing to prokaryotic gene group and be immunized.The gene editing and immunization method can be widely applied to the other application such as bioscience and pharmacy, food, agricultural field.
Description
Technical field
It is exactly a kind of Virginia streptomycete the present invention relates to the genetic engineering field in biotechnology
IBL14type I-B-sv14 type CAS gene editing systems.
Background technology
In biological technical field, CRISPR-Cas9 systems are after zinc finger endonuclease/ZFN (zinc-finger
Nucleases), class activating transcription factor effector nuclease/TALEN (transcription activator-like
Effector nucleases) after occur third generation gene editing technology.Compared with preceding two generation technique, because its low cost,
Easy to operate, quickness and high efficiency and it is rapid fashionable in world's biological technical field, as fields such as scientific research, medical treatment, food
Effective tool (Rath, D.；Amlinger,L.；Rath,A.；Lundgren,M.,The CRISPR-Cas immune
system:Biology,mechanisms and applications.Biochimie 2015,117,119-128)。
CRISPR-Cas systems refer to a kind of adaptive immunity of degraded under the guidance of RNA external source bacteriophage or poisoning intrusion
System, can be divided into I types (type I), П types and Ш types (Gasiunas, G.；Sinkunas,T.；Siksnys,V.,
Molecular mechanisms of CRISPR-mediated microbial immunity.Cell Mol Life Sci
2014,71,(3),449-65).The marker protein of I type CRISPR-Cas systems is Cas3, and it has DNA enzymatic activity and unwindase
Activity, is combined with crRNA with Cascade compounds, and exogeneous target DNA is cracked to reach the purpose for preventing it from infecting
(Gasiunas,G.；Sinkunas,T.；Siksnys,V.,Molecular mechanisms of CRISPR-mediated
microbial immunity.Cell Mol Life Sci 2014,71,(3),449-65；Gong,B.；Shin,M.；Sun,
J.；Jung,C.H.；Bolt,E.L.；van der Oost,J.；Kim,J.S.,Molecular insights into DNA
interference by CRISPR-associated nuclease-helicase Cas3.Proceedings of the
National Academy of Sciences of the United States of America 2014,111,(46),
16359-64)_ENREF_3.The marker protein of П type CRISPR-Cas systems is Cas9, and what is had been commercialized derives from
The Cas9 of Streptococcus pyogenes, by 1368 Amino acid profiles, is that one kind contains two nuclease domain (RuvC-
Like cuts coding strand and HNH cuttings and the noncoding strand of crRNA complementary pairings) DNA restriction endonucleases (Terao, M., et al.
(2016)."Utilization of the CRISPR/Cas9system for the efficient production of
mutant mice using crRNA/tracrRNA with Cas9nickase and FokI-dCas9."
Experimental Animals 65(3):275-283).The marker protein of Ш type CRISPR-Cas systems is Cas10, with Cmr
Complex combines purpose (Gasiunas, the G. for reaching and preventing its exogenous nucleic acid from infecting；Sinkunas,T.；Siksnys,V.,
Molecular mechanisms of CRISPR-mediated microbial immunity.Cell Mol Life
Sci2014,71,(3),449-65).In the CRISPR-Cas systems of three types, in addition to II types, I types and type III are without application
In the report of gene editing.
Virginia streptomycete IBL-14 (the Streptomyces virginiae for obtaining are isolated and purified by this laboratory
IBL14) it is one plant of actinomyces of various steroidal compounds that can degrade.Genome sequencing finds there is a cas7- with analysis
The type I-B-sv14 type Cas systems of 5-3-4-1-2 sequences, have confirmed that itself chromosome can be carried out gene editing (child's prestige
Space；Yong Dexiang；Li Xue；CRISPR-Cas systems in a kind of Virginia streptomycete IBL14 of Qiu Caihua and carry out base using it
Because of the method .2015110028173,2015 for editing；Tong Wangyu；Li Xue；A kind of auspicious Virginia streptomycete IBL14 of Yong De produce blue or green
The construction method .2015109993334,2015 of mycin recombinant bacterial strain).
The present invention connects the gene cas7-5-3-4-1-2 in the type I-B-sv14 type CRISPR-Cas systems and carrier
Connect and obtain a protein expressing plasmid plasmid-cas7-5-3-4-1-2, with reference to the gene editing plasmid (plasmid- of design
) and/or other plasmids realize the gene editing in non-self prokaryotes Escherichia coli and hay bacillus first t/gDNA
And it is immune.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of based on Virginia streptomycete IBL14type I-B-sv14
Type CRISPR-Cas systems and the protein expressing plasmid system set up, realize former with reference to gene editing plasmid and/or other plasmids
The biological gene editing of core with it is immune.
Technical scheme is as follows：
A kind of Virginia streptomycete IBL14type I-B-sv14 type CAS gene editing systems, it is characterised in that：This is
Expression of recombinant proteins plasmid (accompanying drawing 1) that system is constituted comprising six genes (subordinate list 1) of a cas7-5-3-4-1-2 and one
Gene editing plasmid (accompanying drawing 3), can be effectively used for prokaryotic gene editor and be immunized.
A kind of described Virginia streptomycete IBL14type I-B-sv14 type CAS gene editing systems, its feature exists
In：Edit methods are comprised the following steps：
(1) it is (attached according to Virginia streptomycete IBL14 genes cas7-5-3-4-1-2 and relevant information primers
Table 2), it is masterplate with the Virginia streptomycete IBL14 genomes for extracting, with TransTaq DNA Polymerase High
Fidelity polymerases obtain end and carry and the knowledge of carrier restriction enzyme by PCR (PCR reactions) amplification
The cas7-5-3-4-1-2 genes of other and cleavage site 15-25bp overlapping regions, are connected on plasmid (plamisd), obtain albumen
Expression plasmid plamisd-cas7-5-3-4-1-2；
(2) according to target gene DNA sequence dna information design primer (subordinate list 2), it is with the prokaryotic gene group extracted
Masterplate, reacts amplification respectively and obtains with TransTaq DNA Polymerase High Fidelity archaeal dna polymerases by PCR
Target gene upper and lower homology arm of the end with restriction enzyme identification and cleavage site and overlap PCR complementary series
PCR fragment, and upper and lower homology arm is combined into structure gene editing masterplate (t-DNA) with overlap PCR, while according to life
Thing target gene sequence information is designed and is directly synthesized the target gene fragment of head and the tail promoter containing T7 and RNA terminators respectively
(g-DNA) (subordinate list 2), gene editing masterplate and target gene fragment is connected to gene editing plasmid is obtained on plasmid
(plasmid-t/gDNA)；
(3) prokaryote competence is prepared, and the protein expressing plasmid that will be obtained by step (1) and by step (2)
The various target gene editor plasmids for obtaining obtain the restructuring after different gene editings in being transformed into target bacterium competence respectively
Son, performing PCR and gene sequencing and/or functional analysis (accompanying drawing 3) are entered to restructuring daughter chromosome, are recombinated with confirming the purpose after editor
Son.
A kind of described Virginia streptomycete IBL14type I-B-sv14 type CAS gene editing systems, its feature exists
In：The prokaryotes refer to Escherichia coli, hay bacillus and other various prokaryotic micro-organisms.
A kind of described Virginia streptomycete IBL14type I-B-sv14 type CAS gene editing systems, its feature exists
In：Described gene editing refers to can be thin to protokaryon using the protein expressing plasmid combination gene editing plasmid and/or other plasmids
The chromogene of born of the same parents is knocked out, inserted, seamless point mutation and any combination.
A kind of described Virginia streptomycete IBL14type I-B-sv14 type CAS gene editing systems, its feature exists
In：Described immune finger can be applied to biological cell and prevents invading for exogenous nucleic acid by the CRISPR that gene editing masterplate is designed
Enter.
The present invention has the beneficial effect that：
It is first the invention provides a kind of Virginia streptomycete IBL14type I-B-sv14 type CAS gene editing systems
It is secondary realize CRISPR-Cas I types systems to the gene editing of other prokaryotic gene groups with it is immune；The system is given birth to protokaryon
Thing genome can easily and fast, effectively carry out gene editing and be immunized.The gene editing method can be widely applied to biological section
The other application field such as and pharmacy, food, agricultural.
Figure of description
The structure of Fig. 1 protein expressing plasmids pET28a-cas7-5-3-4-1-2.Ori/origin：DNA replication dna start bit
Point；f1ori:The replication origin of f1 bacteriophages origin；T7promoter:T7Promoter starting DNA is transcribed；KanR/
Kanamycin resistance：Kalamycin resistance；T7terminator:T7Terminator, terminates DNA transcriptions, lacI/
lactose repressor：Coding lacrepressor
The structure of Fig. 2 gene editing plasmids pKD46-t/g-DNA.pSC101ori/the origin from pSC:Come from
The replication origin of low-copy (copy) plasmid vector pSC101；Rep101：Replicated based on gene RepA control plasmids pSC101
Copy number；araC/L-arabinose regulatory protein:Arabinose regulatory protein；AmpR/ampicillin
resistance:Amicillin resistance；aBAD promoter/promoter of the L-arabinose operon of
E.coli:By the araB promoters of arabinose regulation and control in Escherichia coli；Exo, bet, gam gene：Red homologous recombinations are bitten by λ
3 genomic constitutions of the exo of thalline, bet, gam, are separately encoded 3 kinds of protein of Exo, Beta, Gam
The knockout result of galM genes in Fig. 3 bacterial strain EC JM109 (DE3).(A) e. coli jm109 (DE3) indigo plant hickie
The selection result, blueness is shown to be original strain, and white is shown to be recombinant bacterial strain；(B) gene galM exterior PCs R electrophoretograms, swimming lane
M：1500bp DNA ladder, swimming lane 1：Blank, swimming lane 2~8：Plasmid pET28a-cas-7-5-3-4-1-2 and
The galM gene PCRs result (970bp shows to knock out successfully) of the transformant after pKD46-galM-g/t-DNA, swimming lane 9：Wild type
(1341bp represents the galM bases of wild type EC JM109 (DE3) genome to the galM gene PCRs of EC JM109 (DE3) genome
Cause)
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, even unless otherwise instructed in listed embodiment use following material：
1) bacterial strain and plasmid
Virginia streptomycete Streptomyces virginiae IBL14/SV IBL14；Escherichia coli
Escherichia coli DH5α/EC DH5α；Escherichia coli JM109/EC JM109(DE3)；Bacillus subtilis
Bacterium Bacillus subtilis168/BS 168, plasmid pET28a, pKD46, pKD3.
2) culture medium
TB culture mediums：
(V/V) the Glycerol 17Mm of 1.2% (W/V) Tryptone, 2.4% (W/V) Yeast Extract 0.4%
K2PO4 72Mm K2HPO4
10 × spizzen salting liquids (100ml)：
K2HPO414g, KH2PO46g, (NH4)2SO42g, sodium citrate 1g, MgSO4·7H2O 0.2g are in order successively
It is dissolved in distilled water, adds water to 100ml.
GM Ⅰ(100ml)：
1 × spizzen salting liquids 95ml, 50% glucose 1ml, 5% caseinhydrolysate 500 μ l, 10% yeast juice 1ml,
2mg/ml L-trp 2.5ml
GM Ⅱ(100ml)：
1 × spizzen salting liquids 97.5ml, 50% glucose 1ml, 5% caseinhydrolysate 80 μ l, the μ of 10% yeast juice 40
The μ l of 500 μ l, 0.5M/L MgCl2 of l, 2mg/ml L-trp, 500 μ l, 0.1M CaCl2 500
Agents useful for same is commercially available product.
Embodiment 1 (EC JM109 (DE3) beta-galactosidase genes galM knockouts)
(1) structure of Cas7-5-3-4-1-2 protein expressing plasmids
According to SV IBL14 genome sequencings information and plasmid pET28a sequence informations, design gene cas7-5-3-4-
1-2 specific primers cas7-5-3-4-1-2-F and cas7-5-3-4-1-2-R (table 2)；SV IBL-14 genomic DNAs are extracted,
Archaeal dna polymerase (the TransTaq DNA Polymerase High produced using Beijing Quanshijin Biotechnology Co., Ltd
Fidelity cas gene PCR amplifications, reaction condition) are carried out：95 DEG C of 2min, 95 DEG C of 20s, 60 DEG C of 20s, 72 DEG C of 3min,
The TransTaq DNA Polymerase High Fidelity (50 μ l reaction systems) of 250units, 30 circulations, 72 DEG C
10min.PCR primer detects that kit is reclaimed, the cas full-length gene fragments for being purified through 1% agarose electrophoresis；By one
With plasmid pET28a be connected cas full-length gene orders by footwork, obtains protein expressing plasmid pET28a-cas7-5-3-4-1-2 standby
With.
(2) structure of gene editing plasmid
(A) gene galM design of primers and amplification
According to EC JM109 (DE3) genome sequencing information, design gene galM specific primers GalM-F and GalM-
R (table 2).EC JM109 (DE3) genomic DNA is extracted, is carried out with TransTaq DNA Polymerase High Fidelity
GalM gene PCRs are expanded, reaction condition：95 DEG C of 5min, 94 DEG C of 30s, 52 DEG C of 30s, 72 DEG C of 2min, the TransTaq of 250units
DNA Polymerase High Fidelitye (50 μ l reaction systems), 30 circulations, 72 DEG C of 10min；PCR primer is through 1% fine jade
Lipolysaccharide electrophoresis detection, kit is reclaimed, and the galM full-length gene fragments for being purified are standby.
(B) upstream and downstream homology arm is prepared
According to galM genes complete sequence (table 1) design galM upstream region of gene homology arm primers galM-UF and galM-UR, under
Trip homology arm primer galM-DF and-galMDR (table 2), and homology arm primer restriction enzyme containing NotI and HindIII up and down
Restriction enzyme site；It is template with the galM gene DNAs for purifying, upstream and downstream homology arm is first expanded respectively, reaction condition is：95℃
The TransTaq DNA Polymerase High Fidelity of 5min, 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min, 250units
(50 μ l reaction systems) 30 circulations, 72 DEG C of 10min；PCR primer detects that kit is reclaimed, and obtains pure through 1% agarose electrophoresis
Upstream and downstream homology arm DNA fragmentation after change is standby.
(C) gene editing masterplate (galM/t-DNA) is prepared
Take homology arm purified product to mix as template with the lower μ l of homology arm purified product 0.5,30 μ l reaction systems are entered
Row overlap PCR, reaction condition is：94 DEG C of predegenerations 5min, 94 DEG C of denaturation l min, 60 DEG C of annealing 1min, 72 DEG C of extensions
30s, people's primer galMUF and each 1 μ l of galMDR are added after a circulation, continue PCR, and reaction condition is：95 DEG C of predegeneration 5min,
94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 2min, carry out 30 circulations, 72 DEG C of 10min；1% agarose gel electrophoresis
Detection amplified production is simultaneously purified, the editor's masterplate fragment for obtaining；The editor's masterplate fragment that will be obtained passes through HindIII restriction enzymes
Digestion goes out cohesive end, and the upstream and downstream homology arm DNA fragmentation for obtaining after purification is standby.
(D) gene editing plasmid pKD46-galM-t/g-DNA is built
The target gene fragment (g-DNA) (table 2) of T7 containing promoter and connection product is direct by the general biotech firm in Chuzhou
Synthesis, head and the tail respectively with carrier restriction enzyme identification and cleavage site 15-25bp NotI restriction enzyme sites and HindIII enzymes
Enzyme site, centre is successively that promoter T7, spacer, repeat and terminator obtain galM-g-DNA；By target gene fragment
G-DNA is connected to obtain gene editing masterplate by the T4 ligases that Beijing Quanshijin Biotechnology Co., Ltd produces
On galM-t-DNA, gene editing masterplate carrier galM-t/g-DNA is obtained；Then by the full formula gold limited public affairs of biotechnology in Beijing
The Peasy-Uni Seamless Cloning and Assembly Kit for taking charge of production are connected on pKD46 plasmids；Obtain base
Because of editor's masterplate carrier pKD46-galM-t/g-DNA.
(3) preparation of recon and inspection
(A) prepared by EC JM109 (DE3) competence
Aseptically trained in 30ml TB liquid with the monoclonal on sterilized toothpick picking E. coli plate
In foster base, 37 DEG C, 220rpm incubated overnights.The μ l of bacterium solution 100 for taking incubated overnight are forwarded in new TB fluid nutrient mediums, and 37
DEG C, 220rpm culture about 2h, to bacterium solution OD600Value about 0.5 or so.In taking the above-mentioned bacterium solutions of 1ml to 1.5ml EP pipes, at 4 DEG C
4000rpm is centrifuged 10min, after eliminating supernatant, takes the SSCS from Generay Biotech productions in precooling on ice
The μ l of solution 100 are uniform by bacterial sediment piping and druming, obtain final product and save backup at the competent cell of Escherichia coli, -80 DEG C that (this process is complete
Journey will be carried out on ice).
(B) cotransformation of protein expressing plasmid and gene editing plasmid
Plasmid pET28a-cas7-5-3-4-1-2 and pKD46-galM-t/g-DNA is added to the competence after melting thin
In born of the same parents, ice bath 30min, 42 DEG C of thermal shock 90s are put into 10~15min on ice immediately after.800 μ l TB fluid nutrient mediums are added,
In 30 DEG C, in 220rpm constant-temperature tables, recover 1 hour.Bacterium solution is applied to containing eventually after taking the μ l of bacterium solution 100 after recovery or concentration
Concentration be 50 μ g/ml kanamycins (Kana) solution and 50 μ g/ml ampicillin (Amp) solution, 20mg/ml X-gal and
On the TB solid plates of 0.84mol/L IPTG, overnight incubation obtains transformant in 30 DEG C of constant incubators.
(C) recon Function detection and genetic analysis
The white monoclonal of screening is recon undetermined, and with its genome as template, enters performing PCR checking and sequencing analysis,
It was found that recon chromosome DNA amplification band reduces expected 371bp compared with wild type, through general biosystem (Anhui)
Co., Ltd's sequencing proves galM genes successful knockout.
(the Cm in EC JM109 (DE3) of embodiment 2RThe insertion of resistant gene)
(1) protein expressing plasmid pET28a-cas7-5-3-4-1-2
With the step of embodiment 1 (1)
(2) structure of gene editing plasmid pKD46-t/g-DNA
Divided by containing chloramphenicol (CmR) resistant maker gene plasmid pKD3 be template, with reference to galM gene knockouts in it is upper
Downstream homology arm enters performing PCR and insertion chloramphenicol (Cm in the middle of upstream and downstream homology arm is synthesizedR) resistance marker PCR primer, even
It is connected on plasmid pKD46, is formed outside gene editing plasmid pKD46-galM-cm-t/g-DNA；Walked in remaining step and embodiment 1
Suddenly (2) are identical.
(3) acquisition of recon and inspection
With gained recon bacterial strain by after induction, then TB fluid nutrient mediums of transferring are cultivated 15 hours under the conditions of 39 DEG C,
Gained bacterium solution is diluted coating afterwards, the single bacterium colony for obtaining is transferred to TB resistant panels containing Cm and is marked resistance survey respectively again
Examination, picking monoclonal on the TB flat boards containing Cm antibiotic enters performing PCR, and product is detected through 1% agarose electrophoresis and verified, is found
Recon chromosome DNA amplification stripe size is consistent with expected, and through the ECJM109 after gene sequencing proof gene editing
(DE3) in recon the knockout of galM and chloramphenicol resistance gene with insert successfully；Remaining step and step (3) in embodiment 1
It is identical.
Embodiment 3 (EC JM109 (DE3) are to the Repeat sequences in immune-EC JM109 (DE3) of plasmid pKD3)
(1) structure of protein expressing plasmid pET28a-cas7-5-3-4-1-2
With the step of embodiment 1 (1)
(2) gene editing plasmid pKD46-galM-pKD3-g/tECThe structure of-DNA
According to DNA gene orders in plasmid pKD3, head and the tail are directly synthesized respectively plus NotI restriction enzyme sites, centre is successively
Promoter T7, spacer (pKD3 base sequences), repeat (the repeat sequences in the CRISPR of EC JM109 (DE3)) and
The pKD3-t of terminatorEC-DNA；Plasmid is connected to by T4 ligases with the galM-g-DNA in the step of embodiment 1 (2)
In pKD46, gene editing plasmid gene editor's plasmid pKD46-galM-pKD3-g/t is obtainedEC-DNA；Remaining step and embodiment 1
Middle step (2) is identical.
(3) acquisition of recon and inspection
With the step of embodiment 1 (3)；Recon ECJM109 is obtained after proving gene editing through DNA cloning and gene sequencing
(DE3)-galM-pKD3。
(4) recon is immune
Plasmid pKD3 is converted respectively to host ECJM109 and recon ECJM109-galM-pKD3, is found
CmRThere is ECJM109 to grow in resistant panel, without recon EC JM109-galM-pKD3 growths, show recon ECJM109-
To plasmid pKD3 immune success rates in galM-pKD3.
Embodiment 4 (EC JM109 (DE3) are to the Repeat sequences in the immune-SV IBL14 of plasmid pKD3)
(1) structure of protein expressing plasmid pET28a-cas7-5-3-4-1-2
With the step of embodiment 1 (1)
(2) gene editing plasmid pKD46-galM-pKD3-g/tSVThe structure of-DNA
Repeat sequences are using the repeat sequences in the CRISPR of SV IBL14；Remaining step and step in embodiment 3
(2) it is identical.
(3) acquisition of recon and inspection
With the step of embodiment 3 (3).
(4) recon is immune
With the step of embodiment 3 (4).
Embodiment 5 (the double-mass model knockout of the gene amyE of BS 168 and the insertion of chloramphenicol resistance gene)
(1) protein expressing plasmid pHT304-cas7-5-3-4-1-2
Cas7-5-3-4-1-2 full-length gene orders are connected with plasmid pHT-304, protein expressing plasmid pET28a- is obtained
Cas7-5-3-4-1-2 is standby；Remaining step is identical with step (3) in embodiment 1.
(2) structure of gene editing plasmid pKC1139-amyE-t/g-DNA
(A) amplification of gene amyE design of primers and amyE full-length genes
Design gene primer is amyE-F and amyE-R (table 2).With BS168 genomic DNAs masterplate, amyE genes are expanded
Fragment.Reaction condition：95 DEG C of 5min, 94 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 1min30s, 250U TransTaq DNA
Polymerase High Fidelity (50 μ l reaction systems), 30 circulations, 72 DEG C of 10min.PCR primer is through 1.0% agar
Sugared electrophoresis detection, kit is reclaimed, and the amyE full-length gene fragments for being purified are standby.
(B) preparation of upstream and downstream homology arm
Separately design upper and lower homology arm primer amyE-UPF, amyE-UPR, amyE-CmF, amyE-CmR, amyE-
DownF, amyE-DownR (table 2).It is template with the amyE genes and Cm gene DNAs that purify, upper, Cm, downstream is first expanded respectively
Homology arm, reaction condition is：95 DEG C of 5min, 94 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 1min 20s, 250U biological limited public affairs of full formula gold
Take charge of the TransTaq DNA Polymerase High Fidelity (50 μ l reaction systems) of production, 30 circulations, 72 DEG C
10min.PCR primer detected through 1.0% agarose electrophoresis, and kit is reclaimed, and obtains upper homology arm after purification, Cm, downstream same
Source arm DNA fragmentation is standby.
(C) structure of gene editing masterplate carrier pKC1139-amyE-t-DNA
Homology arm purified product, cm purified products and the μ l of lower homology arm purified product 1.0 mixing are taken as template, 25 μ l
Reaction system carries out overlap PCR, and reaction condition is：94 DEG C of predegenerations 5min, 94 DEG C of denaturation l min, 55 DEG C of annealing 1min,
72 DEG C of extension 30s, 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 of predegeneration 5min,
94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 2min, carry out 30 circulations, 72 DEG C of 10min.1.0% Ago-Gel electricity
Swimming detection amplified production is simultaneously purified, and obtains gene editing masterplate；The gene editing masterplate that will be obtained is limited by HindIII and XbaI
Property restriction endonuclease cut out cohesive end, be then connected on pKC1139 plasmids by T4 ligases, obtain gene editing masterplate load
Body pKC1139-amyE-t-DNA.
(D) structure of gene editing plasmid pKC1139-amyE-t/g-DNA
Target gene fragment (table 2) containing lactose operon promoter and guide DNA-amyE connection products is led to by Chuzhou
It is directly synthesized with biotech firm, 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
On carrier pKC1139-amyE-t-DNA gene editing plasmid pKC1139-amyE-t/g-DNA.
(3) acquisition of recon and inspection
(A) preparation of BS168 competence
The fresh BS168 single bacteriums of inoculation are fallen within 5ml GM I, and 30 DEG C, 100~150 revs/min of shaking table cultures are overnight.It is secondary
In the culture mediums of 9ml GM I, 37 DEG C in shaking table, 200 revs/min are cultivated 3~4h to the day inoculation above-mentioned cultures of 1ml.Take 5ml
Second step culture is transferred to be carried out second pass generation in the culture mediums of 45ml GM II, 37 DEG C, 100~150 revs/min of shaking table trainings
Support 90min.Whole cultures are taken, 4000 revs/min of room temperatures are centrifuged 5min, with the 1/10 resuspended thalline of volume supernatant, obtain final product
BS168 competent cells.
(B) cotransformation of plasmid pHT304-cas7-5-3-4-1-2 and pKC1139-amyE-t/g-DNA
Plasmid pHT304-cas7-5-3-4-1-2 and pKC1139-amyE-t/g-DNA are added into 1ml BS168 competence
Mixed in cell suspension, 30~60min is stood in 37 DEG C of waters bath with thermostatic control, then with 37 DEG C, 200 revs/min of vibrations in shaking table
2~4h of culture.Conversion fluid is coated with (the μ g/ml of final concentration 5) the TB solid mediums containing chlorampenicol resistant, in 37 DEG C of constant temperature trainings
Overnight incubation is inverted in foster case, secondary daily inspection transformant is drawn appropriate bacterium solution and is applied on the TB culture mediums containing Cm resistances.
(C) restructuring daughter chromosome PCR and gene sequencing analysis
Picking contains CmRThe single bacterium colony grown in resistant panel, extracts its genome or with heat treated bacterium colony conduct
Template, knocks out the upstream and downstream primer of gene for template enters performing PCR amplification, and gained genetic fragment is carried out into electrophoresis, and product is through 1% fine jade
Lipolysaccharide electrophoresis detection, the change for observing recon chromosome DNA amplification stripe size is consistent with expection, and is proved through gene sequencing
BS168-amyE gene knockouts and chloramphenicol resistance gene are inserted successfully.
Embodiment 6 (the simple substance grain knockout of the gene amyE of BS 168 and the insertion of chloramphenicol resistance gene)
(1) structure of protein expressing plasmid pHT304-cas7-5-3-4-1-2
With the step of embodiment 1 (1)
(2) structure of gene editing plasmid pHT304-cas7-5-3-4-1-2-amyE-t/g-DNA
In addition to amyE-t/g-DNA target fragments are connected to pHT304-cas7-5-3-4-1-2 plasmids, remaining step with
Step (2) is identical in embodiment 5.
(3) acquisition of recon and inspection
With the step of embodiment 5 (3)
The above only further illustrates technology contents of the invention with embodiment, is easier to understand in order to reader,
But embodiments of the present invention not being represented and being only limitted to this, any technology done according to the present invention extends or recreates, and is sent out by this
Bright protection.
The Escherichia coli galM of table 1, hay bacillus amyE and Virginia streptomycete IBL14 cas7-5-3-4-1-2 gene orders
Gene order
galM
(1041bp） gtgctgaacgaaactcccgcactggcacccgatggtcagccgtaccgactgttaactttgcgta
acaacgcagggatggtagtcacgctgatggactggggtgcgactttactttccgcccgtattccgctttccgatggc
agcgtccgcgaggcgctgctcggctgtgccagcccggaatgctatcaggatcaggccgcgtttctgggggcctctat
tggtcgttatgccaaccgtatcgccaatagccgttatacctttgacggtgaaaccgtgacgctttcgccaagtcagg
gcgttaaccagctgcacggcgggccggaagggttcgacaaacgtcgctggcagattgtgaaccagaacgatcgtcag
gtgctgtttgccctgagttcagatgatggtgatcagggcttcccgggtaatctcggcgcgacggtgcaatatcgtct
gaccgacgataaccgtatctccattacttatcgcgccacagttgataaaccttgcccggtgaatatgactaatcacg
tctatttcaatcttgacggcgagcagtctgacgtgcgcaatcacaagttgcagattctggcggacgaatatctgccg
gttgatgaaggcggcattccgcacgacggcctgaaatctgtcgccggaacgtcttttgatttccgcagcgccaaaat
catcgccagtgagtttcttgccgacgacgatcagcgcaaagtgaaaggttacgatcacgcattcttgttacaggcca
aaggcgatggcaagaaagtggcggcgcatgtctggtcagcagatgaaaaattgcagctgaaggtctacaccaccgct
ccggctctgcaattctactccggcaacttcctcggcggcacaccgtcgcggggaaccgaaccttacgccgactggca
agggctggctctggaaagcgagtttctaccggacagcccgaaccaccctgaatggccgcaaccggactgcttcctgc
gtcctggcgaagagtattccagcctgacggaatatcagtttattgctgagtaa
amyE
(1980bp) atgtttgcaaaacgattcaaaacctctttactgccgttattcgctggatttttattgctgtttc
atttggttctggcaggaccggcggctgcgagtgctgaaacggcgaacaaatcgaatgagcttacagcaccgtcgatc
aaaagcggaaccattcttcatgcatggaattggtcgttcaatacgttaaaacacaatatgaaggatattcatgatgc
aggatatacagccattcagacatctccgattaaccaagtaaaggaagggaatcaaggagataaaagcatgtcgaact
ggtactggctgtatcagccgacatcgtatcaaattggcaaccgttacttaggtactgaacaagaatttaaagaaatg
tgtgcagccgctgaagaatatggcataaaggtcattgttgacgcggtcatcaatcataccaccagtgattatgccgc
gatttccaatgaggttaagagtattccaaactggacacatggaaacacacaaattaaaaactggtctgatcgatggg
atgtcacgcagaattcattgctcgggctgtatgactggaatacacaaaatacacaagtacagtcctatctgaaacgg
ttcttagacagggcattgaatgacggggcagacggttttcgatttgatgccgccaaacatatagagcttccagatga
tggcagttacggcagtcaattttggccgaatatcacaaatacatctgcagagttccaatacggagaaatcctgcagg
atagtgcctccagagatgctgcatatgcgaattatatggatgtgacagcgtctaactatgggcattccataaggtcc
gctttaaagaatcgtaatctgggcgtgtcgaatatctcccactatgcatctgatgtgtctgcggacaagctagtgac
atgggtagagtcgcatgatacgtatgccaatgatgatgaagagtcgacatggatgagcgatgatgatatccgtttag
gctgggcggtgatagcttctcgttcaggcagtacgcctcttttcttttccagacctgagggaggcggaaatggtgtg
aggttcccggggaaaagccaaataggcgatcgcgggagtgctttatttgaagatcaggctatcactgcggtcaatag
atttcacaatgtgatggctggacagcctgaggaactctcgaacccgaatggaaacaaccagatatttatgaatcagc
gcggctcacatggcgttgtgctggcaaatgcaggttcatcctctgtctctatcaatacggcaacaaaattgcctgat
ggcaggtatgacaataaagctggagcgggttcatttcaagtgaacgatggtaaactgacaggcacgatcaatgccag
gtctgtagctgtgctttatcctgatgatattgcaaaagcgcctcatgttttccttgagaattacaaaacaggtgtaa
cacattctttcaatgatcaactgacgattaccttgcgtgcagatgcgaatacaacaaaagccgtttatcaaatcaat
aatggaccagagacggcgtttaaggatggagatcaattcacaatcggaaaaggagatccatttggcaaaacatacac
catcatgttaaaaggaacgaacagtgatggtgtaacgaggaccgagaaatacagttttgttaaaagagatccagcgt
cggccaaaaccatcggctatcaaaatccgaatcattggagccaggtaaatgcttatatctataaacatgatgggagc
cgagtaattgaattgaccggatcttggcctggaaaaccaatgactaaaaatgcagacggaatttacacgctgacgct
gcctgcggacacggatacaaccaacgcaaaagtgatttttaataatggcagcgcccaagtgcccggtcagaatcagc
ctggctttgattacgtgctaaatggtttatataatgactcgggcttaagcggttctcttccccattga
cas7-5-3-4-1-2
(5882bp) gtggtcgccggtgccccgaacaacggggagggcgaggacaacacggggcgtgtgaagaagctga
gggtcgggcgggaggagttcccgtacgtgtccgcgcaggcgttccgtcggtggttgcgtgactcgctgccggcgcag
gagccgcgttcggtggtcactcgctcgggcagcggtgccaagcagcaggcacacaccgcgggccggccggacctgca
cctggacgatgatctgttcggctacatggtcgcggtgaaggggaaggggggaagctaccagcgggacaccgtgctgg
ctaccgggactttagtttcagtggtgccgcagcgtccgacgttggacttcggcacgatgagccgggacttcccggct
ggtgagcacccggtgattcactcgcacgagctgtacagcgcgaccctggccggcgatgttctgctggatctgccgcg
ggctggggtcttcgagacggacggcaacgggttgcgcgtggcgatcagccctgccgtcgctgaggaagcggcgaaga
acggggcggaggtcaccacgctgcggggcagtgcggccattcggttgccgcttactgagcggcaccggcggatcggc
acgctgttgcggacgctggcgtcggtgcgtggtggggccaagcaggctctgcactacggggaccgggccccttcatt
ggtcttgttggctcctctcaagggtggcgtcaatccgttcacccgtgttctgggcgcccgcgacggtaagcctgtgt
tcctgagcgatgtcctgcgcgaggagctcgaggcgtgggcggatgagctggacgggccggtgctgctgggctgggcc
ccggggtttctcggcgatcagcgtgagcaggtccgccgcgagctcaaggatctgattgacgagggccgtgtcgtcct
gagccatcctcgtgtgctgctgacccagctggccgaccggatcgagcagggtgatcatgacgcgtggttcgaggact
ccgcggcgtgacgggtacggaggtcacggccctgcagatcacggtgacggcgccggttgtctccttccgtaatccgc
tgtatgccggggtgcaggtgacgctgccgtgtccgccgccggccaccgtcggcggcctcctcgccgcagcggctggg
gggtgggagcaggtcaatccggagctgcgtttcgcgatggcgttccacgctggcggcaaggcggtcgatctcgagac
gtaccacccgctggacgcgtctgggaagaaggcgtcgcctgccccgcgtaaccgggagttccttacggcggccgagc
tcaccgtgtggctggtcgacgaccctgaagggtggcagcgccgcctgcgtcggccggtgtggccgctgcggctgggc
cgcagccaggacctggtcggtatccgcaccggcctggttccgttgcgcgcggagcccggcgagcagcggtccgccgt
ggtgccggagacggcggggaggatgggaaccctactgcggctgccgactgcggtctctgggggccgggaccgtaccc
ggtgggacagctaccggttcgacagctcgggccgcagtgaccatgtggtcgtaggcggctggtcgactgccggggga
caggcagtcattctgctgccctcggcccatcccgataccgtcgcgcgttcctgatggttctgccgtcgggccgtacc
gatagggagcccatcgccactatgacggacgtcctgtccacgctgcgggccaagagcgctcaacgggggcgttctgc
ggaccttctcaccgcgcatttgtccgagactcgtgctgcggcagctgggctgcggcagcgtgtgggccgtctggacg
cggtggaggacgtcttcggcggcaggttctggcccgtcgtggaactcgctggcctcacccacgacgccggcaagatt
cccgaaggcttccagcggatgctggcgggatacagccgtgcctggggtgagcgtcacgaagtcgcctcgttgggctt
cctgcccgcgctcatcggcgacccggacgtgctgttgtgggtggcgaccgcggtcgccacccaccatcgtccgctga
ccggccagaacggacgcgacctgcagactctctacagcggtgtcaccatcaccgagctcgcgcaccgtttcgggcct
tttgacccacgcgctgtccccgccttggaggcctggcttcgtgcgagcgccatccgggtcggcctccccgcggccgc
tgttccagacgacggcacgctcaccgacaccggagtggtcgctggcgcccaccagctgctggaggagattttggacc
gttgggcagaccgtgtgaggcctgaggtgggcttggccgctgtactgctgcagggggcggtcaccctggccgaccac
ttgtcctccgcccatcaggctctgcccaccgtccagccgttgggggccgggttccggtcccggttggagaaggagtt
cgctgaacgcggcaggaccctgcgtgcccaccagctggaggccgccaccgttaccggacatcttctgctgcgcgggc
cgaccggcagtgggaagaccgaggctgccctgctgtgggctgccagccaggtcgaggccctgaaggcggaaggccgg
ggcgtgccgcgtgtgtttttcactctcccctacctggcctccatcaacgccatggcaacacggctgggtgacactct
cggcgatggtgaggctgtcggcgttgcccactcccgcgccgcctcctaccaccttgcccaggccatcgccccgcagg
acggcgacgaggaggacgaacacggagccccctgccgtgttgacgcggccgccaaggccttgtcccgggccgctgcc
accaagctgttccgcgagagtgtccgcgtcgccaccccctaccagcttctgcgggccgccctggccgggccggccca
ctccggcatcctcatcgacgccgcgaactcggtgttcatcctggacgaactccacgcctacgacgcccgcaggctcg
gctacatcctggccagtgcccggctgtgggaacgcctcggtggacggatcacagtcctgtccgcgaccctgcccagg
gccctggccgacctgttcgagagcaccctcaccgcccccatcaccttcctcgacacccccgacctcgggctgccggc
gcgccacctcctgcacacccgaggccaccatctcaccgacccggccacactggaggagatccgtctgcggctgtccc
gggacgagtcggtcctggtgatcgccaacaacgtgtcccaggccatcgccctgtacgaacagctcgcacccgacgtg
tgtgaacgcttcggtcaggacgccgcgctactgctgcactcccggtttcgacggatggaccggtcccggattgagca
gaagatcgccgaccggttcgccactgtggcacctgatgcccagaacagccgtaagccgggcctggtcgttgccacgc
aggtggtcgaggtcagtctcgacgtcgacttcgatgtgctgttcactggagcggctccgctcgaggccctcctgcag
cgcttcggccggaccaaccgcgtcggggcccgcccgccggccgacgtcatcgtccaccatcccgcctggaccacacg
ccgccgacagcccggcgagtacgccgacggcatctacccacgggagccggtcgagtccgcgtggcacatcctcaccc
gcaatcacgggcgagtcatcgacgaagcggacgccaccgcgtggctggacgaggtctacgccacggactggggcagg
caatggcaccgcgaggtgctggagcggcgagaaagattcgaccgtgcgttcctgcagttccgctaccccttcgaaga
ccgcactgacctggccgataccttcgacgaactcttcgacggctccgaagccatcctcgccgaagaccaggacgcct
actcagccgcactggccgcaccagacggcgaccaccccggagctggccggctcctcgcagaggaatacctcatcccc
gttccccactgggccagccccctcagccgctacgagaagcagctcaaagtccgcgtcatcaacggcgactaccaccc
cgaccacggcctcatggcggtccgggggctgccccagcccgcctaccgcgccggggaggtcttgtgatcgacaagga
ggacatcggcggcgtccacatcaagtacctctaccactgccgccgccagctgtggctgtacctgcgcggcatccgtc
ccgaacacctcagtgccaccgtccggtccggtgaagccgtccacgacacctcctacacccgcaacacccctgtcgac
ctcggcgcagcacgactcgacttcatcgacggcgcgcactgggtccacgagatcaaatcctccacacgccccagcct
ggccgatcaggcccagggccgccactactgtcaccggctgcgccgcatcggcatcgatgcccggggcgccgttctgc
actaccccaagactcggcgcacacagcgattcccctacacccccgaggctgccgcccaagccgaagaggacatcgcc
gaggccctcatcgttgccgctgctcaaagctccccggagcggctcaaccgctccgcctgccggggctgcagtttcac
cgactactgctggacggaatgagatgcccgctgccgcccaaacatactggctgaccagccccagtcgaatccgccga
aaagaccaatcgctgatcatcgaacgcgaaaccggcggcaacgtccatatccccattaccgatgttcgcgatatcat
cgcctgcgccgaggtcgacatcaacaccgcggtgatctcactgctcaaccagcaccgcatcaacattcacgtcctca
gctactacggcgactacgccggctccctgctcacctcagaaaccagcacctccggggacactgttctggcacaggca
agaatcgcccaggatcgcgatgcgagcctgaatattgcccgcaacatcattgactccaccgccttcaacgtgcgccg
cattgtcgaccgtaagctcctaagccgcccctacgccgtcctcaaaaactccatagtcaccgctgagagcccagccc
agctcatgggcgccgagggaacattccgccgctccgcctgggaagtcatagacaccaaactccccgactggctgcaa
ctcggcggccgcagccgccgcccgcccaagaacgccgggaacgccttcatcagctacgtcaacgggatcacgtatgc
gcgactcctcagcgccattcgtctcaccccactgcacagcggaatctcttttctccacagcagcatggaacgccagc
ggcactccttggtcttggacctctccgagatgttcaagcccctcttcgccgaacggctcctgctacgcatgtcagga
cgaaaccaacttaaggaacaccacttcgaccgcgacagcaaccaagcaatgctcagcgaggcgggtcgcaaactcgt
ggtccaaagtgtccgagacgaattcgccgtcaccgtagcgcaccgcagcctcggccgtaacgtcgcctacgacgaat
tgctgtacctggacgcgcttgccctaacccgccattgcctggaaggcgccgcctacaaacctttccgaatctggtgg
tgaacaccacgtgtatgtagtcgtcgtctacgacacccacgccaaacgaaacgcacggatcctgcgtacctgccgca
agtacctccaccacgtccagcgcagcgtcttcgaaggccacctcagcgacgcccaactccgacactttcaagctgca
gtcgaagatgccctcgacttggagtacgacaacgtcctcgtctacacgttcccagccggcaccgtgccacaacgcct
tgaatggggagcagtcgaacccgcccccagtgacatcctgtga
The primer of table 2 and editor's element sequences table
Primer name Primer Sequence (5, to 3) restriction enzyme site
28acas-AF aagaaggagatataccatggtggtcgccggtgccccgaacaac NdeI
28acas -AR cattatgcggccgcaagcttgcggcttacgactcacaggatgtcact BamHI
304cas -TF accatgattacgccaagcttgtggtcgccggtgccccgaacaac HindIII
304cas -TR gtacccggggatcctctagatgcggcttacgactcacaggatgtcact EcoRI
galMF gtgctgaacgaaactccc
galMR aatactcttcgccaggac
galMUF gcaagctcggttgcggccgcgctgatggactggggtgc NotI
galMUR gcggaatgccgccttcatcatcgttctggttcacaatctg
galMDF cagattgtgaaccagaacgatgatgaaggcggcattccgc
galMDR
galM-check-F
galM-check-R CCCAAGCTTtcagggtggttcgggctgt
cgcgacgaagcaacagcaatgct
gaccggcggcggatttggcg HindIII
amyE-F atgtttgcaaaacgattcaaaa
amyE -R tcaatggggaagagaaccgctta
amyE -UPF cgacggccagtgccaagcttcaaatcgaatgagcttacagc HindIII
amyE -UPR tcttacgtgccgatcaacgtctcagtttttaatttgtgtgtttccat
amyE -CmF atggaaacacacaaattaaaaactgagacgttgatcggcacgtaaga
amyE -CmR tcattggcatacgtatcatgcgataaatgaagttcctattccgaagttc
amyE -DownF gaacttcggaataggaacttcatttatcgcatgatacgtatgccaatga
amyE -DownR gcggccgcggatcctctagaaggataaagcacagctacagacc XbaI
SgRNA sequences Note
guide DNA -galM
guide DNA -pKD3-tEC-DNA gcaagctcggttgcggccgctaatgtgagttagctcactcattaggca
ccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaattgcatcagacatt
gccgtcactgcgtcttttacaccgggcaaggtttatcaactgtggcgcgataagtaatggttgcatcagacattgcc
gtcactgcgtcctagcataaccccttggggcctctaaacgggtcttgaggggttttttgAAGCTTCCC
gcaagctcggttgcggccgctaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttc
cggctcgtatgttgtgtggaattgtgagcggataacaattgcatcagacattgccgtcactgcgtccggatgagcat
tcatcaggcgggcaagaatgtgaataaagttgcatcagacattgccgtcactgcgtcctagcataaccccttggggc
ctctaaacgggtcttgaggggttttttgtttgcccgattgcggccgc NotI
HindIII
NotI
guide DNA -pKD3-tSV-DNA gcaagctcggttgcggccgctaatgtgagttagctcactcattaggca
ccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaagtcctcatcgcccc
ttcgaggggtcgcaaccggatgagcattcatcaggcgggcaagaatgtgaataaaggtcctcatcgccccttcgagg
ggtcgcaacctagcataaccccttggggcctctaaacgggtcttgaggggttttttgtttgcccgattgcggccgc
NotI
guide DNA -amyE aggtcgactctagaggatcctaatgtgagttagctcactcattaggcaccccaggct
ttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaattgcatcagacattgccgtcact
gcgtctgttagacagggcattgaatgacggggcagacggttttcgatttgcatcagacattgccgtcactgcgtcct
agcataaccccttggggcctctaaacgggtcttgaggggttttttgtatgacatgattacgaattc BamHI
EcoRI
Note：Capitalization is restriction enzyme site, and wave is protection base, and the overstriking of primer black matrix is complementary region, and single underscore is to open
Mover promoter, italic is spacer, and black matrix overstriking is repeat, and double underline is terminator terminator
Claims (6)
1. a kind of Virginia streptomycete IBL14 type I-B-sv14 type CAS gene editing systems, it is characterised in that：This is
System includes onecasExpression of recombinant proteins plasmid and a gene editing plasmid that six genes of 7-5-3-4-1-2 are constituted.
2. a kind of Virginia streptomycete IBL14 type I-B-sv14 type CAS gene editings system according to claim 1
System, it is characterised in that：Edit methods are comprised the following steps：
（1）According to Virginia streptomycete IBL14 genescas7-5-3-4-1-2 and relevant information primers, to extract
The Virginia streptomycete IBL14 genomes for arriving are masterplate, with TransTaq DNA Polymerase High Fidelity
Polymerase obtains end and carries and the identification of carrier restriction enzyme and cleavage site 15-25 by PCR amplification
Bp overlapping regionscas7-5-3-4-1-2 genes, are connected on plasmid plasmid, obtain protein expressing plasmid plasmid-cas7-5-3-4-1-2；
（2）It is masterplate with the prokaryotic gene group extracted according to target gene DNA sequence dna information design primer, uses
TransTaq DNA Polymerase High Fidelity archaeal dna polymerases react amplification respectively and obtain end band by PCR
Restrictive restriction endonuclease is recognized and the upper and lower homology arm PCR pieces of the target gene of cleavage site and overlap PCR complementary series
Section, and upper and lower homology arm is combined into structure gene editing masterplate template DNA/t-DNA with overlap PCR, while
The target gene of head and the tail promoter containing T7 and RNA terminators respectively is designed and is directly synthesized according to biological targeting gene sequence information
Fragment guide-DNA/g-DNA, gene editing masterplate and target gene fragment is connected to gene editing plasmid is obtained on plasmid
plasmid-t/gDNA；
（3）Prokaryote competence is prepared, and will be by step（1）The protein expressing plasmid that obtains and by step（2）Obtain
Various target gene editor plasmids be transformed into target bacterium competence respectively in obtain the recon after different gene editings, it is right
Restructuring daughter chromosome carries out PCR and gene sequencing and/or functional analysis, to confirm the purpose recon after editor.
3. a kind of Virginia streptomycete IBL14 type I-B-sv14 type CAS gene editings system according to claim 1
System, it is characterised in that：Described gene editing refers to using the protein expressing plasmid combination gene editing plasmid and/or other plasmids
The chromogene of prokaryotic can be knocked out, be inserted, seamless point mutation and any combination.
4. a kind of a kind of Virginia streptomycete IBL14 type I-B-sv14 type CAS gene editings as claimed in claim 1
System, it is characterised in that it can be used for prokaryotic gene editor and is immunized.
5. a kind of Virginia streptomycete IBL14 type I-B-sv14 type CAS gene editings system according to claim 4
System, it is characterised in that：The prokaryotes refer to Escherichia coli, hay bacillus and other various prokaryotic micro-organisms.
6. a kind of Virginia streptomycete IBL14 type I-B-sv14 type CAS gene editings system according to claim 4
System, it is characterised in that：It is outer that described immune finger can be applied to biological cell prevention by the CRISPR that gene editing masterplate is designed
The intrusion of source nucleic acid.
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| CN107630041A (en) * | 2017-09-19 | 2018-01-26 | 安徽大学 | A kind of eukaryotic gene edit methods based on Virginia streptomycete IBL14 I Type B Cas systems |
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| CN110438141A (en) * | 2019-05-13 | 2019-11-12 | 安徽大学 | A kind of gene editing method based on SviCas6-SviCas3 |
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| CN107142272A (en) * | 2017-06-05 | 2017-09-08 | 南京金斯瑞生物科技有限公司 | A kind of method for controlling plasmid replication in Escherichia coli |
| CN107557373A (en) * | 2017-09-19 | 2018-01-09 | 安徽大学 | A kind of gene editing method based on I Type B CRISPR Cas system genes cas3 |
| CN107630041A (en) * | 2017-09-19 | 2018-01-26 | 安徽大学 | A kind of eukaryotic gene edit methods based on Virginia streptomycete IBL14 I Type B Cas systems |
| WO2019056848A1 (en) * | 2017-09-19 | 2019-03-28 | 安徽大学 | Type i-b crispr-cas system gene cas3-based gene editing method |
| EP3556860A4 (en) * | 2017-09-19 | 2020-09-02 | Anhui University | Type i-b crispr-cas system gene cas3-based gene editing method |
| US11286506B2 (en) | 2017-09-19 | 2022-03-29 | Anhui University | Type I-B CRISPR-Cas system gene Cas3-based gene editing method |
| CN110438142A (en) * | 2019-05-13 | 2019-11-12 | 安徽大学 | A kind of transcriptional control method based on SviCas5-6-7 in I-B-Svi type CRISPR-Cas system |
| CN110438141A (en) * | 2019-05-13 | 2019-11-12 | 安徽大学 | A kind of gene editing method based on SviCas6-SviCas3 |
| CN115851664A (en) * | 2022-09-19 | 2023-03-28 | 中国药科大学 | I-B type CRISPR-Cascade-Cas3 gene editing system and application |
| CN115851664B (en) * | 2022-09-19 | 2023-08-25 | 中国药科大学 | I-B CRISPR-Cascade-Cas3 gene editing system and application |
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