CN109868275A - The method of sheep FGF5 gene knockout and site-directed integration MTNR1A gene that CRISPR/Cas9 is mediated - Google Patents
The method of sheep FGF5 gene knockout and site-directed integration MTNR1A gene that CRISPR/Cas9 is mediated Download PDFInfo
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Abstract
The present invention provides a kind of method of sheep FGF5 gene knockout and site-directed integration MTNR1A gene that CRISPR/Cas9 is mediated.The CRISPR/Cas9 targeting vector of special target sheep FGF5 gene third exon and homologous recombination carrier are transferred to jointly in sheep fetal fibroblast, the cell of sheep FGF5 gene knockout and site-directed integration MTNR1A gene is obtained.The present invention is by donor vehicle and targeting vector cotransfection sheep fetal fibroblast by way of consideration convey, and CYP17 promoter can be such that MTNR1A realizes specific expressed in sheep ovary tissue in the unmarked donor vehicle constructed by Gibson Assembly method.Separating the positive monoclonal cell strain identified can be used as the nuclear donor acquisition clone embryos of body-cell neucleus transplanting, and then take a firm foundation to cultivate the transgene clone sheep of reproductive system local strengthening epiphysin signal and voluminous hair.
Description
Technical field
The present invention relates to gene engineering technology fields, specifically, being related to the sheep FGF5 clpp gene of CRISPR/Cas9 mediation
Except the method with site-directed integration MTNR1A gene.
Background technique
Reproductive trait is the important economical trait of aquaculture, and the reproductive trait for effectively improveing sheep is to improve sheep husbandry level
One of key.High-quality meat breed reproductive capacity is relatively low, improves sheep reproductive capacity by traditional Pedigree breeding and improvement hybridization means
Make slow progress.Domestic and foreign scholars had conducted extensive research the Molecular and genetic basis of sheep in recent years, found on a molecular scale
And the major gene resistance of positioning effects sheep breed mechanism, it lays a good foundation for accurate breeding improvement.Presently found prolificacy
Candidate gene has: BMPR-1B, BMP15, GDF9 and melatonin receptors MTNR1A etc., influences the growth point of prlificacy, ovarian follicle respectively
Change, embryonic development and system differentiation and early embryonic development and reproductive rhythm.
Epiphysin (Melatonin, MT) is a kind of important endocrine hormone, especially in the seasonal oestrus animal such as sheep
Key effect is played in breeding regulation.Early period, multiple seminars had extensively studied the effect machine that epiphysin regulation is ovulated both at home and abroad
System finds that egg mother cell mitochondria is the main cell device of epiphysin synthesis for the first time.And it further confirms in its natural state, line
Plastochondria synthesis epiphysin is the effective way that ovulation stress damage is resisted in Oocyte Maturation Process.In addition, also in the world
Luteotropin (LH) induction ovary synthesizes epiphysin and granular cell epiphysin I receptor after reporting heat for the first time
(MTNR1A) increase severely, granular cell leuteinization is regulated and controled by ovarian follicle part epiphysin signal (MT/MTNR1A), promotes progesterone point
It secretes.It has also been found that epiphysin activates MTNR1A receptor in ovulation process, extracellular matrix remodeling and blood are promoted by downstream pathway
Pipe is formed, to promote corpus luteum secreting function, improves pregnancy rate and litter size.In rutting by external source epiphysin
Reason, significantly improves sheep, the conception rate of ox and litter size.
The certain bits for referring to and being transferred to specific exogenous nucleic acid sequences in cell or genes of individuals group are knocked in genome targeting
Point, to realize conditional gene knockout, single base or a variety of accurate genomes targetings such as sequence replacement, cell or genetic marker
Modification.Different from gene knockout, gene insertion will consider the insertion point on genome.The targeting knockout of research discovery before this is to the food in one's mouth
The inhibited fibroblast growth factor 5 of the hair growth of newborn animal (fibroblast growth factor 5,
FGF5) wool production of animal dramatically increases afterwards.In view of the economic value in animal productiong, select to lead using FGF5 as target spot
Enter target gene.
Summary of the invention
The object of the present invention is to provide sheep FGF5 gene knockouts and site-directed integration MTNR1A gene that CRISPR/Cas9 is mediated
Method.
Present inventive concept is as follows: CRISPR/Cas9 technology is utilized, using sheep FGF5 gene as target spot, and fixed point insertion epiphysin
Receptor MTNR1A is overexpressed its specificity in ovary tissue using gonadal steroids synthetic promoter CYP17.The sun of acquisition
Property cell strain can be used as the nuclear donor of body-cell neucleus transplanting, and then cultivate the gene editing clone sheep of prolificacy.
In order to achieve the object of the present invention, in a first aspect, the present invention provides one kind based on CRISPR/Cas9 technology special target
The sgRNA of sheep FGF5 gene, RNA sequence is as shown in SEQ ID NO:1.Encode the DNA sequence dna such as SEQ ID of the sgRNA
Shown in NO:2.
Second aspect, the present invention provide the CRISPR/Cas9 targeting vector for containing above-mentioned sgRNA.
Preferably, skeleton carrier PX458.
The third aspect, the present invention provide a kind of higher donor load of the biological safety without any eukaryon selection markers
Body, the carrier contain ovary-specific promoter CYP17, target gene melatonin receptors MTNR1A, transcription stop signals bGH
Poly (A) signal, for the left and right homology arm sequence of FGF5 target practice site homologous recombination, nucleotide sequence is respectively such as SEQ
Shown in ID NO:3 and SEQ ID NO:4.
Preferably, Gibson Assembly method building donor vehicle can be used to overcome by seamless connection in sequence
In the case that length is big, junction fragment is more, using traditional digestion connection method without the available drawback of suitable restriction enzyme site.
Fourth aspect, the present invention provide a kind of sheep FGF5 gene editing carrier based on CRISPR/Cas9 technological development, packet
Include above-mentioned CRISPR/Cas9 targeting vector and homologous recombination carrier.
Wherein, the homologous recombination carrier includes target gene or destination gene expression box, and being used for will be described
Target gene repairs the element sequences in fixed point insertion sheep FGF5 gene third exon by homologous end.
The target gene is MTNR1A.
The structure of the destination gene expression box are as follows: ovary-specific promoter CYP17-MTNR1A-bGH poly (A).
The element sequences include the left and right homology arm for sheep FGF5 gene targeting site homologous recombination, nucleotide
Sequence is respectively as shown in SEQ ID NO:3 and SEQ ID NO:4.
Further, the homologous recombination carrier includes such as flowering structure: left homology arm-ovary-specific promoter
CYP17-MTNR1A-bGH poly (A)-right homology arm.
In the present invention, the target gene and element sequences come from same species.Guarantee importing in transgenic protocol
Always it is the gene of sheep itself, is equivalent to the copy number for merely adding original gene in its genome, avoids importing heterogenous gene
Express the possible safety issue of heterologous protein.
5th aspect, the present invention provides the cell of sheep FGF5 gene knockout and site-directed integration MTNR1A gene, by above-mentioned sheep
FGF5 gene editing vector introduction (CRISPR/Cas9 targeting vector and homologous recombination carrier cotransfection) sheep placenta is at fiber
It is obtained in cell.
In the specific embodiment of the present invention, (gene is same for the CRISPR/Cas9 targeting vector and donor vehicle
Source recombinant vector) cotransfection mode be consideration convey, compared to traditional rotaring transfecting mode, transfection efficiency is higher.
6th aspect, the present invention provide the sgRNA of targeting sheep FGF5 gene shown in SEQ ID NO:1, contain the sgRNA
CRISPR/Cas9 targeting vector, the sheep FGF5 gene editing carrier or the sheep FGF5 gene knockout and site-directed integration
The cell of MTNR1A gene is preparing the application in gene editing clone sheep.
7th aspect, the present invention provide CRISPR/Cas9 the sheep FGF5 gene knockout and site-directed integration MTNR1A base that mediate
The CRISPR/Cas9 targeting vector and homologous recombination carrier are transferred to sheep fetal fibroblast by the method for cause jointly
In, obtain the cell of sheep FGF5 gene knockout and site-directed integration MTNR1A gene.
By above-mentioned technical proposal, the present invention at least have following advantages and the utility model has the advantages that
(1) the present invention provides not only knock out sheep FGF5 gene but also be overexpressed MTNR1A gene sheep fetal fibroblast,
It lays the foundation to obtain the new varieties gene editing clone sheep of voluminous hair and prolificacy.
(2) the monoclonal positive cell line gone out using CRISPR/Cas9 technology screening, realizes the fixed point of target gene
Integration, has evaded " pollution " of the random integration of marker gene and target gene to genome, thus avoiding to cause
The problems such as gene function exception and biological safety.
(3) present invention is led to by donor vehicle and targeting vector cotransfection sheep fetal fibroblast by way of consideration convey
Crossing CYP17 promoter in the unmarked donor vehicle of Gibson Assembly method building can be such that MTNR1A realizes in sheep ovum
It is specific expressed in nest tissue.Separating the positive monoclonal cell strain identified can be used as the nuclear donor acquisition of body-cell neucleus transplanting
Clone embryos, and then solid base is laid to cultivate the transgene clone sheep of reproductive system local strengthening epiphysin signal and voluminous hair
Plinth.
Detailed description of the invention
Fig. 1 is sheep FGF5 gene targeting site schematic diagram in the embodiment of the present invention 1.
Fig. 2 is targeting vector spectrogram in the embodiment of the present invention 1.
Fig. 3 is MTNR1A fixed point insertion FGF5 target spot schematic diagram in the embodiment of the present invention 2.
Fig. 4 is that MTNR1A donor vehicle constructs schematic diagram in the embodiment of the present invention 2.
Fig. 5 is MTNR1A donor vehicle segment pcr amplification product electrophoretogram in the embodiment of the present invention 2.
Fig. 6 is targeting vector and donor vehicle cotransfection figure in the embodiment of the present invention 3.
Fig. 7 is selected by flow cytometry apoptosis figure in the embodiment of the present invention 4.
Fig. 8 is that cell monoclonal forms figure in the embodiment of the present invention 4.
Fig. 9 is the amplification figure in the embodiment of the present invention 4 near preceding 14 plants of cell target spots；The expression that black surround the is irised out strain
Cell may be that large fragment knockout is knocked in or had occurred to homozygosis.
Figure 10 is that the primer 5'junction for stepping up trip homology arm and CYP17 promoter is utilized in the embodiment of the present invention 4
Long 1-1777bp amplification figure.
Figure 11 is that the primer 3'junction long1- across MT1 and downstream homology arm is utilized in the embodiment of the present invention 4
1628bp amplification figure.
Figure 12 is that the primer 5'junction for stepping up trip homology arm and CYP17 promoter is utilized in the embodiment of the present invention 4
Long 2-1540bp amplification figure.
Figure 13 is that the primer 3'junction long2- across MT1 and downstream homology arm is utilized in the embodiment of the present invention 4
1378bp amplification figure.
Figure 14 is the amplification figure in the embodiment of the present invention 4 near rear 18 plants of cell target spots.
Figure 15 is that the primer 5'junction for stepping up trip homology arm and CYP17 promoter is utilized in the embodiment of the present invention 4
Long 1-1777bp amplification figure, this plant of cell of expression that red frame is irised out may be that heterozygosis is knocked in.
Figure 16 is that the primer 3'junction long1- across MT1 and downstream homology arm is utilized in the embodiment of the present invention 4
1628bp amplification figure, this plant of cell of expression that black surround is irised out may be that heterozygosis is knocked in.
Figure 17 be the embodiment of the present invention 4 in filtered out on the basis of pair of primers qualification result the doubtful positive 13#,
14#, 15# cell strain utilize the primer 5'junction long2-1540bp- for stepping up trip homology arm and CYP17 promoter
Upstream (U) and across the primer 3'junction long2-1378bp-Downstream (D) of MT1 and downstream homology arm expand
Result figure；This plant of cell of expression that red frame is irised out may be that heterozygosis is knocked in.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
According to conventional laboratory conditions, such as Sambrook molecular cloning experiment handbook (Sambrook J&Russell DW,
Molecular Cloning:a Laboratory Manual, 2001), or according to the condition of manufacturer's specification suggestion.
The building of embodiment 1CRISPR/Cas9 targeting vector
Target practice site is located in sheep FGF5 gene (NM_001246263.2) third exon, and corresponding target practice position is as schemed
Shown in 1, wherein identify that the nucleotide sequence of the target spot as shown in SEQ ID NO:1, encodes the above-mentioned sequence in sgRNA sequence
DNA sequence dna as shown in SEQ ID NO:2.According to above-mentioned target sequence, corresponding primer sequence, You Shenggong biotech firm are designed
Synthesis, way of purification HPLC, particular sequence are shown in Table 1.
1 sheep FGF5 gene targeting aligning primer of table
| Nucleotide title | Sequence (5'-3') |
| Ovis aries-FGF5-sgRNA-F | caccgAGGTTCCCCTTTCCGCACCT |
| Ovis aries-FGF5-sgRNA-R | aaacAGGTGCGGAAAGGGGAACCTc |
The formation of Olio nucleic acid sequence: being diluted to 100 μM for primer, phosphoric acid annealing, system: Ovis aries-FGF5-
1 μ L, Ovis aries-FGF5-sgRNA-R (100 μM) of sgRNA-F (100 μM) 1 μ L, T4ligation buffer, 10 ×, 1 μ
L, T4PNK 1 μ L, ddH26 μ L of O, 10 μ L of total volume.Response procedures are as follows: 37 DEG C of 30min；95 DEG C of 5min, it is every in PCR instrument
5~25 DEG C of minute drop.250 times of dilutions, carrier PX458 carry out digestion with BbsI restriction endonuclease after reaction.16 DEG C of connections are overnight.
Connection removes linear DNA residue after reaction, using PlasmidSafe exonuclease.37 DEG C of 30min, 70 DEG C
30min.It is saved in -20 DEG C later, can be reserved at least one week.
The step products therefrom can be directly used for conversion Escherichia coli, it is recommended to use Stbl3 competent cell.Take thermal shock
Method: the plasmid after taking 2 μ L PlasmidSafe is added in 20 μ L competent cells, on ice 10min, 42 DEG C of thermal shock, 30s,
It is immediately placed on 2min on ice, 100 μ L SOC, direct coated plate is added.Use the LB plate containing Amp100.37 DEG C overnight.Second
It observation, control board should without clone, and containing sgRNA insertion plate in clone.Picking monoclonal shakes bacterium 12h, and bacterium solution is sent
Beijing Qing Ke biotech firm is sequenced.After verifying plasmid construction is correct, big upgrading grain carries out subsequent transfection cell experiment.It beats
Targeting vector map is as shown in Figure 2.
The building of 2 donor vehicle of embodiment
As shown in figure 3, MTNR1A is pinpointed insertion FGF5 target spot, (Homology need to be repaired using homologous end
Directed repair, HDR) mode by with homology arm locus be inserted into target site.Since the building of donor vehicle needs
Four long segments are cloned into a purpose carrier, in addition the uncertainty of restriction enzyme site, it is difficult to guarantee that each segment can
Suitable splicing, therefore the Gibson Assembly based on homologous recombination is used, it can be efficiently seamless by more long segments
Be stitched together, do not limited by restriction enzyme site.
The building process of donor vehicle as shown in figure 4, need by upstream homology arm 5-HA, CYP17-promoter,
MTNR1A (MT1)-bGH poly (A) signal, downstream homology arm 3-HA are connected into together in skeleton carrier pUC57-Amp, wherein
Upstream and downstream homology arm and CYP17 promoter are cloned from sheep genome, MTNR1A (MT1)-bGH poly (A) signal clone
The carrier saved from laboratory, each segment is according to Gibson Assembly design of primers principle design primer, and primer sequence is such as
Shown in table 2:
Each fragment amplification primer of 2 donor vehicle of table
The amplification system of each segment are as follows: high-fidelity DNA polymerase PrimeSTAR MAX DNA Polymerase (R045A,
Takara) 0.5 μ l, dNTP mix (2.5mM each) 25 μ l, GC Buffer II (RR02AG, Takara) 8 μ l, forward primer
1 μ l, 1 μ l of reverse primer, sheep genomic templates 2 μ l, 11.5 μ l H2O.Amplification condition are as follows: 98 DEG C of initial denaturation 2min；98 DEG C of 10s,
65 DEG C of 30s, 72 DEG C of 45s, 35 circulations；72 DEG C of 10min after circulation terminates, 4 DEG C of preservations.Amplification is as shown in Figure 5.
Gel: being placed under ultraviolet lamp after electrophoresis, purpose band separated by PCR product purifying, recycling purification DNA, operation
It is as follows: (1) to separate purpose gel fragment in the UV lamp, be placed in clean EP pipe, claim its quality.Blob of viscose volume reduction formula: 1
μ L=lmg.(2) DR-1Buffer of 3 times of volumes is added into EP pipe, being put into 45 DEG C of water-baths after concussion mixing fills blob of viscose
Divide dissolution, time 8-10min.(3) balance columns Spin Column and collecting pipe Collection Tube is taken out, and as required
Correctly place.Solution in (2) is transferred in Spin Column pipe, then 12000rpm is centrifuged 1min, and repeated centrifugation is primary.
(4) it draws Rinse A500 μ L to be added in Spin Column, centrifugal condition is same as above.(5) it draws and had added dehydrated alcohol
Spin Column is added in 700 μ L of Rinse B, and centrifugal condition is same as above, and abandons filtrate, and repeated centrifugation is primary.(6) continue to be centrifuged, item
Part is constant, time 2min.(7) a new Collection Tube is taken, Spin Cohmm is placed, and to Spin
Rnase-free ddH is added in Column20 25 μ L, stand 1-2min under room temperature.(8) 12000rpm is centrifuged 1min and makes
It obtains DNA to be eluted in Collection Tube, -20 DEG C of preservations.
The nucleotide sequence of left and right homology arm is respectively as shown in SEQ ID NO:3 and SEQ ID NO:4, CYP17 promoter
Nucleotide sequence as shown in SEQ ID NO:5, the nucleotide sequence of MTNR1A (MT1)-bGH poly (A) such as SEQ ID NO:
Shown in 6.
Each purified fragments use full formula gold pEASY-Uni Seamless Cloning and Assembly Kit
(CU101-1), it carries out to specifications seamless spliced.
3 cell transfecting of embodiment
1, conventional method prepares fetal fibroblast.
2, using consideration convey mode.
With nuclear transfection-AmaxaTM Basic NucleofectorTM Kit for Primary Mammalian
Fibroblasts (Lonza), using NucleofectorTMThe program V-024 of 2b is transfected, donor vehicle and targeting vector
The step of each 10 microgram, transfection, is referring to kit specification.Transfected condition is shown in Fig. 6.
The screening and identification of 4 positive cell clone of embodiment
Behind cell culture 48 hours after consideration convey, by selected by flow cytometry apoptosis, it is as shown in Figure 7 to sort situation.By number
96 orifice plates are visible after its culture monoclonal appearance, and single cell clone group sees that Fig. 8, picking monoclonal cell to 48 orifice plates continue to train
It supports.
The monoclonal cell for expanding and cultivating is taken, DNA is extracted using Tiangeng kit, carries out positive colony using 3 primer of table
Identification.Wherein, Preliminary Identification is carried out with primer near target spot first, identifies whether be that homozygosis is knocked in, because of Insert Fragment overall length
Degree close to 4000bp, regular-PCR can not expand, be likely to occur if not expanding band near target spot homologous recombination (
It is possible that the site have occurred large fragment knock out), for removal donor plasmid residual and NHEJ mediate radom insertion influence,
Further (to guarantee precise Identification, two pairs of 1 Hes of primer long are designed altogether with the junction PCR primer across homology arm
Across the homology arm upstream long 2,5' and CYP17 promoter, 3' is across MT1 and homology arm downstream) carry out gene expression frame upstream and downstream
Amplification come determine whether fixed point insertion.If short strips (641bp) can be expanded with primer near target spot, it will appear two kinds of feelings
Condition does not carry out site-directed integration one is the site, and another kind is that site-directed integration has occurred in a chain of DNA and another is not sent out
Raw, as heterozygosis is knocked in.
3 positive clone identification primer of table
| Primer | Sequence (5'-3') |
| - 641bp-F near Ovis aries- target spot | TAGGCCAAATTTACAGATGACTGC |
| - 641bp-R near Ovis aries- target spot | AATACATAGTCCTCACCGTTG |
| 5'junction long 1-1777bp-F | TTTGGCACCATCTTGCGCTCT |
| 5'junction long 1-1777bp-R | CCTTGTAAAGCAGGCTGTCTCGTT |
| 3'junction long 1-1628bp-F | CGCAAACCCTCTCCATTAATAGCC |
| 3'junction long 1-1628bp-R | AGTTTCCCATGTCACATAGCCA |
| 5'junction long 2-1540bp-F | GCTTCCTGATGACCTGTGAACA |
| 5'junction long 2-1540bp-R | GGAGTCAGAGGTATGAGCTGGA |
| 3'junction long 2-1378bp-F | CAGCCTCGACTGTGCCTTCT |
| 3'junction long 2-1378bp-R | ATCTCCTTCGGTTCTCCGTGTT |
As a result 14+18 plants of monoclonals are grown altogether.Wherein, preceding 14 plants of qualification result is shown in Fig. 9-Figure 13, near Fig. 9 point of impact on target
Amplification show that 8#, 10# and 12# do not expand product, it may be possible to homozygosis is knocked in, but is reflected through 2 pairs of primers across homology arm
It establishes a capital and does not expand purpose band (Figure 10 and 11 pair of primers qualification result, Figure 12 and 13 are second pair of primer identification knot
Fruit), it was demonstrated that 8#, 10# and 12# are large fragment knockout, and site-directed integration does not all occur in this 14 plants of cells.
18 plants of qualification results are shown in Figure 14-Figure 17 afterwards, wherein the amplification near Figure 14 point of impact on target shows this 18 plants of cells
Band can be expanded, illustrates to knock in without homozygosis.Discovery, only 14# cell strain success are identified by 2 pairs of primers across homology arm
Upstream and downstream target fragment is amplified, therefore 14# cell strain is the heterozygosis positive cell that site-directed integration has occurred in a chain
Strain.
The present invention has been successively inserted into melatonin receptors MTNR1A gene in FGF5 target spot by CRISPR/Cas9 technology, obtains
The positive cell strain obtained can be used as the nuclear donor of body-cell neucleus transplanting, and then cultivate the gene editing clone sheep of prolificacy.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be modified or is improved, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>China Agricultural University
<120>method of CRISPR/Cas9 is mediated sheep FGF5 gene knockout and site-directed integration MTNR1A gene
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agaagccact gcaatgagaa gcctccccac tgcaactaga gtagcccctg ctacccacaa 1080
gtagaggaaa gtctgcacaa caatgaggat ccagtcaaca gtaaataaac tgtttaaaaa 1140
taaacattaa actttttaaa aaagatgtta ttcccattat agaggaggaa actgaaactt 1200
aaaacaggta agtaaccata aagttactat ctgacccagt gattccactt ttaggtattt 1260
accaccccca aaattgaaaa cagagaccca agtagatact atgttcatgc tgtggacatg 1320
tacacccatg tccatagcag cgttattcac agtagccaaa agtggaaaca acctaagtgt 1380
ccatcaactg atgaatgaag aaacaaatgg tggtataccc acttaacaga atgttgttca 1440
gtcacaaaaa ggcatggagg ggccttacct tgtagtccag tggctaagat tctacacttc 1500
taatgcagga ggctcaggtc caatccctgg ccagggaatc agaacccaca tgctgcaatg 1560
aagactgcag atcttgagta ctgcagctaa ctcccagcac aaccaaataa acaaatattt 1620
agaaaatgaa tgaagttctg acatatgcta caaagtagat gaaccttgaa aacatcatgc 1680
ctagagaaac aagccagtca caaaagacca aatgctatat gattccactt acgtgaaata 1740
ggcatattca cagacagatt agagcttacc agggactggg gggaggggaa gggggagtta 1800
ttgcttcatg gatatacttt ctgtttgggg ttgtgaaaac agttttggaa gtagacagcg 1860
gtgatggctg tacaacaact gtgaagcata ttcagtgcta ctgaattgta cactcaaatg 1920
gtaaacttca tgctaaacct tttaagtcat atagctagag agcggttaag ctagaatttg 1980
agcctaaacc catctaacaa cagggcccct gctcttgacg caaaggtttc actccccctg 2040
gcaaaagcag caactaggaa taaagagaac tggattcttg tcatcactat ctgtgtgact 2100
ttattcaagc ctgactctgg gaggcctcac tttcctcagc tgataaaatt ggatagctgt 2160
ctgttgattg tgatttcata gtacagagag tcccgagccg tgattcctga cccagatatc 2220
atttgcttct ggcctactct ggagacgttg atggacaggg agcaagggac agggactagc 2280
tgtctgtgcc cttacctagc ccctccctta ggggtgtgcc ctggtgtgga gccagctctt 2340
gaaagaaacc tcctctcccc ttccctcccc attctgggag aggactactg gctgtgctcc 2400
aggataaact tccaccaagg cgagcaataa cataaagtca aggagaaggt caggggagcc 2460
cttaaaaagg ccttcttgca 2480
<210> 6
<211> 1369
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
atggcggggc ggctgtgggg ctcgccgggc gggaccccca agggcaacgg cagcagcgcg 60
ctgctcaacg tctcgcaggc ggcgcccggc gccggggacg gtgtgcggcc gcggccctcg 120
tggctggccg ccaccctcgc ctccatcctc atcttcacca tcgtggtgga catcgtgggc 180
aacctcctgg tggtcctgtc cgtgtatcgg aacaagaagc tgaggaacgc agggaatgtg 240
tttgtggtga gcctggcagt tgcagacctg ctggtggccg tgtatccgta ccccttggcg 300
ctggcgtcta tagttaacaa tgggtggagc ctgagctccc tgcattgcca acttagtggc 360
ttcctgatgg gcttgagcgt catcgggtcc gttttcagca tcactggaat tgccatcaac 420
cgctattgct gcatctgcca cagcctcaga tacggcaagc tgtatagcgg cacgaattcc 480
ctctgctacg tgttcctgat ctggacgctg acgctcgtgg cgatcgtgcc caacctgtgt 540
gtggggaccc tgcagtacga cccgaggatc tattcctgta ccttcacgca gtccgtcagc 600
tcagcctaca cgatcgccgt ggtggtgttc catttcatag ttccgatgct cgtagtcgtc 660
ttctgttacc tgagaatctg ggccctggtt cttcaggtca gatggaaggt gaaaccggac 720
aacaaaccga aactgaagcc ccaggacttc aggaattttg tcaccatgtt tgtggttttt 780
gtcctctttg ccatttgctg ggctcctctg aacttcattg gtctcgttgt ggcctcggac 840
cccgccagca tggcacccag gatccccgag tggctgtttg tggctagtta ctatatggca 900
tatttcaaca gctgcctcaa tgcgatcata tatggactac tgaaccaaaa tttcaggcag 960
gaatacagaa aaattatagt ctcattgtgt accaccaaga tgttctttgt ggatagctcc 1020
aatcatgtag cagatagaat taaacgcaaa ccctctccat taatagccaa ccataaccta 1080
ataaaggtgg actctgttta actcgagtct agagggcccg tttaaacccg ctgatcagcc 1140
tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg 1200
accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat 1260
tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag 1320
gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatgg 1369
Claims (10)
1. based on the sgRNA of CRISPR/Cas9 technology special target sheep FGF5 gene, RNA sequence such as SEQ ID NO:1 institute
Show.
2. the CRISPR/Cas9 targeting vector containing sgRNA described in claim 1；
Preferably, skeleton carrier PX458.
3. the sheep FGF5 gene editing carrier based on CRISPR/Cas9 technological development, which is characterized in that including claim 2 institute
The CRISPR/Cas9 targeting vector and homologous recombination carrier stated；Wherein, the homologous recombination carrier includes purpose base
Because or destination gene expression box, and for the target gene to be repaired fixed point insertion sheep FGF5 gene the by homologous end
Element sequences in three exons.
4. sheep FGF5 gene editing carrier according to claim 3, which is characterized in that the target gene is MTNR1A.
5. sheep FGF5 gene editing carrier according to claim 4, which is characterized in that the knot of the destination gene expression box
Structure are as follows: ovary-specific promoter CYP17-MTNR1A-bGH poly (A).
6. sheep FGF5 gene editing carrier according to claim 3, which is characterized in that the element sequences include being used for sheep
The left and right homology arm of FGF5 gene targeting site homologous recombination, nucleotide sequence is respectively such as SEQ ID NO:3 and SEQ ID
Shown in NO:4.
7. sheep FGF5 gene editing carrier according to claim 6, which is characterized in that the homologous recombination carrier packet
Containing such as flowering structure: left homology arm-ovary-specific promoter CYP17-MTNR1A-bGH poly (A)-right homology arm；
Preferably, the target gene and element sequences come from same species.
8. the cell of sheep FGF5 gene knockout and site-directed integration MTNR1A gene, which is characterized in that by any one of claim 3-7
It is obtained in the sheep FGF5 gene editing vector introduction sheep fetal fibroblast.
It is carried 9. sgRNA, the CRISPR/Cas9 as claimed in claim 2 of targeting sheep FGF5 gene described in claim 1 practice shooting
The described in any item sheep FGF5 gene editing carriers of body, claim 3-7 or cell according to any one of claims 8 are preparing gene volume
Collect the application in clone sheep.
10.CRISPR/Cas9 the method for the sheep FGF5 gene knockout and site-directed integration MTNR1A gene that mediate, which is characterized in that
CRISPR/Cas9 targeting vector as claimed in claim 2 and homologous recombination carrier are transferred to sheep placenta into fiber finer jointly
In born of the same parents, the cell of sheep FGF5 gene knockout and site-directed integration MTNR1A gene is obtained；
Wherein, the definition of the homologous recombination carrier is the same as described in claim any one of 3-7.
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| CN112553207A (en) * | 2020-12-30 | 2021-03-26 | 新疆畜牧科学院生物技术研究所（新疆畜牧科学院中国-澳大利亚绵羊育种研究中心） | sgRNA for realizing accurate mutation of sheep FGF5 gene, kit and application |
| CN112553207B (en) * | 2020-12-30 | 2023-05-16 | 新疆畜牧科学院生物技术研究所（新疆畜牧科学院中国-澳大利亚绵羊育种研究中心） | sgRNA, kit and application for realizing accurate mutation of sheep FGF5 gene |
| CN112779340A (en) * | 2021-02-01 | 2021-05-11 | 新疆农垦科学院 | Haplotype molecular marker related to sheep high fertility, screening method and application |
| CN112779340B (en) * | 2021-02-01 | 2023-05-16 | 新疆农垦科学院 | Haplotype molecular marker related to sheep high fertility, screening method and application |
| CN114525350A (en) * | 2022-04-22 | 2022-05-24 | 三亚中国农业大学研究院 | SNP (Single nucleotide polymorphism) marker related to Hu sheep melatonin character and application thereof |
| CN115305256A (en) * | 2022-06-07 | 2022-11-08 | 中国农业大学 | Method for improving sheep ovary reserve |
| CN118562881A (en) * | 2024-05-19 | 2024-08-30 | 中国人民解放军军事科学院军事医学研究院 | Method for preparing goat fetal fibroblast strain with directionally integrated recombinant human butyrylcholinesterase by targeting milk goat CSN2 locus |
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