CN104531705A - Method for knocking off animal myostatin gene by using CRISPR-Cas9 system - Google Patents
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Abstract
The invention provides a method for knocking off an animal myostatin gene by using a CRISPR-Cas9 system. The method comprises the following steps: firstly, acquiring a DNA sequence aiming at an sgRNA recognition area of a second myostatin exon, wherein the base sequence of the DNA sequence is as shown in SEQ ID NO.1; secondly, establishing an sgRNA expression structure of the second myostatin exon, inserting a T7 starter before an sgRNA transcriptional start site, establishing an in-vitro transcription carrier of Cas9 protein, and regulating and controlling by using the T7 starter. Cas9 mRNA and sgRNA are obtained through the in-vitro transcription carrier of Cas9 and sgRNA, and the method can be used for knocking off the animal myostatin gene.
Description
Technical field
The invention belongs to animal genetic engineering and genetic modification field, specifically, relate to a kind of method utilizing CRISPR-Cas9 system knock-out animal myostatin gene.
Background technology
Since the eighties in last century, genetically engineered was risen, lots of genes editing technique occurs to meet scientific research needs, and gene targeting is wherein a kind of technology of meticulous modification of fixing a point to gene in higher animal.Tradition gene targeting relies on homologous recombination (HR, homologous recombination) spontaneous in body, and efficiency approximately only has 1/10
6.In recent years in order to solve the low problem of homologous recombination efficiency, people are cut specific DNA sequence dna by artificial constructed hybrid molecule, improve the efficiency of gene targeting with this, are wherein that the artificial compound molecule of core is of greatest concern with endonuclease.
Myostatin gene (GDF8) is the member of transforming growth factor superfamily, and it is in animal body as the negative regulatory factor of muscle growth, plays a significant role in the formation and differentiation and development process of muscle.The natural spontaneous mutation type animal that there is myostatin gene of occurring in nature, as Belgian Blue ox, pyrmont beef etc., they all have the extreme flourishing phenotype of muscle, therefore have very important practical value at production field.And by traditional genetic engineering technique to Mammals, especially large animal carries out myostatin gene and carries out knocking out the very large technical difficulty of existence, and thus the myostatin gene knockout efficiency of large animal is very low all the time.
CRISPR/Cas9 system comprises one to be possessed DNA combination and the Cas9 albumen cut and responsible specific recognition DNA sequence dna and guides Cas9 protein-specific to be attached to the sgRNA in target DNA site.In animal body, first Cas9 albumen and sgRNA are combined into a protein complexes, then by the specific recognition effect of sgRNA, recognize specific target dna sequence and also one be attached on DNA chain to protein complexes in genome.Then at target site, DNA is cut by the endonuclease activity of Cas9, form a DNA double splitting of chain (DSB).Played a role by the DNA repair mechanism of inducing self-body, as homologous recombination or non-homologous end joining (NHEJ, Non-homologous end joining), thus produce transgenation in this site, thus arrive the object of gene targeting.
Compared with the gene targeting mediated with traditional homologous recombination, utilizing CRISPR/Cas9 system to carry out gene targeting to Mammals has easy and simple to handle, efficiency is high, the advantages such as applicable species are extensive, especially disposablely can realize polygene to practice shooting, this has extremely wide application prospect in animal genetic modification and disease model research.
Traditional gene targeting relies on homologous recombination phnomena spontaneous in organism, thus efficiency is very low, along with technical development had just occurred Zinc finger nuclease (ZFNs afterwards, zinc fingernucleases) and transcriptional activation increment effector mediation nuclease (TALENs, transcriptionactivator-like effector nucleases).These two kinds of system structure comparison are similar, each protein molecular is made up of FolkI endonuclease enzyme domains and DNA recognition structure territory two portions, by the specific purpose sequence in the specific DNA differential threshold identification genome in each protein molecular, thus FolkI restriction endonuclease is positioned to target site.Because FolkI endonuclease enzyme require forms the function of dimeric forms competence exertion endonuclease on DNA double chain, thus ZFNs and TALENs needs two molecules to be positioned on two DNA chains of target site respectively when gene targeting, just can play the effect of endonuclease, cutting be carried out to DNA and produces double-strand break (DSB).
Although ZFNs and TALENs has the high advantage of target practice efficiency compared to traditional homologous recombination, but still there is a lot of defects in them, mainly comprise: 1, the DNA cutting structure territory of ZFNs and TALENs is FolkI, it must play a role with dimeric forms, at least will adopt two DNA expression structures when thus carrying out gene targeting to Mammals, this can have higher requirement to transfection efficiency when cell transfecting; 2, the design of ZFNs and production relative complex, cost is higher, is applied to cost when a large amount of mammalian genes is practiced shooting and is difficult to control the scope bearing; 3, the DNA recognition rule of ZFNs and TALENs and design requirements are comparatively strict, may occur finding suitable ZFNs, TALENs cog region in target-gene sequence, thus cannot apply the situation that it carries out gene targeting; When 4, carrying out gene targeting for the same gene of different genes or different plant species, all need, from newly designing and build new ZFNs, TALENs expression plasmid or mRNA, to operate complicated; 5, ZFNs and TALENs is when carrying out polygene and practicing shooting, by the restriction of carrier and mRNA molecular size, and the target practice efficiency that very difficult acquisition is higher.
Summary of the invention
The object of this invention is to provide a kind of method of CRISPR-Cas9 system knock-out animal myostatin gene.
Another object of the present invention is to provide the sgRNA of selectively targeted myostatin gene.
The present invention is first than right different plant species (mankind, mouse, pig, ox, sheep, goat) myostatin gene order, therefrom have found a relatively conservative region, carry out the design of sgRNA in this region and obtain the sequence information of a sgRNA.The sgRNA of this selectively targeted myostatin gene Second Exon, its DNA sequence dna is as shown in SEQ ID NO.1.The invention provides the application of sgRNA in knock-out animal myostatin gene of this selectively targeted myostatin gene Second Exon.
Present invention also offers the carrier of the DNA sequence dna containing above-mentioned sgRNA.
In an embodiment of the present invention, the above-mentioned carrier provided is pX330-M2.
Particularly, the construction process of pX330-M2 is: (1) designs and synthesizes the sgRNA cog region DNA sequence dna identifying myostatin Second Exon, as shown in SEQ ID NO.1; (2) after the sgRNA sequence after synthesis carries out phosphorylation, gradient cooling is annealed, concrete steps are the water polishing system adding 3 times of volumes after being mixed with the ratio of 2:2:1 with 10X T4Ligation Buffer and T4PNK by the oligo DNA of synthesis again, then 30min is hatched for 37 DEG C, 95 DEG C of 5min sex change again, complete reaction to produce phosphorylation sticky end being cooled to 25 DEG C with the speed of 5 DEG C of per minutes afterwards, simultaneously BbsI enzyme cuts carrier pX330 generation sticky end; (3) with T4 ligase enzyme, this fragment is connected with pX330, obtains eucaryon CRISPR-Cas9 systemic vectors pX330-M2.
Present invention also offers in-vitro transcription carrier pIVT-M2-T carrier.
The construction process of described in-vitro transcription carrier pIVT-M2-T is: the upstream primer of design containing T7 promotor, the downstream primer that sequence is mated as shown in SEQ ID NO.2 and with it, sequence is as shown in SEQ ID NO.3, with carrier pX330-M2 for template PCR amplifications obtains the IVT-M2 fragment that may be used for in-vitro transcription, in the mode of TA clone, this fragment is inserted in pMD18-T carrier again, obtain the carrier pIVT-M2-T being used for in-vitro transcription.
The invention provides the CRISPR-Cas9 system for knock-out animal myostatin gene, the expression vector of sgRNA containing selectively targeted myostatin gene Second Exon and the in-vitro transcription carrier of Cas9 albumen.
In embodiments of the invention, the described expression vector containing the sgRNA of selectively targeted myostatin gene Second Exon is the in-vitro transcription carrier of pIVT-M2-T, Cas9 albumen is pCas9-puro3.
Wherein, pCas9-puro3 builds by the following method and obtains: the Cas9 that (1) cuts in carrier pX330-U6-Chimeric_BB-CBh-hSpCas9 acquisition pX330 carrier with restriction endonuclease AgeI and NotI enzyme expresses section; (2) with AgeI and NotI linearized vector pIRES-puro3; (3) Cas9 of step (1) is expressed section to carry out being connected with the linearized vector pIRES-puro3 of step (2) and obtain whole carrier pCas9-puro3.
The invention provides the method utilizing CRISPR-Cas9 system knock-out animal myostatin gene, comprise the following steps:
(1) expression vector of the sgRNA of selectively targeted myostatin gene Second Exon is built; Expressed by in-vitro transcription and obtain myostatin gene Second Exon sgRNA;
(2) build the in-vitro transcription carrier of Cas9 albumen, obtain Cas9mRNA;
(3) by after the Cas9mRNA purifying of the sgRNA of step (1) and step (2), mixing, be injected in fertilised non-human eggs tenuigenin or nucleus, be implanted in jenny uterine tube of the same race after external short-term culture, or vitro culture to blastula stage be transplanted in female uterus of the same race again, to produce the animal knocking out myostatin gene.Described short-term culture refers to 30min-48h.
In the inventive method, the expression vector of the sgRNA of step (1) selectively targeted myostatin gene Second Exon is pIVT-M2-T.The in-vitro transcription carrier of the Cas9 albumen of step (2) is pCas9-puro3.
The mRNA that generation is transcribed in step (1) and (2) carries out adsorption column purifying, and the mRNA after purifying is utilized spectrophotometric determination concentration.The sgRNA concentration obtained in an embodiment of the present invention is the concentration of 103ng/ μ l, Cas9mRNA is 775ng/ μ l, and according to, after making mixing, sgRNA and Cas9mRNA final concentration is respectively 20ng/ μ l and 150ng/ μ l.
In step (3), before sgRNA and Cas9mRNA mixing, the concentration range of sgRNA is the concentration range of 100 ~ 200ng/ μ l, Cas9mRNA be the mixed mass ratio of 500 ~ 2000ng/ μ l, sgRNA and Cas9mRNA is 1:10 ~ 1:2 mixing.
Preferably, the two mixed mass ratio is 1:7.5.
Present invention also offers aforesaid method and prepare the application knocked out in the animal of myostatin gene.
The present invention utilizes CRISPR-Cas9 system to carry out mammalian genes target practice, its advantage is: tri-base pairs of the NGG 1, in sgRNA specific recognition DNA sequence dna, recognition rule simply and easily analyze, multiple sgRNA recognition site can be found in target gene simultaneously, thus can select according to target practice requirement, suitability is extensive; 2, compared to the complicated expression structure of ZFNs and TALENs, Cas9 expression structure in CRISPR-Cas9 system is changeless, only need the recognition sequence of 23bp to be inserted in sgRNA expression structure and can set up by completion system for different genes, simple to operate, cost is low, is applicable to large-scale mammalian genes target practice work; When 3, utilizing CRISPR-Cas9 system to continue cell transfecting, because sgRNA expression structure is very short, so greatly transfection efficiency can be improved, also Cas9 and sgRNA expression structure can be incorporated in a carrier, further raising transfection efficiency, this is all that ZFNs and TALENs is difficult to accomplish; When 4, carrying out gene targeting for the different genes of same family or the same gene of different plant species, conserved regions in Select gene can carry out sgRNA design, thus realize same sgRNA to the target practice of multiple gene or modification, compare other technologies easier, efficient; 5, can easily realize polygene by the mode importing a Cas9 expression structure and multiple sgRNA in Mammals simultaneously to practice shooting, be all that other technologies are incomparable simultaneously from target practice efficiency or degree easy and simple to handle.
The present invention is by building the expression vector of sgRNA and the in-vitro transcription carrier of Cas9 albumen of selectively targeted myostatin gene Second Exon, obtain the mRNA of sgRNA and the Cas9 albumen of target myostatin gene Second Exon, fertilised non-human eggs tenuigenin or nucleus is injected after being mixed, through external short-term culture or cultivate after a couple of days and be transplanted in jenny uterine tube of the same race or uterus, the animal obtaining knocking out myostatin gene can be produced, efficiency and the adaptability of gene targeting can be improved.Another outstanding advantages of the present invention is, the sgRNA of target myostatin gene Second Exon provided by the invention is the contrast mankind, mouse, pig, ox, sheep, the conserved sequence of multiple species such as goat obtains, and therefore sgRNA of the present invention can be common to the myostatin gene knockout of multiple species.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that technique means used in embodiment is well known to those skilled in the art, is raw materials usedly commercial goods.
The test materials related in following examples and reagent: pX330-U6-Chimeric_BB-CBh-hSpCas9 is purchased from addgene, pMD18-T purchased from takara company, and pIRES-puro3 is purchased from Clontech company.B6D2F1 mouse is C57BL/6 and DBA/2 mouse hybrid 1 generation mouse, C57BL/6 and DBA/2 is all purchased from Beijing Vital River Experimental Animals Technology Co., Ltd..Sheep is purchased from Shunyi, Beijing Jin Xin modern agricultural development company limited.
Do not make the experimental methods of molecular biology illustrated in following examples, all carry out with reference to the concrete grammar described in " Molecular Cloning: A Laboratory guide " (third edition) J. Pehanorm Brooker one book, or carry out according to test kit and product description.
Embodiment 1 is for the structure of the CRISPR-Cas9 system of myostatin gene
1, than the myostatin gene order of right different plant species (mankind, mouse, pig, ox, sheep, goat), therefrom have found a relatively conservative region, carry out the design of sgRNA in this region and obtain the sequence information of a sgRNA.The wherein sgRNA of selectively targeted myostatin gene Second Exon, its DNA sequence dna is as shown in SEQ ID NO.1.
2, the structure of pX330-M2: (1) designs and synthesizes the sgRNA cog region DNA sequence dna identifying myostatin Second Exon, as shown in SEQ ID NO.1; (2) after the sgRNA sequence after synthesis carries out phosphorylation, gradient cooling is annealed, concrete steps are the water polishing system adding 3 times of volumes after being mixed with the ratio of 2:2:1 with 10X T4Ligation Buffer and T4PNK by the oligo DNA of synthesis again, then 30min is hatched for 37 DEG C, 95 DEG C again, 5min sex change, complete reaction to produce phosphorylation sticky end being cooled to 25 DEG C with the speed of 5 DEG C of per minutes afterwards, simultaneously BbsI enzyme cuts carrier pX330 generation sticky end; (3) respectively these two fragments are connected with pX330 with T4 ligase enzyme, obtain eucaryon CRISPR-Cas9 systemic vectors pX330-M2.
3, the structure of transcription vector pIVT-M2-T: the upstream primer of design containing T7 promotor, the downstream primer that sequence is mated as shown in SEQ ID NO.2 and with it, sequence is as shown in SEQ IDNO.3, with carrier pX330-M2 for template PCR amplifications obtains the IVT-M2 fragment that may be used for in-vitro transcription, in the mode of TA clone, this fragment is inserted in pMD18-T carrier again, obtain the carrier pIVT-M2-T being used for in-vitro transcription.
4, transcription vector pCas9-puro3 builds by the following method and obtains: the Cas9 that (1) cuts in carrier pX330-U6-Chimeric_BB-CBh-hSpCas9 acquisition pX330 carrier with restriction endonuclease AgeI and NotI enzyme expresses section; (2) with AgeI and NotI linearized vector pIRES-puro3; (3) Cas9 of step (1) is expressed section to carry out being connected with the linearized vector pIRES-puro3 of step (2) and obtain whole carrier pCas9-puro3.
CRISPR-Cas9 system for myostatin gene is: transcription vector pIVT-M2-T and transcription vector pCas9-puro3.
Embodiment 2 in-vitro transcription
In-vitro transcription carrier pIVT-M2-T and pCas9-puro3 built is utilized to carry out the in-vitro transcription of T7 promotor mediation, namely using T7 promotor as the promotor of in-vitro transcription, utilize the transcription that RNA polymerase realizes in vitro from DNA to mRNA, concrete grammar is: respectively with SalI and NotI linearized vector pIVT-M2-T and pCas9-puro3, then with linearizing in-vitro transcription carrier for template, add T7 transcriptase, buffer and rNTPs, hatch 6h for 37 DEG C, then DNA enzymatic 37 DEG C is added, template DNA is removed in 15min digestion, after imitating extracting removal protein impurities with phenol again, alcohol settling obtains the mRNA after transcribing, the mRNA transcribing generation is carried out adsorption column purifying, concrete grammar is: add the binding buffer liquid of 3.5 times of volumes and the dehydrated alcohol of 2.5 times of volumes to mRNA, add after mixing in adsorption column, the centrifugal 10min of 12000rpm, adsorption column is washed twice again with washings, finally dissolve mRNA with the pure water of 100 μ l, mRNA after purifying is utilized spectrophotometric determination concentration, sgRNA concentration is 103ng/ μ l, the concentration of Cas9mRNA is 775ng/ μ l.
Embodiment 3 utilizes the CRISPR-Cas9 system mRNA producer gene target practice mouse for myostatin gene
1, procaryotic injection and embryo transfer
Get the protokaryon phase zygote of B6D2F1 mouse, (Cas9mRNA final concentration is 150ng/ μ l by Cas9mRNA/sgRNA mixture mixed in advance to utilize microinjection instrument, sgRNA final concentration is 20ng/ μ l), be injected in mouse fertilized egg tenuigenin or nucleus.Zygote after injection is transferred to of short duration cultivation in nutrient solution, then migrates in the uterine tube of the female mouse of acceptor, producer gene target practice mouse.
2, the qualification of gene targeted mice
After the female mouse of replace-conceive produces, treat that newborn mouse grows to clip about 1cm mouse tail in 2 week age, Proteinase K, after 55 DEG C of digestion, phenol is imitated extracting and is extracted mouse coda gene group.With mouse coda gene group for template, designing the primer for myostatin Second Exon, increases in sequence such as Seq No.4 ~ 5, checks order to the PCR primer obtained, as bimodal situation appears in sequencing result target practice location proximate, then and may for practice shooting successfully.Select bimodal sample PCR again, after product glue reclaims, TA is cloned in carrier T, after transforming, picking positive colony checks order again, insert or base deletion as there is base near myostatin gene target site in sequencing result, cause frame shift mutation, then can be judged as myostatin gene knockout.
Embodiment 4 utilizes the CRISPR-Cas9 system mRNA for myostatin gene to carry out sheep embryo gene targeting
1, procaryotic injection and vitro culture
From Sheep Ovary, gather ovum carry out maturation in vitro, choosing ripe ovum carries out in vitro fertilization, (Cas9mRNA final concentration is 150ng/ μ l by Cas9mRNA/sgRNA mixture mixed in advance to utilize microinjection instrument in the protokaryon phase, sgRNA final concentration is 20ng/ μ l), be injected in fertilized egg cell's core.Zygote after injection is transferred in nutrient solution continues to cultivate.After zygote after to be injected is cultured to blastula stage, gets embryo and add lysate and carry out cracking process, to carry out next step identification experiment.
2, the detection of gene targeting result
With the sheep embryo after growth for template carries out PCR, primer is for the Second Exon of myostatin, and primer sequence is as Seq No.6 ~ 7, and PCR primer checks order, as bimodal situation appears in sequencing result target practice location proximate, then and may for practice shooting successfully.Select bimodal sample PCR again, after product glue reclaims, TA is cloned in carrier T, after transforming, picking positive colony checks order again, insert or base deletion as there is base near myostatin gene target site in sequencing result, cause frame shift mutation, then can be judged as gene knockout, be gene targeting positive findings.
Although above with general explanation, embodiment and test, the present invention is described in detail, and on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (10)
1. the sgRNA of selectively targeted myostatin gene Second Exon, is characterized in that, its DNA sequence dna is as shown in SEQ ID NO.1.
2. the carrier of the DNA sequence dna containing sgRNA described in claim 1.
3. the carrier as shown in claim 2, it is in-vitro transcription carrier pIVT-M2-T.
4. for the CRISPR-Cas9 system of knock-out animal myostatin gene, it is characterized in that, the expression vector of sgRNA containing selectively targeted myostatin gene Second Exon and the in-vitro transcription carrier of Cas9 albumen.
5. CRISPR-Cas9 system as claimed in claim 4, is characterized in that, the expression vector of the sgRNA of described selectively targeted myostatin gene Second Exon is the in-vitro transcription carrier of pIVT-M2-T, Cas9 albumen is pCas9-puro3.
6. CRISPR-Cas9 system as claimed in claim 5, it is characterized in that, pCas9-puro3 builds by the following method and obtains: the Cas9 that (1) cuts in pX330-U6-Chimeric_BB-CBh-hSpCas9 acquisition pX330 carrier with restriction endonuclease AgeI and NotI enzyme expresses section; (2) with AgeI and NotI linearized vector pIRES-puro3; (3) two terminal sequences are connected the whole carrier pCas9-puro3 of acquisition.
7. utilize the method for the arbitrary described CRISPR-Cas9 system knock-out animal myostatin gene of claim 4-6, it is characterized in that, comprise the following steps:
(1) expression vector of the sgRNA of selectively targeted myostatin gene Second Exon is built; Expressed by in-vitro transcription and obtain myostatin gene Second Exon sgRNA;
(2) build the in-vitro transcription carrier of Cas9 albumen, obtain Cas9mRNA;
(3) by after the Cas9mRNA purifying of the sgRNA of step (1) and step (2), mixing, be injected in fertilised non-human eggs tenuigenin or nucleus, then be implanted into after external short-term culture in jenny uterine tube of the same race, or vitro culture to blastula stage be transplanted in female uterus of the same race, to produce the animal knocking out myostatin gene.
8. method as claimed in claim 7, is characterized in that, in step (3), before sgRNA and Cas9mRNA mixing, the concentration of sgRNA is the concentration of 100 ~ 200ng/ μ l, Cas9mRNA is 500 ~ 2000ng/ μ l, and the two mixed mass ratio is 1:10 ~ 1:2.
9. method as claimed in claim 8, it is characterized in that, in step (3), it is 1:7.5 that sgRNA and Cas9mRNA mixes the latter two mass ratioes.
10. the method described in claim 8 or 9 is preparing the application knocked out in the animal of myostatin gene.
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