CN106957858A - A kind of method that utilization CRISPR/Cas9 systems knock out sheep MSTN, ASIP, BCO2 gene jointly - Google Patents
A kind of method that utilization CRISPR/Cas9 systems knock out sheep MSTN, ASIP, BCO2 gene jointly Download PDFInfo
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- CN106957858A CN106957858A CN201610854587.1A CN201610854587A CN106957858A CN 106957858 A CN106957858 A CN 106957858A CN 201610854587 A CN201610854587 A CN 201610854587A CN 106957858 A CN106957858 A CN 106957858A
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
- A01K67/0276—Knock-out vertebrates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention provides a kind of method that utilization CRISPR/Cas9 systems knock out sheep MSTN, ASIP, BCO2 gene jointly.The present invention obtain first the sgRNA cog regions for sheep MSTN Second Exons and the 3rd extron two segment DNA sequences, for the extrons of sheep ASIP the 5th sgRNA cog regions two segment DNA sequences, for sheep BCO2 Second Exons sgRNA cog regions two segment DNA sequences, separately design and with into corresponding sgRNA oligonucleotide sequences；Then build respectively for sheep MSTN Second Exons and the 3rd extron, for the extrons of sheep ASIP the 5th, for sheep BCO2 Second Exons, and the saRNA in-vitro transcription carriers of the promoter containing T7；Cas9 mRNA and sgRNA are obtained by in-vitro transcription using Cas9 and sgRNA in-vitro transcription carrier, the Transgenic Sheep that can be used for production MSTN, ASIP, BCO2 to knock out jointly is injected by embryonated egg.
Description
Technical field
The present invention relates to animal genetic engineering and genetic modification field, and in particular to one kind utilizes CRISPR/Cas9 systems
The common method for knocking out sheep MSTN, ASIP, BCO2 gene.
Background technology
CRISPR/Cas systems are to cut the acquisition of exogenous genetic material in bacterium and archeobacteria by the RNA specificity mediated
Property immune system.II type CRISPR/Cas systems be CRISPR/Cas9 be proved to can in test tube high efficiency cutting it is any
Given DNA.CRISPR/Cas9 efficiency high, sequence selection compared with traditional ZFN and TALEN technologies limit small (only need to
Occur GG on genome), and its building process is simple, and suitable sgRNA need to be only built for each gene.But
CRISPR/Cas9 can cause serious effect of missing the target in mammalian cell, be carried using Cas9 nickases plus two phases
PAM, apart from closer and sgRNA on different chains can be combined can substantially reduce efficiency of missing the target.
The shortcomings of traditional breeding method has breeding year limit for length, a selection traits limited amount, molecular labeling is auxiliary
Help the problem of selection is still difficult to apply to production practices rapidly still in theoretical developments, transgenic breeding technology and traditional breeding method
The combination of technology is particularly important.
Important gene --- the MSTN related to growth, i.e. flesh generate inhibin, also known as growth/(GDF- of differentiation factor -8
8).MSTN genes are main in mammal is expressed in skeletal muscle, and the gene is produced in the variant sites (A ＞ G) of non-translational region
The target site of interference, it is suppressed that the translation process and adjusting function of MSTN genes, so as to have impact on the development of animal muscle.
ASIP is that Agouti signal protein gene is proved to its important function in B16 cell signal path, ASIP bases
Because height expression is white significantly correlated by hair with sheep.The ASIP albumen that ASIP is expressed in skin by with α-MSH (melanin
Cytositimulation hormone) competitive binding MC1R (melanin cortical hormone receptor 1), make MC1R structural changes, suppress cycli phosphate gland
Glycosides enzyme system causes cAMP levels to decline, and suppresses the formation of eumelanin by cascade reaction, produces brown-black element.Too high expression
ASIP genes may also cause B16 cell reduce so that showing as white by hair color.The agronomical value of sheep
Offer mutton is not only in that, wool and sheepskin are also the important product of sheep, but the irregular of Mianyang hair color has a strong impact on hair
The dyeing process processing in skin quality and later stage.
By CRISPR/Cas9 systems on suppressing the MSTN genes of muscle growth in ovine genome, influenceing fur quality
ASIP genes and bring the BCO2 genes of yellow fat disease to be knocked out jointly by CRISPR/Cas9, produce it is muscular,
The sheep that fur quality is high, vigor is good is significant to the tremendous development of China's animal husbandry.
The content of the invention
Sheep MSTN, ASIP, BCO2 gene is knocked out jointly the invention provides a kind of utilization CRISPR/Cas9 systems
Method.
To achieve the above object, the technical scheme taken of the present invention is：One kind is knocked out jointly using CRISPR/Cas9 systems
The method of sheep MSTN, ASIP, BCO2 gene, it is characterised in that comprise the following steps：
(1) selectively targeted MSTN Second Exons and the 3rd extron, the extrons of ASIP the 5th are built, outside BCO2 second
Show the sgRNA of son in-vitro transcription carrier；Obtained by in-vitro transcription for MSTN Second Exons and the 3rd extron
SgRNA-1M, sgRNA-2M, it is outer aobvious for sheep BCO2 second for sgRNA-1A, sgRNA-2A of the extrons of ASIP the 5th
SgRNA-1B, sgRNA-2B of son；
(2) the in-vitro transcription carrier of in-vitro transcription Cas9 albumen, obtains Cas9mRNA；
(3) by the sgRNA-1M of step (1) and step (2), sgRNA-2M, sgRNA-1A, sgRNA-2A, sgRNA-1B,
SgRNA-2B and Cas9mRNA survey concentration after purification, mixing, are injected into sheep zygotes cytoplasm, then after in vitro culture
It is implanted into female sheep fallopian tubal of the same race, for producing the common Transgenic Sheep for knocking out MSTN, ASIP, BCO2 gene.
A kind of utilization CRISPR/Cas9 systems of the present invention knock out sheep MSTN, ASIP, BCO2 gene jointly
Method, it is characterised in that sgRNA-1M, sgRNA-2M, sgRNA-1A, sgRNA-2A, sgRNA-1B, sgRNA-2B with
Cas9mRNA mixing after, final concentration of Cas9 mRNA 20ng/ μ L, sgRNA-1M 5ng/ μ L, sgRNA-2M 5ng/ μ L,
sgRNA-1A 5ng/μL、 sgRNA-2A 5ng/μL、sgRNA-1B 5ng/μL、sgRNA-2B 5ng/μL。
A kind of utilization CRISPR/Cas9 systems of the present invention knock out sheep MSTN, ASIP, BCO2 gene jointly
Method, it is characterised in that described sgRNA-1M, sgRNA-2M, sgRNA-1A, sgRNA-2A, sgRNA-1B, sgRNA-2B's
Expression vector is pUC57-T7-gRNA, and the in-vitro transcription carrier of Cas9 albumen is pST1374-NLS-flag-linker-Cas9.
A kind of utilization CRISPR/Cas9 systems of the present invention knock out sheep MSTN, ASIP, BCO2 gene jointly
Method, it is characterised in that the selectively targeted sgRNA-1M for sheep MSTN Second Exons and the 3rd extron,
SgRNA-2M, for sgRNA-1A, sgRNA-2A of the extrons of sheep ASIP the 5th, for sheep BCO2 Second Exons
sgRNA-1B、sgRNA-2B。
Sheep MSTN, ASIP, BCO2 gene is knocked out jointly present invention also offers above-mentioned utilization CRISPR/Cas9 systems
Method application.
(1) sgRNA-1M, sgRNA-2M are collectively or individually transfected to cell simultaneously with Cas9mRNA, for studying MSTN
The function of gene；
(2) sgRNA-1M, sgRNA-2M are collectively or individually injected into embryonated egg simultaneously with Cas9mRNA, then pass through embryo
Tire transplants the Transgenic Sheep for producing targeting knock out MSTN genes；
(3) sgRNA-1M, sgRNA-2M are collectively or individually transfected to cell simultaneously with Cas9mRNA, with the positive after screening
Cell produces the Transgenic Sheep of targeting knock out MSTN genes as donor cell by the method for nuclear transfer；
(4) sgRNA-1A, sgRNA-2A are collectively or individually transfected to cell simultaneously with Cas9mRNA, for studying ASIP
The function of gene；
(5) sgRNA-1A, sgRNA-2A are collectively or individually injected into embryonated egg simultaneously with Cas9mRNA, then pass through embryo
Tire transplants the Transgenic Sheep for producing targeting knock out ASIP genes；
(6) sgRNA-1A, sgRNA-2A are collectively or individually transfected to cell simultaneously with Cas9mRNA, with the positive after screening
Cell produces the Transgenic Sheep of targeting knock out ASIP genes as donor cell by the method for nuclear transfer.
(7) sgRNA-1B, sgRNA-2B are collectively or individually transfected to cell simultaneously with Cas9mRNA, for studying BCO2
The function of gene；
(8) sgRNA-1B, sgRNA-2B are collectively or individually injected into embryonated egg simultaneously with Cas9mRNA, then pass through embryo
Tire transplants the Transgenic Sheep for producing targeting knock out BCO2 genes；
(9) sgRNA-1B, sgRNA-2B are collectively or individually transfected to cell simultaneously with Cas9mRNA, with the positive after screening
Cell produces the Transgenic Sheep of targeting knock out BCO2 genes as donor cell by the method for nuclear transfer；
(10) by sgRNA-1M, sgRNA-2M, sgRNA-1A, sgRNA-2A, sgRNA-1B, sgRNA-2B with
Cas9mRNA is injected into embryonated egg simultaneously, is then used to produce common knockout MSTN, ASIP, BCO2 gene by embryo transfer
Transgenic Sheep；
(11) by sgRNA-1M, sgRNA-2M, sgRNA-1A, sgRNA-2A, sgRNA-1B, sgRNA-2B with
Cas9mRNA is transfected to cell simultaneously, is used to produce by embryo transfer as donor cell with positive cell after screening and is struck jointly
Except the Transgenic Sheep of MSTN, ASIP, BCO2 gene；
(12) by sgRNA-1M, sgRNA-2M, sgRNA-1A, sgRNA-2A, sgRNA-1B, sgRNA-2B any two
Individual or two or more sgRNA and Cas9mRNA is injected into embryonated egg simultaneously, is then used to produce special target by embryo transfer
The Transgenic Sheep of knockout；
(13) by sgRNA-1M, sgRNA-2M, sgRNA-1A, sgRNA-2A, sgRNA-1B, sgRNA-2B any two
Individual or two or more sgRNA and Cas9mRNA is transfected to cell simultaneously, and embryo is passed through as donor cell with positive cell after screening
Tire transplants the Transgenic Sheep for producing special target knockout.
The operation principle of CRISPR/Cas9 systems be crRNA (CRISPR-derived RNA) by base pairing with
TracrRNA (trans-activating RNA) combines to form tracrRNA/crRNA compounds, this compound guiding nuclease
Cas9 albumen shears double-stranded DNA in the sequence target site matched with crRNA.And by both RNA of engineer, can transform
Form the sgRNA (singleguide RNA) with guiding function, it is sufficient to guide Cas9 to cut DNA fixed point.It is used as one
The dsDNA associated proteins that RNA is oriented to are planted, Cas9 effector nucleases are the known first unified factor (unifying
Factor), can common location RNA, DNA and albumen, so as to possess huge transformation potentiality.By albumen and the Cas9 of nuclease free
(Cas9nuclease-null) merge, and express appropriate sgRNA, any dsDNA sequences can be targeted, and sgRNA end can
Target dna is connected to, Cas9 combination is not influenceed.Therefore, Cas9 can bring any fusion protein at any dsDNA sequences
And RNA, this brings great potential for the research and transformation of organism.
The gene order of the present invention：
1st, the method for knocking out sheep MSTN, ASIP, BCO2 gene jointly using CRISPR/Cas9 systems, its described MSTN,
ASIP, BCO2 gene specific target site sequence are shown in Table 1.
Table 1
2nd, the method for knocking out sheep MSTN, ASIP, BCO2 gene jointly using CRISPR/Cas9 systems, its described MSTN,
The sgRNA sequences of ASIP, BCO2 gene design are shown in Table 2.
| MSTN sgRNA-1top strand | tagGTCTCAGATATATCCACAGT |
| MSTN sgRNA-1bottom strand | aaacACTGTGGATATATCTGAGA |
| MSTN sgRNA-2top strand | TAGGATTTTGAAGCTTTTGGAT |
| MSTN sgRNA-2bottom strand | aaacATCCAAAAGCTTCAAAAT |
| ASIP sgRNA-1top strand | ccggCTTCAGGTTCCTTTCATCTC |
| ASIP sgRNA-1bottom strand | aaacGAGATGAAAGGAACCTGAAG |
| ASIP sgRNA-2top strand | CCGGCAATTCTTCCATGAACCTGT |
| ASIP sgRNA-2bottom strand | aaacACAGGTTCATGGAAGAATTG |
| BCO2sgRNA-1top strand | ccgGTTAGAAGCGGTGCAATGCA |
| BCO2sgRNA-1bottom strand | aaacTGCATTGCACCGCTTCTAA |
| BCO2sgRNA-2top strand | ccGGTCGTCTCAGCTCGAGTCC |
| BCO2sgRNA-2bottom strand | aaacGGACTCGAGCTGAGACGA |
Table 2
3rd, the method for knocking out sheep MSTN, ASIP, BCO2 gene jointly using CRISPR/Cas9 systems, its knockout MSTN,
The primer sequence is shown in Table 3 during the lamb detection that the sheep of ASIP, BCO2 gene is produced.
Table 3
Embodiment
In order that objects and advantages of the present invention are more clearly understood, the present invention is carried out with reference to embodiments further
Describe in detail.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to limit this hair
It is bright.
Sheep MSTN, ASIP, BCO2 are knocked out jointly using CRISPR/Cas9 systems the embodiments of the invention provide one kind
The method of gene, comprises the following steps：
(1) selectively targeted MSTN Second Exons and the 3rd extron, the extrons of ASIP the 5th are built, outside BCO2 second
Show the sgRNA of son in-vitro transcription carrier；Obtained by in-vitro transcription for MSTN Second Exons and the 3rd extron
SgRNA-1M, sgRNA-2M, it is outer aobvious for sheep BCO2 second for sgRNA-1A, sgRNA-2A of the extrons of ASIP the 5th
SgRNA-1B, sgRNA-2B of son；
(2) the in-vitro transcription carrier of in-vitro transcription Cas9 albumen, obtains Cas9mRNA；
(3) by the sgRNA-1M of step (1) and step (2), sgRNA-2M, sgRNA-1A, sgRNA-2A, sgRNA-1B,
SgRNA-2B and Cas9mRNA survey concentration after purification, mixing, are injected into sheep zygotes cytoplasm, then after in vitro culture
It is implanted into female sheep fallopian tubal of the same race, for producing the common Transgenic Sheep for knocking out MSTN, ASIP, BCO2 gene.
Wherein sgRNA-1M, sgRNA-2M, sgRNA-1A, sgRNA-2A, sgRNA-1B, sgRNA-2B and Cas9mRNA
After mixing, final concentration of Cas9mRNA 20ng/ μ L, sgRNA-1M 5ng/ μ L, sgRNA-2M 5ng/ μ L, sgRNA-1A 5ng/
μL、sgRNA-2A 5ng/μL、sgRNA-1B 5ng/μL、sgRNA-2B 5ng/μL。
Described sgRNA-1M, sgRNA-2M, sgRNA-1A, sgRNA-2A, sgRNA-1B, sgRNA-2B expression vector
For pUC57-T7-gRNA, the in-vitro transcription carrier of Cas9 albumen is pST1374-NLS-flag-linker-Cas9.
It is described selectively targeted for sheep MSTN Second Exons and sgRNA-1M, sgRNA-2M of the 3rd extron, pin
To sgRNA-1A, sgRNA-2A of the extrons of sheep ASIP the 5th, sgRNA-1B for sheep BCO2 Second Exons,
sgRNA-2B。
Step one：For the structure of the CRISPR/Cas9 systems of MSTN, ASIP, BCO2 gene
1st, according to ovine genome sequence in NCBI, in selection ovine genome outside MSTN genes Second Exon and the 3rd
Aobvious son designs sgRNA-1M, sgRNA-2M as target site, and the 5th extron of ASIP genes designs sgRNA- as target site
1A, sgRNA-2A, the Second Exon of BCO2 genes design sgRNA-1B, sgRNA-2B its target site sequence such as target site
Shown in table 1, sgRNA sequences are as shown in table 2.
Table 1
Table 2
2nd, the structure of the pUC57-T7-gRNA carriers containing specific sgRNA sequences：(1) identification is designed and synthesized
SgRNA-1M, sgRNA-2M of MSTN genes Second Exon and the 3rd extron, recognize the 5th extron of ASIP genes
SgRNA-1A, sgRNA-2A, recognize sgRNA-1B, sgRNA-2B of the Second Exon of BCO2 genes；(2) after synthesizing
The paired oligonucleotides of sgRNA-1M, sgRNA-2M, sgRNA-1A, sgRNA-2A, sgRNA-1B, sgRNA-2B carries out body respectively
Outer annealing；(3) sgRNA-1M, sgRNA-2M, sgRNA-1A, sgRNA-2A, sgRNA-1B, sgRNA-2B are passed through BsaI
Point carries out digestion, connection, is inserted into pUC57-T7-gRNA, is respectively designated as pUC57-T7-sgRNA-1M, pUC57-T7-
sgRNA-2M、 pUC57-T7-sgRNA-1A、pUC57-T7-sgRNA-2A、pUC57-T7-sgRNA-1B、 pUC57-T7-
sgRNA-2B；
CRISPR/Cas9 systems for MSTN, ASIP, BCO2 gene are：In-vitro transcription carrier pUC57-T7-
sgRNA-1M、pUC57-T7-sgRNA-2M、pUC57-T7-sgRNA-1A、 pUC57-T7-sgRNA-2A、pUC57-T7-
SgRNA-1B, pUC57-T7-sgRNA-2B and pST1374-NLS-flag-linker-Cas9.
Step 2：In-vitro transcription
Utilize carrier pUC57-T7-sgRNA-1M, pUC57-T7-sgRNA-2M, the pUC57-T7-sgRNA- built
1A, pUC57-T7-sgRNA-2A, pUC57-T7-sgRNA-1B, pUC57-T7-sgRNA-2B and Cas9mRNA in-vitro transcription
Carrier pST1374-NLS-flag-linker-Cas9 carries out the in-vitro transcription mediated with T7 promoters.(1) by pUC57-T7-
sgRNA-1M、pUC57-T7-sgRNA-2M、pUC57-T7-sgRNA-1A、 pUC57-T7-sgRNA-2A、pUC57-T7-
SgRNA-1B, pUC57-T7-sgRNA-2B are respectively with Dra I linearisations, pST1374-NLS-flag-linker-Cas9 use
Age1 is linearized；(2) MEGAshortscript kit (Ambion) kit is used, pUC57-T7- is carried out to specifications
sgRNA-1M、 pUC57-T7-sgRNA-2M、pUC57-T7-sgRNA-1A、pUC57-T7-sgRNA-2A、 pUC57-T7-
SgRNA-1B, pUC57-T7-sgRNA-2B, pST1374-NLS-flag-linker-Cas9 in-vitro transcription；(3) use
MEGAClear kit (Ambion) kit, carries out pUC57-T7-sgRNA-1M, pUC57-T7-sgRNA- to specifications
2M、pUC57-T7-sgRNA-1A、 pUC57-T7-sgRNA-2A、pUC57-T7-sgRNA-1B、pUC57-T7-sgRNA-2B、
Cas9 mRNA purifying.
Step 3：Using the CRISPR/Cas9 systems production for MSTN, ASIP, BCO2 gene it is common knock out MSTN,
The gene targeting sheep of ASIP, BCO2 gene
1st, procaryotic injection and embryo transfer
Single stage is in by surgical collection out of the natural mating after estrus synchronization donor ewe body
Embryo, using microinjection instrument by pre- mixed sgRNA-1M, sgRNA-2M, sgRNA-1A, sgRNA-2A, sgRNA-1B,
SgRNA-2B, Cas9mRNA mixture (final concentration of Cas9mRNA 20ng/ μ L, sgRNA-1M after mixing, sgRNA-2M,
The respective 5ng/ μ L of sgRNA-1A, sgRNA-2A, sgRNA-1B, sgRNA-2B) it is injected into the cytoplasm of sheep zygotes.Note
Embryonated egg after penetrating is transferred to Quinn ' s Advantage Cleavage Medium (Sage Biopharma, NJ, USA) body
Outer 37 DEG C of cultures 24h, then migrates to the ampulla of uterine tube of recipient sheep and isthmus junction, production is common knock out MSTN,
The gene targeting sheep of ASIP, BCO2 gene.
2nd, the identification of the gene targeting sheep of MSTN, ASIP, BCO2 gene is knocked out jointly
After acceptor ewe production, after the blood sample that lamb is adopted after lamb length to 1 week old, lamb poba gene group DNA is extracted.With
Lamb poba gene group is masterplate, for sheep MSTN Second Exons and the 3rd extron, the extrons of ASIP the 5th, BCO2 the
The primer of 2 extrons, sequence such as table 3 is expanded, and row agarose gel electrophoresis are entered to the PCR primer of acquisition and detects and produces
Objects system is reclaimed, and the PCR primer after recovery carries out T7EN1 digestions, carries out electrophoresis detection after digestion is complete, testing result shows two
The possibility of bar or many band is gene targeting success；PCR primer after recovery is sent and is sequenced and carries out sequence analysis, with reference to
T7EN1 digestions result and sequencing result analysis determine positive individuals；PCR primer to positive individuals is cloned into carrier T, after conversion
Picking positive colony is sequenced again, and the successful positive individuals of gene knockout and the positive are determined according to sequencing result is further
The variation pattern of base in individual.
Table 3
Although being described in detail above with generality explanation, embodiment to the present invention, it is only
The preferred embodiment of the present invention.It should be pointed out that for those skilled in the art, it is former not departing from invention
On the premise of reason, some improvements and modifications can also be made, these improvements and modifications also should be regarded as protection scope of the present invention.
Claims (4)
1. a kind of method that utilization CRISPR/Cas9 systems knock out sheep MSTN, ASIP, BCO2 gene jointly, it is characterised in that
Comprise the following steps：
(1) build selectively targeted：MSTN Second Exons and the 3rd extron, the extrons of ASIP the 5th, BCO2 second are outer aobvious
The sgRNA of son in-vitro transcription carrier；Obtained by in-vitro transcription for MSTN Second Exons and the 3rd extron
SgRNA-1M, sgRNA-2M, it is outer aobvious for sheep BCO2 second for sgRNA-1A, sgRNA-2A of the extrons of ASIP the 5th
SgRNA-1B, sgRNA-2B of son；
(2) the in-vitro transcription carrier of in-vitro transcription Cas9 albumen, obtains Cas9 mRNA；
(3) by the sgRNA-1M of step (1) and step (2), sgRNA-2M, sgRNA-1A, sgRNA-2A, sgRNA-1B,
SgRNA-2B and Cas9 mRNA survey concentration after purification, then by sgRNA-1M, sgRNA-2M, sgRNA-1A, sgRNA-2A,
SgRNA-1B, sgRNA-2B are mixed with Cas9 mRNA, are injected into sheep zygotes cytoplasm, are then moved after in vitro culture
It is implanted into female sheep fallopian tubal of the same race, for producing the common Transgenic Sheep for knocking out MSTN, ASIP, BCO2 gene.
2. one kind according to claim 1 knocks out sheep MSTN, ASIP, BCO2 base jointly using CRISPR/Cas9 systems
The method of cause, it is characterised in that sgRNA-1M, sgRNA-2M, sgRNA-1A, sgRNA-2A, sgRNA-1B, sgRNA-2B with
Cas9 mRNA mixing after, final concentration of Cas9 mRNA 20ng/ μ L, sgRNA-1M 5ng/ μ L, sgRNA-2M 5ng/ μ L,
sgRNA-1A 5ng/μL、sgRNA-2A 5ng/μL、sgRNA-1B 5ng/μL、sgRNA-2B 5ng/μL。
3. one kind according to claim 1 knocks out sheep MSTN, ASIP, BCO2 base jointly using CRISPR/Cas9 systems
The method of cause, it is characterised in that described sgRNA-1M, sgRNA-2M, sgRNA-1A, sgRNA-2A, sgRNA-1B, sgRNA-2B
Expression vector be pUC57-T7-gRNA, the in-vitro transcription carrier of Cas9 albumen is pST1374-NLS-flag-linker-
Cas9。
4. one kind according to claim 1 knocks out sheep MSTN, ASIP, BCO2 base jointly using CRISPR/Cas9 systems
The method of cause, it is characterised in that the selectively targeted sgRNA-1M for sheep MSTN Second Exons and the 3rd extron,
SgRNA-2M, for sgRNA-1A, sgRNA-2A of the extrons of sheep ASIP the 5th, for sheep BCO2 exon 2s
sgRNA-1B、sgRNA-2B。
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