CN104651398A - Method for knocking out microRNA gene family by utilizing CRISPR-Cas9 specificity - Google Patents
Method for knocking out microRNA gene family by utilizing CRISPR-Cas9 specificity Download PDFInfo
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Abstract
The invention discloses a method for knocking out a microRNA gene family by utilizing CRISPR-Cas9 specificity, wherein a target gene is knocked out by mainly adopting a CRISPR/Cas9 system. The microRNA family is knocked out by utilizing the specificity of the CRISPR/Cas9 system for the first time; the disadvantage that only one gene can be knocked out for one time in traditional transgenosis can be overcome by utilizing the novel method; the time for establishing a model organism is reduced to three weeks; furthermore, the construction step is simple; an expensive molecular reagent is also reduced; the period for constructing a genetically modified mouse is greatly shortened to four months; multiple genes constructed for one time can be knocked out simultaneously; the fund investment is obviously reduced; F1 generations of mice can be obtained only in need of 50000 Yuan; the gene modification efficiency can be above 90%; the unreliability of the traditional technology is reduced; the operation technology is simple; and a series of complex steps including constructing a targeting vector, screening ES (Embryonic Stem) cells, selectively breeding chimeric mice and the like are unnecessary.
Description
(1) technical field
The present invention relates to that CRISPR-Cas9 is special pounds out the method for microRNA gene and the design of special target sgRNA.
(2) background technology
In recent years, China's pharmaceutical industries development is very rapid, but also there is a lot of problem simultaneously, such as, research and develop weak foundation, imitated foreign patent is spread unchecked, lacked independent intellectual property right etc., and these all seriously govern the pharmaceutical industries development of China.Therefore, the drug research and development ability strengthening China is very urgent.Drug development process is exactly the process of disease pathogenesis and drug targets screening, the process of screening is mostly by means of the animal disease model of all kinds of human diseases, and particularly nearer with people's sibship mouse model (comprising transgenosis and gene knockout) comes the pathogenesis of analysis of disease and the research and development of newtype drug.Be decided by that the own characteristic of mouse is similar to the mankind as allelotaxis, biochemical physiological metabolism etc., also depend on that MOUSE REPRODUCTION rate is high, draw materials easy, greatly facilitate research work.At present, China also starts to walk evening in disease mice model is set up, particularly in genetic modification mouse as in transgenosis/gene knockout, only have model animals center, south, Shanghai and model animals institute of Nanjing University, their production capacity is limited, and most of investigator goes back dependence on import, and costly every genetic modification mouse approximately needs 200,000, and the cycle is long, greatly constrain the development of China's medicinal industry.Therefore, under this background, how to set up and research and develop a kind of method that simple efficient cheapness fast prepares genetic modification mouse model to have great significance to China's Development of pharmaceutical industry strategy.
In animals and plants growth course, MicroRNA participates in the expression regulation of gene as the regulatory factor that a class is important.MiRNA, by combining with the 3 ' non-translational region (UTR) of specific mRNA, causes mRNA to degrade or is that the translation process of masterplate is suppressed with mRNA, thus reaches the effect of a certain genetic expression of negative regulation.The research of miRNA function is significant for the generation development disclosing disease further.
Research microRNA method mainly utilize knock out microRNA make its afunction to study its effect.But most of microRNA is with the form regulate gene expression of microRNA family; Knocking out separately certain microRNA can not make target gene function change completely.And utilize traditional way to build microRNA family to pound out the numerous and diverse and labor intensive material resources of animal model step; Utilize the microRNA antagonist of synthetic to carry out transfection or intravenous injection in animal body to cell, its application is obviously not enough: cost is high, needs repeatedly to inject, and wherein, comparatively outstanding is action time is short and cannot go down to posterity.Have experiment to show, after cell transfecting miRNA inhibitor, can act on 7 days at most, this obviously cannot meet the requirement of some disease research.Cannot go down to posterity and constrain the research of some fetal developments especially.Therefore need a kind of new genomic modification technology to pound out multiple microRNA simultaneously.
CRISPR/Cas9 technology is an emerging genetic modification technology, a kind of adaptive immunity defence that this scientific discovery is formed in long-term evolution process in bacterium and archeobacteria, can be used to the virus and the foreign DNA that resist invasion.Its principle mainly crRNA (CRISPR-derived RNA) is combined by base pairing and tracrRNA (trans-activating RNA) and forms tracrRNA/crRNA mixture, and this mixture guides the sequence target site that nuclease Cas9 albumen is matching with crRNA to shear double-stranded DNA.Utilize this system not only can modify goal gene efficiently, once can accurately modify multiple goal gene simultaneously.
At present, CRISPR/Cas9 system has very large advantage in genome editor, can fix a point to delete goal gene.The present invention utilizes this technology to knock out gene site-directed for miR-382 family miR-382 and miR-154, and it is high that this technology knocks out efficiency; Operation steps is simple simultaneously.Utilize this technology to carry out transgenosis to there is unique advantage there is high controllability and security, reduce the risk of genetically modified organism.
(3) summary of the invention
The object of the invention is to provide a kind of method that efficient fixed point knocks out microRNA family gene, and the main CRISPR/Cas9 system that adopts knocks out goal gene.This utilizes micrRNA family being knocked out of CRISPR/Cas9 systemic characteristic first.Utilize this new method can overcome the genetically modified drawback that once only can knock out a gene of tradition, the foundation accelerating model organism is reduced to 3 weeks; Construction step is simple simultaneously, decreases expensive molecular agents.
The technical solution used in the present invention is:
The invention provides a kind of special method knocking out microRNA gene of CRISPR-Cas9 of utilization, described method is: (1) with px330 plasmid for template, primer Cas9-R and containing T7 promotor primer Cas9-F effect under carry out PCR reaction, PCR reaction product is connected with pGME-T carrier, obtain plasmid pGEM-Cas9, again linearized for plasmid pGEM-Cas9, in-vitro transcription and RNA purification kit are reclaimed, obtain Cas9mRNA; (2) with px330 plasmid for template, under primer sgRNA-R and the effect containing the primer sgRNA-F of T7 promotor, carry out PCR reaction, then pcr amplification product to be connected with carrier pGEM-T, acquisition carrier pGEM-T7-sgRNA; (3) miR-382sgRNA shown in miR-154sgRNA with the SEQ ID NO.4 shown in SEQ ID NO.3 is connected with carrier pGEM-T7-sgRNA respectively, obtain plasmid T7-sgRNA-miR-382 and plasmid T7-sgRNA-miR-154 respectively, linearized, in-vitro transcription and RNA purification kit reclaim, and obtain T7-sgRNA-miR-382mRNA and T7-sgRNA-miR-154mRNA; (4) Cas9mRNA, T7-sgRNA-miR-382mRNA and T7-sgRNA-miR-154mRNA are mixed into mixed solution, injection mammal zygote, realizes gene site-directed for the microRNA object knocked out;
Cas9-F：5’-TTAATACGACTCACTATAGGATGGACTATAAGGACCACGAC-3’；
Cas9-R：5’-GCGAGCTCTAGGAATTCTTAC-3’；
sgRNA-F：5’-TTAATACGACTCACTATAGGGTGGAAAGGACGAAACACCGGGTCTTCGAGAAGACCT-3’；
sgRNA-R：5’-AAAAGCACCGACTCGGTGCC-3’。
Further, in described every 20 μ l mixed solutions, the concentration of Cas9mRNA, T7-sgRNA-miR-382mRNA and T7-sgRNA-miR-154mRNA is respectively 50ng/ μ l, 100ng/ μ l and 10ng/ μ l.
Further, described mammal zygote is mouse fertilized egg.
Compared with prior art, beneficial effect of the present invention is mainly reflected in:
1) build genetic modification mouse greatly to shorten on the cycle, be reduced to 4 months;
2) knock out while can once building multiple gene;
3) fund input obviously reduces, and only needs 50,000 yuan and can obtain F1 generation mouse;
4) genetic modification efficiency can reach more than 90%, reduces the unreliability of conventional art;
5) operative technique is simple, without the need to the series of steps such as targeting vector structure, ES cell screening, allophenic mice seed selection by complexity.
Therefore, CRISPR/Cas9 technology is prepared genetic modification mouse and is had very large advantage, and simple and fast material benefit, can prepare mouse model completely when funds are limited.
(4) accompanying drawing explanation
Fig. 1 knocks out miR-382 family gene forming types figure for utilizing CRISPR/cas9.
Fig. 2 is Cas9 expression vector pcr amplification product electrophorogram, and swimming lane M is standard molecule, and swimming lane cas9 is Cas9 expression vector pcr amplification product.
Fig. 3 is Cas9mRNA in-vitro transcription product electrophorogram, and swimming lane M is standard molecule, and swimming lane cas9mRNA is pGEM-Cas9 vector in vitro transcript.
Fig. 4 is T7-sgRNA expression vector PCR primer electrophorogram, and swimming lane M is standard molecule, and swimming lane pGEM-sgRNA is the pcr amplification product of sgRNA.
Fig. 5 is miR-382 family sgRNA in-vitro transcription electrophorogram, and swimming lane M is standard molecule, and swimming lane miR-154 is T7-sgRNA-miR-154 in-vitro transcription product electrophorogram, and swimming lane miR-382 is T7-sgRNA-miR-382 in-vitro transcription product electrophorogram.
Fig. 6 is miR-382 family deletion sequence.
Fig. 7 is that miR-382 knocks out rear heart picture.
(5) embodiment
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
Material therefor, reagent etc. in following embodiment, if no special instructions, all can obtain from commercial channels.
PGEM-T carrier purchased from Promega company, catalog number A3600.LA Taq purchased from TAKARA, catalog number RR02MA.
Embodiment 1
1, the outer Cas9 expression vector of construct, called after pGEM-Cas9, sequence is as SEQ ID NO.1, and the time is 3 days.
(1) design of primers
With px330 (Addgene:42330) sequence for template, design primer Cas9-F (underscore part is T7 promoter sequence) and primer Cas9-R.
Cas9-F：
5’-
ATGGACTATAAGGACCACGAC-3’；
Cas9-R：5’-GCGAGCTCTAGGAATTCTTAC-3’。
(2) pcr amplification
Take px330 as template, under the effect of primer Cas9-F and primer Cas9-R, carry out PCR reaction, amplified production carries out gel electrophoresis analysis, and obtaining object clip size is 5309bp, as shown in Figure 2.
PCR reaction system is:
PCR reaction conditions is:
(3) pGEM-Cas9 expression vector establishment
Get 1 μ l step (2) PCR primer to be connected with pGEM-T carrier, room temperature (25 DEG C) places 1h, and linked system is as follows:
Get connecting fluid 2 μ l and transform 50 μ l bacillus coli DH 5 alpha competent cells, ice bath 30min, 42 DEG C of thermal shock 60s, 500 μ l LB liquid nutrient mediums are added after ice bath 2min, be placed in 37 DEG C, 200rpm shaking table 1h, get 200 μ l nutrient solutions and coat X-gal+ final concentration 24mg/ml IPTG+ final concentration 100mg/ml Amp containing final concentration 50mg/ml
+lB dull and stereotyped, 37 DEG C of overnight incubation.Picking hickie, is inoculated in 5ml containing final concentration 100mg/ml Amp
+lB liquid nutrient medium in, 37 DEG C, 200rpm incubator overnight, extract plasmid, sequencing result show pGEM-Cas9 expression vector establishment success.
(4) linearizing pGEM-Cas9 carrier
PGEM-Cas9 expression vector prepared by step (3) is spent the night at 37 DEG C, carries out linearization process.
Reaction system is as follows:
(5) in-vitro transcription Cas9
Getting the linearizing pGEM-Cas9 carrier of 1 μ g utilizes in-vitro transcription test kit (ambionAM1340) to carry out in-vitro transcription, 37 DEG C of reaction 3h, and obtain pGEM-Cas9 vector in vitro transcript, system of specifically transcribing is as follows:
(6) utilize RNA purification kit (ambion AM1908) purifying in-vitro transcription thing, concrete purification system is as follows:
Refined solution crosses RNA purification kit adsorption column, and effluent liquid is the centrifugal 30s of 12000rpm again, and precipitation washes twice with 70% alcohol, finally uses 80 μ l without RNase water elution, collects liquid and carries out gel electrophoresis analysis, and as Fig. 3, result shows to obtain Cas9mRNA.
2, the vector construction of in-vitro transcription sgRNA skeletal coding sequence and preparation, name pGEM-sgRNA, sequence is as SEQ ID NO.2.
The T7-sgRNA nucleic acid fragment of synthetic, with the sgRNA frame sequence (i.e. T7-sgRNA nucleic acid fragment) of T7 promotor, is cloned on pGEM-T carrier by synthetic.Specific as follows:
(1) primer is designed
SgRNA-F (underscore is T7 promotor):
5’-
GTGGAAAGGACGAAACACCGGGTCTTCGAGAAGACCT-3’
sgRNA-R：5’-AAAAGCACCGACTCGGTGCC-3’
(2) pcr amplification
Take px330 as template, under containing the primer sgRNA-F of T7 promotor and the effect of primer sgRNA-R, carry out PCR reaction, pcr amplification product carries out gel electrophoresis analysis, and obtaining object clip size is 120bp, as Fig. 4.
PCR reaction system is:
PCR reaction conditions is:
(3) get 1 μ l step (2) pcr amplification product to be connected with pGEM-T, room temperature places 1h.
Get connecting fluid 2 μ l and transform 50 μ l bacillus coli DH 5 alpha competent cells, ice bath 30min, 42 DEG C of thermal shock 60s, 500 μ l LB liquid nutrient mediums are added after ice bath 2min, be placed in 37 DEG C, 200rpm shaking table 1h, get 200 μ l nutrient solutions and coat X-gal+ final concentration 24mg/ml IPTG+ final concentration 100mg/ml Amp containing final concentration 50mg/ml
+lB dull and stereotyped, 37 DEG C of overnight incubation.Picking hickie, is inoculated in 5ml containing final concentration 100mg/ml Amp
+lB liquid nutrient medium in, 37 DEG C, 200rpm incubator overnight, extract plasmid, sequencing result shows that pGEM-T7-sgRNA successfully constructs, for subsequent use.Containing two BbsI restriction enzyme sites between T7 and sgRNA sequence, the recognition sequence for certain gene can be inserted between two restriction enzyme sites.
Embodiment 2
1, for mmu-miR-154, mmu-miR-382 gene design sgRNA, BbsI restriction enzyme site (underscore) is added at primer 5 ' end, obtain miR-154sgRNA (sequence is as SEQ ID NO.3) and miR-382sgRNA (sequence is as SEQ ID NO.4) through sex change, annealing reaction, the time is 2 days.
sgRNA-miR-382-F:5’-
TACTTGTGACGAATCATTCA-3’
sgRNA-miR-382-R:5’-
TGAATGATTCGTCACAAGTAC-3’
sgRNA-miR-154-F:5’-
TATTCGTGACGAATCATACA-3’
sgRNA-miR154-R:5’-
TGTATGATTCGTCACGAATAC-3’
Sex change, annealing reaction system is as follows:
In PCR instrument, reaction system is as follows: 37 DEG C of 30min; 95 DEG C of 5min; 95-25 DEG C, 5 DEG C/min, product is miR-382sgRNA.
Sex change, annealing reaction system is as follows:
In PCR instrument, reaction system is as follows: 37 DEG C, 30min; 95 DEG C, 5min; 95-25 DEG C, 5 DEG C/min, product is miR-154sgRNA.
MiR-382sgRNA and miR-154sgRNA after sex change being annealed is connected respectively to pGEM-sgRNA carrier, reacts 12 hours under room temperature, and obtain plasmid T7-sgRNA-miR-382 and plasmid T7-sgRNA-miR-154 respectively, reaction system is as follows:
2, difference linearization plasmid T7-sgRNA-miR-382, T7-sgRNA-miR-154, the reaction times is 3 hours.
Reaction system is as follows:
Use phenol chloroform purify DNA again.
3, in-vitro transcription T7-sgRNA-miR-382/miR-154
Respectively get 1 μ g linearization plasmid (T7-sgRNA-miR-382, T7-sgRNA-miR-154), utilize in-vitro transcription test kit (ambion AM1340) to carry out in-vitro transcription, 37 DEG C of reaction 3h, concrete system is as follows:
4, RNA purification kit (ambion AM1908) purification step 3 is utilized) in-vitro transcription product, concrete system is as follows:
Cross RNA purification kit adsorption column, 12000rpm, 30s, 70% alcohol washes twice, finally uses without RNase water elution, and run glue qualification, as Fig. 5, result shows to obtain T7-sgRNA-miR-382mRNA, T7-sgRNA-miR-154mRNA.
5, transgenic mice preparation (3 hours time)
1) CRISPR/Cas9 injection mouse system is as follows:
2) inject
Eppendorf 2xTransferMan NK2 microinjection instrument is utilized to draw 2 μ l steps 1) mixed solution injection 60 ~ 80 zygotes.
After mouse is born 5 days, clip mouse nail extracts genomic dna.PCR identifies miR-154, miR-382 gene and order-checking, as Fig. 6.Result shows, after miR-382 knocks out, mouse heart diminishes, as shown in Figure 7.
Claims (3)
1. one kind utilizes the special method knocking out microRNA gene family of CRISPR-Cas9, it is characterized in that described method is: (1) with px330 plasmid for template, primer Cas9-R and containing T7 promotor primer Cas9-F effect under carry out PCR reaction, PCR reaction product is connected with pGME-T carrier, obtain plasmid pGEM-Cas9, again linearized for plasmid pGEM-Cas9, in-vitro transcription and RNA purification kit are reclaimed, obtain Cas9mRNA; (2) with px330 plasmid for template, under primer sgRNA-R and the effect containing the primer sgRNA-F of T7 promotor, carry out PCR reaction, then pcr amplification product to be connected with carrier pGEM-T, acquisition carrier pGEM-T7-sgRNA; (3) miR-382sgRNA shown in miR-154sgRNA with the SEQ ID NO.4 shown in SEQ ID NO.3 is connected with carrier pGEM-T7-sgRNA respectively, obtain plasmid T7-sgRNA-miR-382 and plasmid T7-sgRNA-miR-154 respectively, linearized, in-vitro transcription and RNA purification kit reclaim, and obtain T7-sgRNA-miR-382mRNA and T7-sgRNA-miR-154mRNA; (4) Cas9mRNA, T7-sgRNA-miR-382mRNA and T7-sgRNA-miR-154mRNA are mixed into mixed solution, injection mammal zygote, realizes gene site-directed for the microRNA object knocked out;
Cas9-F：5’-TTAATACGACTCACTATAGGATGGACTATAAG
GACCACGAC-3’；
Cas9-R：5’-GCGAGCTCTAGGAATTCTTAC-3’；
sgRNA-F：5’-TTAATACGACTCACTATAGGGTGGAAAGGACGAAACA
CCGGGTCTTCGAGAAGACCT-3’；
sgRNA-R：5’-AAAAGCACCGACTCGGTGCC-3’。
2. the method for claim 1, is characterized in that the concentration of Cas9mRNA, T7-sgRNA-miR-382mRNA and T7-sgRNA-miR-154mRNA in described every 20 μ l mixed solutions is respectively 50ng/ μ l, 100ng/ μ l and 10ng/ μ l.
3. the method for claim 1, is characterized in that described mammal zygote is mouse fertilized egg.
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