CN110438142A - A kind of transcriptional control method based on SviCas5-6-7 in I-B-Svi type CRISPR-Cas system - Google Patents
A kind of transcriptional control method based on SviCas5-6-7 in I-B-Svi type CRISPR-Cas system Download PDFInfo
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- CN110438142A CN110438142A CN201910420999.8A CN201910420999A CN110438142A CN 110438142 A CN110438142 A CN 110438142A CN 201910420999 A CN201910420999 A CN 201910420999A CN 110438142 A CN110438142 A CN 110438142A
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Abstract
The invention discloses a kind of transcriptional control methods based on 1 class I type CRISPR-Cas system.SviCas5 in the crRNA guidance I-B-Svi type CRISPR-Cas system generated after targeting g-DNA transcription is utilized for the first time, the Trinitarian R-loop compound that the Cascade and target DNA that SviCas6 and SviCas7 are formed are formed realizes and carries out accurate, quickly and effectively gene transcription regulation to cellular genome.The exploitation of this transcriptional control tool proposes challenge and supplement to the transcriptional control based on 2 class CRISPR-Cas systems.
Description
The present invention relates to the technique for gene engineerings of field of biotechnology, are exactly a kind of based on I-B-Svi type
The transcriptional control method of SviCas5, SviCas6 and SviCas7 in CRISPR-Cas system.
Background technique
Gene regulation refers to the controlling mechanism of biological gene expression in vivo, mainly includes that the synthesis of (1) semiconservative DNA replication is new
Regulation (synthesis regulation), (2) DNA on DNA level are transcribed into the regulation (transcriptional control) in messenger RNA mRNA level in-site
(3) mRNA translates into the regulation (translational control) (being often related to the modification after protein translation in eukaryon) on protein level.
Biology changes metabolic way by gene regulation to adapt to the variation of environment, is that molecular genetics and the important of development genetics are ground
Study carefully field.Present invention is primarily concerned be using I type CRISPR-Cas system to biological cell carry out mRNA level in-site on transcription
Regulation.
The transcriptional control principle of I type CRISPR-Cas system is: the crRNA guidance Cascade and target that targeting g-DNA is generated
Mark DNA forms Trinitarian R-loop compound, and in the case where absence of endonuclease Cas3, specificity interference transcription is prolonged
Stretch, RNA polymerase combine or transcription factor combine [Yibei Xiao, Min Luo, Adam E.Dolan, Maofu Liao,
Ailong Ke.(2018)Structure basis for RNA-guided DNA degradation by Cascade and
Cas3.Science (New York, N.Y.) 361, doi:10.1126/science.aat0839；Yibei Xiao, Min
Luo, RobertP.Hayes, Jonathan Kim, Sherwin Ng, Fang Ding, Maofu Liao, and Ailong Ke
(2017)Structure Basis for Directional R-loop Formation and Substrate Handover
Mechanisms in Type I CRISPR-Cas System.Cell 170,48-60 ell, doi:10.1016/
j.cell.2017.06.012].It has been found that CRISPR-Cas system be divided into two classes, six type, wherein 1 class I type CRISPR-Cas
System is the most extensive.So far, carrying out gene regulation report using CRISPR-Cas system is preferably at most 2 class II type CRISPR-
In Cas system dCas9 [Matthew H Larson, Luke A Gilbert, Xiaowo Wang, Wendell A Lim,
Jonathan S Weissman&Lei S Qi(2013)CRISPR interference(CRISPRi)for sequence-
Specific control of gene expression.Nat Protoc 8,2180-2196, doi:10.1038/
nprot.2013.132].In addition, the mRNA that 2 class VI type Cas13a (C2c2 of the past) can also be used for specificity knocks out [Omar
O.Abudayyeh, Jonathan S.Gootenberg, Silvana Konermann, Julia Joung, Ian
M.Slaymaker, David B.T.Cox, Sergey Shmakov, Kira S.Makarova, Ekaterina Semenova,
Leonid Minakhin, Konstantin Severinov, Aviv Regev, Eric S.Lander, Eugene
V.Koonin, and Feng Zhang (2016) C2c2 is a single-component programmable RNA-
Guided RNA-targeting CRISPR effector.Science (New York, N.Y.) 353, aaf5573, doi:
10.1126/science.aaf5573]。
Gene editing is carried out about application SviCas3 combination SviCas5, SviCas6 and SviCas7 to have been reported by us
A kind of [prokaryotic gene editor derived from I type Cas 4 cas genes of system of Tong Wangyu, Cao Suli, Yang Xingwang (20170919)
Method, application number: 201710847172.6；Tong Wangyu, Wang Anjing, Sun Yan (20170919) are a kind of to be based on Virginia streptomycete
The eukaryotic gene edit methods of IBL14 I-B type Cas system, application number: 201710847164.1], but applying SviCas5,
The SviCas6 gene regulation that g-DNA is carried out in conjunction with SviCas7 has not been reported.It is reported that: in I-E type CRISPR-Cas system
In, Cas5, Cas6 and Cas7 molecule participate in the formation of Cascade compound jointly, also participate in the production (RNA shearing) of crRNA
And promote combination [Prarthana Mohanraju, the Kira S.Makarova, Bernd of crRNA and target DNA sequence
Zetsche, Feng Zhang, Eugene V.Koonin, John van der Oost (2016) Diverse
evolutionary roots and mechanistic variations of the CRISPR-Cas
Systems.Science (New York, N.Y.) 353, aad5147, doi:10.1126/science.aad5147；Yibei
Xiao, Min Luo, Adam E.Dolan, Maofu Liao, Ailong Ke. (2018) Structure basis for RNA-
Guided DNA degradation by Cascade and Cas3.Science (New York, N.Y.) 361, doi:
10.1126/science.aat0839].It has been observed that SviCas5 and SviCas6 can be to target in conjunction with g-DNA with SviCas7
Gene carries out effective transcriptional control.
The present invention it is first public it is a kind of with SviCRISPR feature in I-B-Svi subtype C RIPSPR-Cas system (g-DNA's
Protospacer adjacent motif/PAM selection) and SviCas5, SviCas6 and SviCas7 amino acid sequence based on
Design regulates and controls carrier Vector by protein expressioniThe transcriptional control method that-cas6-7-5-g-DNA is executed.We utilize targeting
The Cascade and target DNA that crRNA the guidance SviCas5, SviCas6 and SviCas7 generated after g-DNA transcription is formed is formed
Trinitarian R-loop compound to change the transcriptional level of target gene and the expression of albumen, and then changes cell
Physiological and biochemical property.There is nuclear membrane due to eucaryote and only carry out protein translation in a manner of monocistron, thus it is of the invention
Eukaryotic protein expression regulation carrier in base optimization after 3 cas genes be respectively provided with transcripting promoter, terminator
And nuclear localization sequence.The appearance of this transcriptional control tool proposes the transcriptional control of dCas9 and Cas13a (C2c2 of the past)
Supplement and challenge, while foundation is provided further to develop other new transcriptional control tools.
Summary of the invention
The technical problem to be solved in the present invention is to provide one kind based on SviCas5- in I-B-Svi type CRISPR-Cas system
The transcriptional control method of 6-7, to realize that it changes the function of cell nongenetic.
For this purpose, the technical scheme adopted by the invention is that:
A kind of transcriptional control method based on SviCas5-6-7 in I-B-Svi type CRISPR-Cas system, feature exist
In: protein is separately encoded containing 3 genes cas5, cas6 and cas7 in the streptomycete IBL14 genome of Virginia
SviCas5, SviCas6 and SviCas7 (protein sequence is seen attached list), three protein combine targeting after expressing in organism
DNA (guide-DNA/g-DNA: by transcripting promoter/promoter, repetitive sequence/repeat, intervening sequence/spacer and turns
Record terminator/terminator composition；Wherein intervening sequence is the DNA fragmentation complementary with target gene particular sequence) or with target base
Because having the template DNA (template DNA/t-DNA) of complementary series that can have to the genetic transcription in organism genome
Effect regulation.
A kind of transcriptional control method based on SviCas5-6-7 in I-B-Svi type CRISPR-Cas system, it is special
Sign is:
(1) regulate and control the building of carrier
According to SviCas5, SviCas6 and SviCas7 amino acid sequence synthesizes the DNA sequence of the base optimization of corresponding cell
Column, and g-DNA and expression vector dna primers are combined, through expression vector establishment, (PCR amplification and ligase respectively will
Protein coding gene cas6-7-5 and g-DNA are connect with expression vector) obtain the regulation carrier Vector of expression albumeni-cas6-
7-5-g-DNA。
(2) acquisition and inspection of gene transcription regulation transformant
By the method for conversion, transfection or electrotransformation, carrier Vector will be regulated and controled by the protein expression that step (1) obtainsi-
Cas6-7-5-g-DNA is imported in biological cell, and selection culture obtains candidate gene transcription regulation transformant；Candidate is converted
Son carries out Function Identification, target sequence PCR and sequencing analysis, obtains correct gene transcription regulation transformant.
SviCas5, SviCas6 and the SviCas7 amino acid sequence refer to it is original or engineered after
SviCas5, SviCas6 and SviCas7 amino acid sequence.
The gene transcription regulation transformant, which refers to, leads to target protein expression levels because of the regulation of gene transcription level
Up-regulation is lowered or is not expressed, and then the apparent of host cell, function and the changed transformant of physiology.
The DNA sequence dna of the corresponding base optimization refers to according to SviCas5, SviCas6 and SviCas7 amino acid sequence
And the cDNA sequence for being suitable for expressing in different cells optimized.
The organism refers to any one of protokaryon and eucaryote, such as: Escherichia coli, corynebacterium glutamicum,
Saccharomyces cerevisiae, mouse embryonic fibroblasts.
Beneficial effect
The present invention is a kind of transcriptional control method based on SviCas5-6-7 in I-B-Svi type CRISPR-Cas system.It answers
With SviCas5, SviCas6, SviCas7 combination g-DNA, I-B-Svi type CRISPR-Cas system is realized for the first time to biology base
Because of the orientation transcriptional control of gene in group, also for based on base constructed by dCas9 and Cas13a in II type CRISPR-Cas system
Because regulator control system provides new supplement and selection.The system can carry out gene in protokaryon and eukaryotic gene group effective
, accurately, the gene transcription regulation of nongenetic.
Specification subordinate list, attached drawing
The amino acid sequence of subordinate list, SviCas5, SviCas6 and SviCas7
Fig. 1, transcriptional control carrier pRS415i- cas6-7-5-g-crtE structure figures.Ori/origin:DNA replication initiation
Site；The replication origin of f1 ori:f1 bacteriophage origin；CEN/ARS: centriole/autonomous duplication；AmpR/
Ampicillin resistance: amicillin resistance；LEU2 promoter: leucine promoter.
The regulation result of crtE gene in Fig. 2, SC BY4741 genome.(A) (a: red is original bacteria for erythroleukoplakia screening
Strain, b: white is to regulate and control successfully bacterial strain)；(B) crtE gene PCR electrophoresis (M:5000bp DNA ladder, 1: blank control, 2:
CrtE gene PCR in original SC BY4741 genome, 3~6:pRS415i- cas6-7-5-g-crtE transcriptional control converts subbase
Because of a group PCR product).(red disappears, and through crtE-vF/vR primer amplification, DNA band and original strain crtE Genome Size
It is unanimously 3072bp, shows to regulate and control successfully.)
Specific embodiment
In order to more fully understand technology contents of the invention, technical solution of the present invention is made combined with specific embodiments below
It is further described and illustrates, it is intended to preferably explain that the contents of the present invention, following embodiment do not limit the scope of the invention.
In addition, being all made of following material unless otherwise instructed in listed embodiment:
1) bacterial strain, cell and plasmid
Escherichia coli Escherichia coli DH5 α/EC DH5 α, Escherichia coli JM109 (DE3)/EC
JM109 (DE3), corynebacterium glutamicum Corynebacterium glutamicum B253/CG B253；Saccharomyces cerevisiae
Saccharomyces cerevisiae BY4741/SC BY4741；Mouse embryonic fibroblasts/NIH3T3, plasmid pCas,
PEC-XK99E, pRS415, pAIO.
2) solution and culture medium
1.0M LiAc solution
The LiAc solid powder for weighing 6.6g adds 90mL distilled water to dissolve, is settled to 100mL.
100mM LiAc solution
The LiAc solid powder for weighing 0.66g adds 90mL distilled water to dissolve, is settled to 100mL.
400mL LiAc/DTT/TE buffer
0.48456g Tris (10mM, pH7.5), 4.088g LiAc2H2O (0.1M), 0.1169g EDTA (1mM),
300mL ddH2O dissolution, HCl tune pH to 7.5 are settled to 400mL (being added after 0.617g DTT filtration sterilization).
HEPES buffer solution
Weigh 0.8g NaCl, 0.0135g Na2HPO4.H2O, 0.037g KCl, 0.5g glucose, 0.5g HEPES add
The dissolution of 90mL distilled water, NaOH tune pH to 7.5 are settled to 100mL.
50mg/mL ampicillin
The ampicillin powder for weighing 0.5g is placed in 10mL centrifuge tube, and 8mL ddH is added2O, it is fixed after mixing well
Appearance be dispensed into the EP pipe of 2mL after 0.2 μm of sterile water system membrane filtration to 10mL, be put in -20 DEG C of refrigerators save it is standby
With.
10mg/mL l-Alanine titer
L-Alanine 1g is weighed, is dissolved with a small amount of distilled water, 100mL volumetric flask constant volume, room temperature preservation is stand-by.
LB culture medium
NaCl 10g, yeast powder 5g, peptone 10g, be added it is appropriate single steam water dissolution after, be settled to 1L, pH is adjusted to 7.0~
7.2, after packing wrapping, 15g agar is added in 121 DEG C/20min sterilizing, plate such as processed.
LBPET culture medium (1000mL LB culture medium)
20mM MgCl2·6H2O, 40mg/L arabinose, that is mould for 50mg/L X-gal, 50mg/L IPTG, 100mg/L card
Element and 100mg/L apramycin (keep out of light)
LBG culture medium (1000mL)
Yeast extract 5g, peptone 10g, NaCl 10g, glucose 5g are settled to 1L, pH after appropriate single steaming water dissolution is added
7.0, after packing wrapping are adjusted to, 115 DEG C/30min sterilizing.
LBGT culture medium
Yeast extract 5g, peptone 10g, NaCl 10g, glycine 30g, Tween80 1g are settled to 1L, pH is adjusted to 7.0-
7.2 115 DEG C/30min sterilizing.
LBHIS culture medium (1000mL)
Sorbierite 91g, yeast extract 2.5g, peptone 5g, NaCl 5g, brain heart infusion 18.5g are settled to 1L, 121 DEG C/
15g agar is added in 20min sterilizing, plate such as processed.
YNB culture medium
Peptone 20g, yeast extract 10g, glucose 20g after appropriate tap water dissolution is added, are settled to 1L, packing wrapping
Afterwards, 115 DEG C/30min sterilizes, and 15g agar is added in plate such as processed.
SD-LEU Selective agar medium (100mL)
Appropriate ddH is added in 0.5g ammonium sulfate, 2g glucose, 0.17gYNB2After O dissolution, it is settled to 99.6mL, packing packet
After bundle, 115 DEG C/30min sterilizing.400 μ l amino acid (methionine 0.5g/100mL, histidine 0.5g/ of filtration sterilization are added
100mL, uracil 0.5g/100mL), 2g agar and 18.2g sorbierite is added in plate such as processed.
SCCM culture medium (1 liter DMEM)
100mL fetal bovine serum, 100mg penicillin, 100mg streptomysin, 10g glutamine
DMEM in high glucose culture medium (GibcoTM)
Purchased from Thermo Fisher Scientific Inc., 168 Third Avenue, Waltham, MA USA
02451。
Agents useful for same is commercially available product.
PCas in embodiment 1, EC JM109 (DE3)iThe transcriptional control of-cas6-7-5-g-lacI
(1) gene transcription regulation carrier pCasiThe building of-cas6-7-5-g-lacI
(A) preparation of cas6-7-5-g-lacI segment
Target gene segment g-lacI (table 1) is directly synthesized by the general biotech firm in Chuzhou, and stem is with pCas plasmid
Complementary series, tail portion have plasmid cas5 complementary series, and centre is successively lactose operon promoter, repeat (30bp),
Spacer (40bp), repeat (30bp) and terminator, selection ttc are the site PAM.Cas6-7-5 genome sequence from
PCR amplification obtains on SVIBL14 genome, and primer cas6-F/pCas-R (table 1) makes cas6-7-5 head end with pCas plasmid
Complementary series, tail end have the complementary series of gRNA.Cas6-7-5 connect that [PCR is anti-with g-lacI segment by overlap PCR
Answering system (50 μ L) includes: 5 μ 10 × Pfu of l buffer, 5 μ l dNTPs (2.5mM each), 1 10 μM of μ l cas6-F, 1 μ l
10 μM of pCas-R, 5 μ l DMSO, 0.5 μ l Pfu DNA Polymerase, 0.5 μ l SV IBL14 genomic DNA, 32 μ l without
Bacterium water (nuclease-free), reaction condition: 95 DEG C of 10min, 95 DEG C of 30s, 61 ± 2 DEG C of 30 ± 30s, 72 DEG C of 90 ± 30s,
The Pfu DNA Polymerase of 2.5U Sangon Biotech (Shanghai) Co., Ltd. production, 30 circulations, 72 DEG C
10min].PCR product is detected through 1% agarose electrophoresis, kit recycling, the cas6-7-5-g-lacI segment purified.
(B) carrier pCasiThe building of-cas6-7-5-g-lacI
The cas6-7-5-g-lacI segment that purifying obtains is connected (seamless gram with pCas plasmid backbone by one-step cloning
Grand reaction system includes: 4 μ l 5 × CE II buffer, 1 μ l pCas plasmid backbone, the cas6-7-5-g-lacI of 3 μ l amplification,
2 μ L Exnase II, 10 μ L sterile waters are placed in 37 DEG C of reactions 30min, ice bath 5min) then connection product is converted to EC
In DH5 α competence, bacterium colony PCR is carried out by primer pCas-F/cas6-R (table 1) after monoclonal is grown, by PCR positive bacteria
Overnight incubation in the remaining colony inoculation fallen to the LB liquid medium containing Kana resistance extracts plasmid identification and obtains pCasi-
Cas6-7-5-g-lacI plasmid.
Primer involved in the present embodiment and the g-lacI sequence of synthesis are as shown in table 1.Dotted line represents complementary series in table；
Single underscore represents promoter；Double underline represents terminator；Tilted letter represents spacer；Bold-type letter represents repeat.
1 primer of table and g-lacI sequence
(2) gene transcription regulation transformant EC JM109 (DE3)-g-lacIiBuilding and verifying
(A) prepared by EC JM109 (DE3) competence
By sterile working specification with the Escherichia coli clones on toothpick picking plate in 30 ± 10mL LB liquid medium
In, 37 ± 5 DEG C, 220 ± 100rpm is incubated overnight.Transferring by 1% inoculum concentration, this is incubated overnight liquid to new 30 ± 10mL LB
In fluid nutrient medium, 37 ± 5 DEG C, 220 ± 100rpm is cultivated again to bacterium solution OD600Value about 0.5 ± 0.2 or so (2 ±
1.5h), culture solution is gone in 50mL centrifuge tube, and 4000 ± 1000rpm is centrifuged 8min at 4 DEG C, supernatant is removed, with being pre-chilled on ice
The SSCS solution 1mL for the Generay Biotech company production crossed blows and beats the thallus of precipitating uniformly to get the sense of Escherichia coli
By state cell, (this process whole process carries out on ice) is saved backup at -80 DEG C.
(B) plasmid pCasi- cas6-7-5-g-lacI conversion
By 10 μ l plasmid pCasi- cas6-7-5-g-lacI is added in 100 μ l EC JM109 competence, is uniformly mixed,
It is uniformly coated in LBPET solid medium in 30 ± 10min on ice, 45 ± 5 DEG C of water-bath 90s then at 15 ± 5min on ice,
30 ± 2 DEG C are incubated overnight 24-36h, obtain candidate transformant.
(C) subfunction and genetic analysis are converted
Extracting white monoclonal genomic DNA is template, and lacI-vF/lacI-vR (table 1) is that verifying primer is carried out with real
Apply the Standard PCR of example 1 (1-A), the lacI in amplification gene group.Product is through 1% agarose gel electrophoresis and sequencing (general biology
System (Anhui) Co., Ltd) it analyzes, gene lacI gene in gene lacI and starting strain EC JM109 (DE3) in transformant
Sequence is completely the same, shows the success of lacI gene regulation.
PEC-XK99E in embodiment 2, CG B253iThe transcriptional control of-cas6-7-5-g-ldh
(1) gene transcription regulation carrier pEC-XK99EiThe building of-cas6-7-5-g-ldh
(A) preparation of cas6-7-5-g-ldh segment
Except the sequence of target gene segment g-ldh and pEC-XK99E plasmid, and the corresponding primer pEC-cas6-F/ of design
R, pEC-F, cas675-R and ldh-vF/vR are outer (table 2), remaining step is the same as embodiment 1 (1-A).
(B) plasmid pEC-XK99EiThe building of-cas6-7-5-g-ldh
Except cas6-7-5-g-ldh segment is connect with pEC-XK99E by one-step cloning, verifying primer is pEC-F/
Outside, remaining step is the same as embodiment 1 (1-B) by cas675-R (table 2).
Primer involved in the present embodiment and g-ldh sequence are as shown in table 2.Dotted line represents complementary series in table；Single lower stroke
Line represents promoter；Double underline represents terminator；Tilted letter represents spacer；Bold-type letter represents repeat.
2 primer of table and g-ldh sequence
(2) gene editing transformant CG B253-g-ldhiBuilding and verifying
(A) preparation of CG B253 competence
It is trained with the CG B253 monoclonal that sterile toothpick aseptically picks from the plate in 30 ± 10mL LBG seed
It supports in base, 30 ± 3 DEG C, 220 ± 100rpm is incubated overnight.Fresh 50mL LBGT Liquid Culture is forwarded to by 1% inoculum concentration
In base, 30 ± 5 DEG C, 220 ± 100rpm is cultivated to bacterium solution OD600Value be about 0.6~0.9 or so (4 ± 1h), be transferred to 50mL from
In heart pipe, ice bath 10-15min, 4000 ± 1000rpm is centrifuged 8min at 4 DEG C, removes supernatant, with 10% glycerol of pre-cooling by bacterium
Uniformly, 4000 ± 1000rpm is centrifuged 8min to the piping and druming of body precipitating at 4 DEG C, washes repeatedly 3 ± 1 times, finally pre- on ice with 1 ± 0.5mL
Bacterial sediment is blown and beaten uniformly CG B253 competent cell to obtain the final product by 10% cold glycerol, is saved backup or is directly used at -80 DEG C
It converts (this process whole process carries out on ice).
(B) plasmid pEC-XK99EiThe conversion of-cas6-7-5-g-ldh
By 6~10 μ l plasmid pEC-XK99Ei- cas6-7-5-g-ldh is added to 80~100 μ l CG B253 competence
In, gently piping and druming makes its mixing, and 10 ± 5min of ice bath is added in the electric revolving cup of the 0.2cm of pre-cooling, 1.8kv, and 5ms electric shock adds
Enter 900 ± 100 μ l LBHIS culture mediums, mixes 46 DEG C of water-bath 6min, 30 DEG C of 100rpm cultivate 2 ± 1h, containing chloramphenicol
30 DEG C of culture 24-48h in LBHIS solid medium obtain CG B253 containing pEC-XK99EiThe candidate of-cas6-7-5-g-ldh turns
Beggar CG B253-g-ldhi。
(C) subfunction and genetic analysis are converted
To candidate transformant CG B253-g-ldhiCarry out fermentation and HPLC analysis, after fermentation optimization alanine yield from
0.06g/L is increased to 0.67g/L；Simultaneously with candidate transformant CG B253-g-ldhiGenome is template, ldh-vF/vR is to draw
Object (table 2) carries out PCR amplification, by the genetic analysis method of same embodiment 1 (2-C), finds candidate transformant gene ldh and goes out
Gene ldh gene order is completely the same in bacterium germination strain CG B253, shows the success of ldh gene regulation.
PRS415 in embodiment 3, SC BY4741iThe transcriptional control of-cas6-7-5-g-crtE
(1) gene transcription regulation carrier pRS415iThe building of-cas6-7-5-g-crtE
(A)pRS415iThe building of-cas6-7-5
(a) preparation of the cas6 genetic fragment with promoter, terminator and nuclear localization sequence
The cas6 gene optimized using pMD-18T plasmid and base is template, with what is optimized according to pMD-18T and yeast base
Cas6 gene order designs and synthesizes cas6-yF/yR the and pMD-18T-cas6-yF/yR primer (table 3) of specificity, with same reality
The PCR method for applying example 1 (1-A) expands pMD-18T plasmid backbone and the cas6 gene containing TEF2p and TEF2t respectively.It will purifying
The pMD-18T and cas6 of recycling carry out one-step cloning seamless connection by same 1 (1-B) method of embodiment.Connection product convert to
In EC DH5 α competence, the monoclonal grown is cultivated through PCR and sequencing analysis, positive bacterium colony passes through the LB solid culture containing Amp again
Overnight incubation in base, extracting and purifying must construct successful pMD-18T-cas6 plasmid.Using pMD-18T-cas6 as template,
PRS415-cas6-F/pRS415-cas6-R is that primer (table 3) is expanded with the PCR method of embodiment 1 (1-A) again, and product is pure
DNA fragmentation TEF2p-cas6-TEF2t is obtained after change.
(b) preparation of the cas7 genetic fragment with promoter, terminator and nuclear localization sequence
Except the cas7 gene order according to CDC19p, CDC19t and the optimization of yeast base, and the corresponding primer cas7- of design
YF/yR, pMD-18T-cas7-yF/yR, pRS415-cas7-F/R are outer (table 3), DNA fragmentation CDC19p-cas7-CDC19t synthesis
In remaining step with embodiment 3 (1-A-a).
(c) preparation of the cas5 genetic fragment with promoter, terminator and nuclear localization sequence
Except the cas5 gene order according to ENO2p and ENO2t and the optimization of yeast base, and the corresponding primer cas5- of design
YF/yR, pMD-18T-cas5-yF/yR, pRS415-cas5-F/R are outer (table 3), in DNA fragmentation ENO2p-cas5-ENO2t synthesis
Remaining step is the same as embodiment 3 (1-A-a).
(d)pRS415iThe building of-cas6-7-5 plasmid
With TEF2p-cas6-TEF2t after purification, CDC19p-cas7-CDC19t, ENO2p-cas5-ENO2t are template,
PRS415-cas6-F/pRS415-cas5-R is primer (table 3), is expanded by the overlap PCR method of same embodiment 1 (1-A)
Increasing connection, (20 μ L amplification systems include: 4 μ l 5 × CE II buffer, 1 μ l TEF2p-cas6-TEF2t, 1 μ l CDC19p-
Cas7-CDC19t, 1 μ l ENO2p-cas5-ENO2t, 2 μ l Exnase II, 11 μ l sterile waters) obtain cas6-7-5 segment.With
PRS415 is template, and pRS415-cas675-F/pRS415-cas675-R is primer (table 3), with the PCR of same embodiment 1 (1-A)
Method expands plasmid pRS415 skeleton.Cas6-7-5 segment and plasmid pRS415 skeleton embodiment 1 (1-A) after purification
After the connection of overlap PCR method, convert into EC DH5 α competence.Select monoclonal and correct through PCR and sequence verification
Afterwards, pRS415 is obtainedi- cas6-7-5 plasmid.
(B) plasmid pRS415iThe building of-cas6-7-5-g-crtE
Except the sequence of target gene segment g-crtE and pRS415 plasmid, and the corresponding primer pRS415- designed and synthesized
Cas675-g-F/R and pRS415-vF/vR is outer (table 3), plasmid pRS415iRemaining step of-cas6-7-5-g-crtE building is same
Embodiment 1 (1-A).Finally obtain the successful pRS415 of buildingi- cas6-7-5-g-crtE plasmid.(attached drawing 1)
Primer involved in the present embodiment and g-crtE sequence are as shown in table 3.Dotted line represents complementary series in table；Single lower stroke
Line represents promoter；Double underline represents terminator；Tilted letter represents spacer；Bold-type letter represents repeat.
3 primer of table and g-crtE sequence
(2) gene transcription regulation transformant SC BY4741-g-crtEiBuilding and verifying
(A) prepared by SCBY4741 competence
It picks from the plate red single bacterium to fall in 50mL YPD culture medium, shaken cultivation is to OD600 at 30 ± 3 DEG C
1.3-1.5；Place 10min on ice；In transposition 50mL sterile centrifugation tube, 4000rpm is centrifuged 8min, abandons supernatant；Cell is resuspended
In 25mL ddH2In O, the speed of 4000rpm is centrifuged 5min；Supernatant is abandoned, 25mL 1M sorbierite, 4000rpm is added in sediment fraction
Centrifuge washing 8min, the precipitating after washing are first added the suspension of 2mL LiAc/DTT/TE buffer, add the 25mL buffer,
With ParafilmTM, 30 ± 3 DEG C of culture 30min, thalline were collected by centrifugation by rear 4000rpm 5min；Thallus 1mL ddH2O suspends
And it is forwarded in 2mLEP pipe, 4000rpm 3min centrifugation；Precipitating is suspended with 1mL ice sorbierite, and 4000rpm 3min is centrifuged again
Washing, cell after washing are added 100-200 μ l sorbierite and suspend and dispense up to competent cell, be placed in -80 DEG C of preservations to
With (whole operation carries out under the conditions of 4 DEG C).
(B)pRS415i- cas6-7-5-g-crtE conversion
Draw 5 μ l pRS415i- cas6-7-5-g-crtE is added in 50 μ l SCBY4741 competence, after mixing well
It goes in 0.2cm electricity revolving cup, carries out electricity after standing 8-10min on ice and turn (condition: 1.5 ± 0.5kV, 5ms), then turn at once
Into the test tube equipped with 950 μ l pre-cooling sorbierite；100 μ l are taken to be coated on the selection plate of SD-LEU defect, in 30 DEG C of insulating boxs
2-4d is cultivated, transformant SC BY4741-g-crtE is obtainedi。
(C) subfunction and genetic analysis are converted
The colourless monoclonal transformant SC BY4741-g-crtE of pickingi, and extract its genomic DNA as template, crtE-
VF/vR is primer (table 3) by PCR amplification is carried out, and by the genetic analysis method of same embodiment 1 (2-C), finds transformant gene
Gene crtE gene order is completely the same in crt and starting strain SC BY4741, shows crtE gene regulation success (attached drawing 2).
Embodiment 4, pAIOiTranscriptional control of-the cas6-7-5-g-vegfa in NIH3T3
(1) plasmid pAIOiThe building of-cas6-7-5-g-vegfa
(A) plasmid pAIOiThe building of-cas6-7-5
Except according to cas6, the cas7 for carrying out base optimization by mammalian cell, cas5 sequence and pAIO plasmid sequence information are set
Meter synthesizes corresponding primer pMD-18T-cas6-mF/R, cas6-mF/R, pAIO-cas6-mF/R, cas7-mF/R, pMD-18T-
Cas7-mF/R, pAIO-cas7-mF/R, cas5-mF/R, pMD-18T-cas5-mF/R, pAIO-cas5-mF/R and synthesis are drawn
Object pAIO-cas675-mF/R is used to amplify the gene of cas6-7-5, and primer pAIO-F/R has for expanding with cas6-7-5
The pAIO plasmid backbone of complementary series, pAIO-vF/vR are used to verify the pAIO of buildingi- cas6-7-5 plasmid is outer (table 4), plasmid
pAIOiRemaining step of-cas6-7-5 building is the same as embodiment 3 (1-A).
(B) plasmid pAIOiThe building of-cas6-7-5-g-vegfa
It is directly closed by the method for same embodiment 1 (1-A) by the general biotech firm in Chuzhou according to gene vegfa sequence information
At g-vegfa (table 4).Target gene the segment g-vegfa and pAIO of synthesisi- cas6-7-5-vegfa by Hind III and
EcoR I restriction enzyme cuts out cohesive end, is then connected to pAIO by T4 ligasei-cas6-7-5-vegfa
On, connection product is converted into EC DH5 α competence, positive bacterium colony verifying is selected, finally obtains the correct pAIO of verifyingi-
cas6-7-5-g-vegfa。
Primer involved in the present embodiment and the g-vegfa sequence of synthesis are as shown in table 4.Capitalization represents digestion in table
Site；Dotted line represents complementary series；Single underscore represents promoter；Double underline represents terminator；Tilted letter represents
space；Bold-type letter represents repeat.
4 primer of table and g-vegfa sequence
(2) gene transcription regulation transformant pAIOi-cas6-7-5-g-vegfaiBuilding and verifying
5 ± 1 × 10 are added in each hole of 6 orifice plates5A NIH3T3 cell and SCCM (every hole 2mL), 37 DEG C of 5%CO2Training
It supports in case and cultivates 8-12h (cell confluency rate 70-90%), Lip2000-DMEM mixed liquor (5 μ l Lip2000 are added
Transfection Reagent and 25 μ l DMEM) and vector-DMEM mixed liquor (2 μ g pAIOi-cas6-7-5-g-
125 μ l DMEM of vegfa and), 37 DEG C of 5%CO2Continue in incubator cultivate 48h (SCCM replaces secondary daily) be added afterwards it is fast
Purine mycin (1 μ g/mL) continues to cultivate 6-8d (SCCM is replaced once daily), obtains transformed cells pAIOi-cas6-7-5-g-
vegfai。
(3) transformed cells function and genetic analysis
It is covered with when on resistance plate when cell culture, to transformant cell pAIOi-cas6-7-5-g-vegfaiIt carries out
NASBA and SDS-PAGE analysis, finds mRNA band and protein band without vegfa gene；Simultaneously with transformant cytogene
Group be template, vegfa-vF/vR is primer (table 4) progress PCR amplification, by the genetic analysis method of same embodiment 1 (1-C),
It was found that candidate transformant cytogene vegfa and gene vegfa sequence in host's NIH3T3 cell are completely the same, show vegfa
Gene regulation success.
Embodiment 5, pAIOiTranscriptional control of-the cas6-7-5-t/g-vegfa in NIH3T3
(1) plasmid pAIOiThe building of-cas6-7-5-g-vegfa
(A) plasmid pAIOiThe building of-cas6-7-5
With embodiment 4 (1-A).
(B) plasmid pAIOiThe building of-cas6-7-5-g-vegfa
With embodiment 4 (1-B).
(C) plasmid pAIOiThe building of-cas6-7-5-t/g-vegfa
(a) preparation of t-vegfa
According to vegfa gene and pAIOi- cas6-7-5-g-vegfa plasmid sequence information, designs and synthesizes primer
Vegfa-F/R, vegfa-UF/UR and vegfa-DF/DR (table 5) pass through same implementation using the NIH3T3 genome of extraction as template
Standard PCR and overlap PCR synthesis in example 1 (1-A), obtain t-vegfa after purification.
(b)pAIOiThe building of-cas6-7-5-t/g-vegfa
Except using pAIOiOutside-cas6-7-5-g-vegfa and t-vegfa substitution pCas and cas6-7-5-g-RNA, remaining step
Suddenly with embodiment 1 (1-B).
Primer involved in the present embodiment and the t-vegfa sequence of synthesis are as shown in table 5.Dotted line represents complementary series.
5 primer of table and t-vegfa sequence
(2) gene transcription regulation transformant pAIOi-cas6-7-5-t/g-vegfaiBuilding and verifying
Except using pAIOi- cas6-7-5-t/g-vegfa substitutes pAIOiOutside-cas6-7-5-g-vegfa, remaining step is the same as real
Apply example 4 (2).
(3) transformed cells function and genetic analysis
With embodiment 4 (3).
It is described above that technology contents of the invention are only further illustrated with embodiment, in order to which reader is easier to understand,
But embodiments of the present invention are not represented and are only limitted to this, any technology done according to the present invention extends or recreation, is sent out by this
Bright protection.
Claims (6)
1. a kind of transcriptional control method based on SviCas5-6-7 in I-B-Svi type CRISPR-Cas system, it is characterised in that:
It is separately encoded protein s viCas5 containing 3 genes cas5, cas6 and cas7 in the streptomycete IBL14 genome of Virginia,
SviCas6 and SviCas7 (protein sequence is seen attached list), three protein combine targeting DNA after expressing in organism
(guide-DNA/g-DNA: by transcripting promoter/promoter, repetitive sequence/repeat, intervening sequence/spacer and transcription
Terminator/terminator composition；Wherein intervening sequence is the DNA fragmentation complementary with target gene particular sequence) or and target gene
Template DNA (template DNA/t-DNA) with complementary series can carry out the genetic transcription in organism genome effective
Regulation.
2. a kind of transcription tune based on SviCas5-6-7 in I-B-Svi type CRISPR-Cas system according to claim 1
Prosecutor method, it is characterised in that:
(1) regulate and control the building of carrier
According to SviCas5, SviCas6 and SviCas7 amino acid sequence synthesizes the DNA sequence dna of the base optimization of corresponding cell, and
In conjunction with g-DNA and expression vector dna primers, through expression vector establishment, (PCR amplification respectively compiles albumen with ligase
Code gene cas6-7-5 and g-DNA are connect with expression vector) obtain the regulation carrier Vector of expression albumeni-cas6-7-5-g-
DNA。
(2) acquisition and inspection of gene transcription regulation transformant
By the method for conversion, transfection or electrotransformation, carrier Vector will be regulated and controled by the protein expression that step (1) obtainsi-cas6-
7-5-g-DNA is imported in biological cell, and selection culture obtains candidate gene transcription regulation transformant；Candidate transformant is carried out
Function Identification, target sequence PCR and sequencing analysis obtain correct gene transcription regulation transformant.
3. a kind of transcription based on SiCas5-6-7 in I-B-Svi type CRISPR-Cas system according to claim 1 or 2
Regulation method, it is characterised in that: SviCas5, SviCas6 and the SviCas7 amino acid sequence refers to that original or engineering changes
SviCas5, SviCas6 and SviCas7 amino acid sequence after making.
4. a kind of turn based on SviCas5-6-7 in I-B-Svi type CRISPR-Cas system according to claim 1 or 2
Record regulation method, it is characterised in that: the gene transcription regulation transformant, which refers to, leads to target because of the regulation of gene transcription level
The up-regulation of protein expression level is lowered or is not expressed, and then the apparent of host cell, function and the changed conversion of physiology
Son.
5. a kind of turn based on SviCas5-6-7 in I-B-Svi type CRISPR-Cas system according to claim 1 or 2
Record regulation method, it is characterised in that: the DNA sequence dna of the described corresponding base optimization refers to according to SviCas5, SviCas6 and
SviCas7 amino acid sequence and the cDNA sequence for being suitable for expressing in different cells optimized.
6. a kind of turn based on SviCas5-6-7 in I-B-Svi type CRISPR-Cas system according to claim 1 or 2
Record regulation method, it is characterised in that: the organism refers to any one of protokaryon and eucaryote, such as: Escherichia coli, paddy
Propylhomoserin corynebacteria, saccharomyces cerevisiae, mouse embryonic fibroblasts.
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