CN105950626B - The method of different hair color sheep is obtained based on CRISPR/Cas9 and targets the sgRNA of ASIP genes - Google Patents
The method of different hair color sheep is obtained based on CRISPR/Cas9 and targets the sgRNA of ASIP genes Download PDFInfo
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Abstract
The invention discloses a kind of to obtain the method for different hair color sheep based on CRISPR/Cas9 and targeting the sgRNA of ASIP genes.The present invention provides a kind of sgRNA (ASIP sgRNA) of targeting modification sheep ASIP genes that can be special, be sequence table the RNA shown in the nucleotide of 5 ' end the 3rd to 22 of sequence 4 or the sequence 4 with sequence table from the RNA of 5 ' the 3rd to 22 nucleotide in end.In the embodiment of the present invention, the ASIP sgRNA concretely RNA shown in the sequence 4 of sequence table.The present invention also protects a kind of method for the sheep that acquisition hair color changes, and includes the following steps：By sgRNA the and Cas9mRNA cotransfection ovine cells of the targeting modification sheep ASIP genes that can be special, to knock out sheep ASIP genes, the sheep of hair color change is obtained.New CRISPR/Cas9 genome editing techniques are combined by the present invention with microinjection technique, and effective technological means is provided for the artificial sheep hair color that changes.
Description
Technical field
The invention belongs to animal genetic engineering fields, are related to CRISPR/Cas9 technologies, and in particular to one kind is based on
CRISPR/Cas9 gene Knockouts obtain the method for different hair color sheep and for selectively targeted ASIP genetic modifications
sgRNA。
Background technology
Genome manipulation technology be in recent years based on genome and gene information technology grow up by engineer reality
The cutting edge technology that accurate edits are now carried out to specific gene or genome target site has become biomedical, agricultural and moves at present
The research hotspot in the fields such as object breeding and model animal.In animal breeding field, to cultivate a kind or establish one specially
Change strain, it is necessary to improve its production performance, premised on stablizing its hereditary basis.In the breeding work of sheep, Coat Color is
A kind of important varietal characteristic and the production traits are determining that cross combination, variety and affiliation etc. are significant,
It acts on and protruding especially on obtaining required hair color type by crossbreeding.Therefore, it is grasped using the genome of efficient stable
It is most important that work is educated as technology changing sheep hair color to sheep's wool color sorting.
In recent years, scientists have invented the genome based on CRISPR/Cas9 according to the principle of bacterium acquired immunity
New technology is edited, the difficulty for carrying out gene knockout, genetic modification to animal is not only greatly reduced, even more by Animal Transgenic skill
Art has been pushed to the precisely modifications such as the deletion of genome orientation, insertion or the replacement of high precision by traditional random integration, is started
The new era of transgenic animals production.CRISPR/Cas9 systems are that a ribonucleoprotein being made of nucleic acid and protein is answered
Object is closed, it depends on identification of the nucleic acid to nucleic acid to the identification of target spot, is completed by the complementary pairing of base.One site of target practice
It only needs to design the oligonucleotides for synthesizing 20bp or so according to target sequence.This identification based on base pair complementarity principle,
Being compared to the interaction between protein and DNA will more stablize and simply, can realize once-through operation simultaneous mutation two
A above gene or site substantially increase genome editing technique efficiency.
Core component due to playing active function in CRISPR/Cas9 systems is sgRNA and protein, can be led to
Cross carrier construction, after in-vitro transcription obtains RNA, microinjection fertilised non-human eggs and obtain target practice animal, in entire target practice process
In be not present exogenous DNA integration., will not be in long-term existence organism and due to the unstability of mRNA, it will not be to ring
Border, which generates, further to be influenced, thus can be to avoid bio-safety problem caused by traditional transgenosis.That is, CRISPR/
What Cas9 technologies were modified is the gene of a species itself, takes mRNA or RNA as gene targeting raw material, without any sieve
Resistant gene is selected, because biosafety issues may be not present.Moreover, the final products obtained have passed through genetic modification
(by transgene method pointed decoration, because this system is in bacterium, dynamic plant is gone to by transgene method
In object), but the ingredient of any transgenosis can not included in finished product, substantially increase safety.So being planted dynamic at present
In the preparation of object rearing new variety, especially transgenic animals, the targeting system that CRISPR/Cas9 is mediated widely is adopted by researcher
With.
ASIP (agouti signalling protein, wild ash site signal albumen) gene is compiled by the sites agouti
A kind of signal protein of code is the main candidate for influencing sheep hair color, important regulating and controlling effect is played in pigment is formed, it
With α-melanocyte-stimulating hormone (α-MSH) competitive binding Melanocortin receptor 1 (melanocortin-1, MC1R), make
MC1R structures change, and inhibit cyclic adenosine monophosphate (cAMP) enzyme system, and cAMP levels is caused to decline, and by cascade reaction, promote brown
The generation of melanocyte.In sheep, ASIP genes are positioned on No. 13 chromosomes, and there are multiple in ovine genome for the gene
Copy and multiple allele, coded sequence is mainly by 3 ', 5 ' untranslated district's groups of 3 exons, 2 intrones and part
At.The ASIP albumen of sheep Agouti gene codes is by 133 Amino acid profiles, by a segment signal peptide and function amino acid group
At.Numerous studies have demonstrated that there are close correlations for the hair color of ASIP genes and sheep.Norris and Whan is disclosed
The white hair of sheep is with the repetition of 190kbp in ASIP genomes related, and the black hair of sheep is then in Second Exon
On missing it is related with the missense mutation on the 4th exon.In addition, change of the different sheep varieties due to ASIP gene copy numbers
Different and missense mutation will lead to the appearance of different type hair color.
Since fine-wool sheep is albino through long-term breeding, by novel gene group editing technique knock out ASIP genes from
And change fine-wool sheep hair color, to cultivating colored fine-wool sheep new varieties or new type, the molecular basis for furtheing investigate the gene genetic,
Sheep variety improvement, selection and breeding is instructed to have important use.
Invention content
The object of the present invention is to provide a kind of sides obtaining different hair color sheep based on CRISPR/Cas9 gene Knockouts
Method and sgRNA for selectively targeted ASIP genetic modifications.
The present invention provides a kind of sgRNA (ASIP-sgRNA) of targeting modification sheep ASIP genes that can be special, are
The sequence 4 of sequence table from RNA shown in the nucleotide of 5 ' end the 3rd to 22 or the sequence 4 with sequence table from 5 ' ends the 3rd to
The RNA of 22 nucleotide.In the embodiment of the present invention, the ASIP-sgRNA is concretely shown in the sequence 4 of sequence table
RNA。
The present invention also protects the DNA molecular for encoding the ASIP-sgRNA.Encode the DNA molecular tool of the ASIP-sgRNA
Body can be DNA molecular shown in the sequence 3 of sequence table.
The present invention also protects a kind of targeting sequence of targeting modification sheep ASIP genes that can be special, is the sequence of sequence table
Row 1 are from the nucleotide of 5 ' end the 359th to 378.
The present invention also protects a kind of complete nucleic acid molecules of specific knockdown sheep ASIP genes, including the sgRNA.
The complete nucleic acid molecules may also include Cas9mRNA.The Cas9mRNA is Cas9 albumen shown in the sequence 6 of polynucleotide
RNA.The Cas9mRNA concretely has the sequence 7 of sequence table from the RNA of 5 ' the 7th to 4278 nucleotide in end, more
Concretely RNA shown in the sequence 7 of sequence table.
The present invention also protects a kind of complete nucleic acid molecules of specific knockdown sheep ASIP genes, including the DNA points
Son.The complete DNA molecule may also include the DNA molecular of coding Cas9mRNA.The Cas9mRNA is code sequence
The RNA of Cas9 albumen shown in the sequence 6 of list.The Cas9mRNA concretely the sequence 7 with sequence table from 5 ' ends the 7th
More specifically can be RNA shown in the sequence 7 of sequence table to the RNA of 4278 nucleotide.Encode the DNA molecular tool of Cas9mRNA
Body can be DNA molecular of the sequence 5 with sequence table from 5 ' the 24th to 4295 nucleotide in end, more specifically can be sequence table
Molecule shown in sequence 5.
The present invention also protects a kind of method of specific knockdown sheep ASIP genes, includes the following steps：It can by described in
SgRNA the and Cas9mRNA cotransfection ovine cells of special targeting modification sheep ASIP genes, to knock out sheep ASIP bases
Cause.The Cas9mRNA is the RNA of Cas9 albumen shown in the sequence 6 of polynucleotide.The Cas9mRNA concretely has
The sequence 7 of sequence table more specifically can be RNA shown in the sequence 7 of sequence table from the RNA of 5 ' the 7th to 4278 nucleotide in end.
The concretely co-injection of the mode of the cotransfection.Concretely sheep zygotes are unicellular for the ovine cells.
The present invention also protects a kind of method for the sheep that acquisition hair color changes, and includes the following steps：It can be special by described in
SgRNA the and Cas9mRNA cotransfection ovine cells of targeting modification sheep ASIP genes obtained to knock out sheep ASIP genes
The sheep changed to hair color.
The sheep that the hair color changes is specific as follows：Hair color is the sheep of black, hair color is that black-and-white flower (spend by black/white spot
Line) sheep or sheep that hair color is brown white flower (brown/white decorative pattern).
Any description above sheep ASIP genes can be the gene of protein shown in the sequence 2 of polynucleotide.More than
Any sheep ASIP genes concretely code area DNA molecular or the sequence of sequence table 1 as shown in the sequence 8 of sequence table
Shown in DNA molecular or the sequence of sequence table 1 DNA molecular shown in the nucleotide of 5 ' end the 222nd to 1398 or such as
(VERSION NC_019470.1 shown in GENBANK ACCESSION NO.NC_019470；GI:417531912；linear
CON 03-APR-2015) DNA molecular.
Any description above sheep concretely Xinjiang Merino.
It is still very high to realize that the cost of modification and transformation and technology require on big Animal genome at present, therefore obtains special
Different, efficient sgRNA becomes the key that ovine genome editor cultivates.SgRNA specificity provided by the invention is high and being capable of essence
True targeting modification sheep ASIP genes, realize gene mutation.
In the present invention, CRISPR/Cas9 technologies is used to realize the accurate modification of ASIP genes in sheep zygotes for the first time
It is mutated and obtains the gene editing sheep of different hair colors, not only construction step is simple with this method, safe, Er Qie great
It is big to reduce expensive experimental cost and shorten experimental period, realize the accurate editor of wool color genes of sheep, obtain black,
Black/white grivelle and brown/white decorative pattern fine-wool sheep.
New CRISPR/Cas9 genome editing techniques are combined by the present invention with microinjection technique, are changed to be artificial
Sheep hair color provides effective technological means.
Description of the drawings
Fig. 1 is 1% agarose gel electrophoresis figure after restriction enzyme BbsI single endonuclease digestion px330 plasmids；Swimming lane M is
1kbDNA Marker。
Fig. 2 is the gel electrophoresis figure of in-vitro transcription product ASIP-sgRNA；Swimming lane M is RNA Marker, and swimming lane 1 is
sgRNAASIP。
Fig. 3 is the amplified production electrophoretogram that PCR amplification is carried out using the primer pair that Cas9-F and Cas9-R is formed；Swimming lane M
For 1kb DNA Marker.
Fig. 4 is the gel electrophoresis figure of Cas9mRNA；Swimming lane M is RNA Marker.
Fig. 5 is that sheep embryo PCR product carries out the electrophoretogram after T7EN1 digestions；1-19 is test process group, and Con1-5 is
Control treatment group, swimming lane M are 100bp DNA Marker.
Fig. 6 is sheep embryo PCR product sequencing result；In wild type (WT), red base sequence (lower stroke of straight line) is marked
For the target sequence of sgRNA, green base sequence TGG (lower stroke of wave) is the PAM sequences that ASIP gene Cs as9 practices shooting；Under " ^ "
Fang Hongse bases indicate that insertion base, "-" indicate to delete base, and mutating alkali yl (box) is indicated with red.
Fig. 7 is to inject the different hair color lamb photos being born after ASIP-Cas9mRNA.
Fig. 8 is the electrophoretogram of lamb ASIP gene PCR products；Con1-3 represents the lamb of non-injection group of life, and swimming lane M is
100bp DNA Marker。
Fig. 9 is the edit format for being mutated lamb；In wild type (WT), marking red base sequence (lower stroke of straight line) is
The target sequence of sgRNA, green base sequence TGG (lower stroke of wave) are the PAM sequences that ASIP gene Cs as9 practices shooting；Below " ^ "
Red base indicates that insertion base, "-" indicate to delete base, and mutating alkali yl (box) is indicated with blue.
Specific implementation mode
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention, in addition, it should also be understood that, reading
After the content that the present invention lectures, those skilled in the art can make various modifications or changes to the present invention, such equivalent forms
It also falls within the scope of the appended claims of the present application.Experimental method in following embodiments, unless otherwise specified,
It is conventional method, refers to《Molecular cloning (third edition)》.Test material as used in the following examples, unless otherwise specified,
It is to be commercially available from conventional biochemical reagent company.Px330 plasmids：Addgene companies, article No. 42330.RNA purifying examinations
Agent box：Life Technologies companies, article No. AM1908.
The nucleotide sequence of relevant primer in embodiment is shown in Table 1.
The nucleotide sequence of 1 ASIP gene-correlation primers of table
| Primer | Sequence (5 ' to 3 ') |
| ASIP-CF | caccgTTTCCCTTCTGTCTCTATCG |
| ASIP-CR | aaacCGATAGAGACAGAAGGGAAAc |
| ASIP-TF | ttaatacgactcactataggTTTCCCTTCTGTCTCTATCG |
| ASIP-TR | aaaagcaccgactcggtgcc |
| ASIP-S | CCAAGGAAACAAAGAAAGCAG |
| ASIP-AS | AACCAAACAAGTTAAGGGACA |
| ASIP-in-S | CCTTCTCTGTCGCTCTCAAGCCTCC |
| ASIP-in-AS | CTGAGGAATGAGCACAAAGGA |
Note：Each primer is single strand dna.
Embodiment 1 prepares sgRNA and Cas9mRNA
One, it designs sheep ASIP genes target sequence and identifies the sgRNA of target sequence
Sheep ASIP full length genes sequence (VERSION as shown in GENBANK ACCESSION NO.NC_019470
NC_019470.1；GI:417531912；linear CON 03-APR-2015).
The sequence 1 of sequence table is the partial sector of sheep ASIP genes, wherein there are three exons (222-381 for tool
Nucleotide, 795-859 nucleotide, 1222-1398 nucleotide), protein shown in the sequence 2 of polynucleotide.
By a large amount of trial tests and verification test, select in the sequence 1 of sequence table 359-378 nucleotide as
SgRNA is directed to the target sequence of sheep ASIP genes.
Two, sgRNA is preparedASIP
1, with restriction enzyme BbsI enzyme single endonuclease digestion px330 plasmids, 1% agarose gel electrophoresis (digestion is then carried out
1% agarose gel electrophoresis figure of product is shown in Fig. 1), the plasmid of gel extraction and purified linear.
2, the purified product of step 1 is subjected to dephosphorylation.
3, by primer ASIP-CF and primer ASIP-CR annealing, obtain to both ends be viscous end double chain DNA molecule.
4, the product of step 2 is connect with the double chain DNA molecule that step 3 obtains, obtains recombinant plasmid.
5, the recombinant plasmid obtained using step 4 carries out PCR as template using the primer pair that ASIP-TF and ASIP-TR is formed
Amplification, obtains pcr amplification product.Through sequencing, pcr amplification product is as shown in the sequence 3 of sequence table.
6, the pcr amplification product for taking step 5 to obtain, using in-vitro transcription kit (
T7Kit, Life Technologies companies, article No. AM1354) in-vitro transcription is carried out, then use RNA purification kits pure
Change recycling, obtains ASIP-sgRNA.The gel electrophoresis figure of ASIP-sgRNA is shown in Fig. 2.4 institute of sequence of ASIP-sgRNA such as sequence tables
Show.
Three, Cas9mRNA is prepared
1, using px330 plasmids as template, using the primer of Cas9-F (underscore marks T7 promoters) and Cas9-R compositions
To carrying out PCR amplification, pcr amplification product (4311bp) is obtained.The electrophoretogram of pcr amplification product is shown in Fig. 3.Through sequencing, PCR amplification
Product is as shown in the sequence 5 of sequence table.In the sequence 5 of sequence table, from the opening that the nucleotide of 5 ' end 24-4295 is Cas9
Reading frame.Cas9 albumen shown in the sequence 6 of polynucleotide.
Cas9-F：5’-TAATACGACTCACTATAGGGAGAATGGACTATAAGGACCACGAC-3’；
Cas9-R：5’-GCGAGCTCTAGGAATTCTTAC-3’.
2, the pcr amplification product for taking step 1 to obtain, using in-vitro transcription kit (Life Technologies companiesT7Ultra Kit, article No. AM1345) in-vitro transcription is carried out, then use RNA purified reagents
Box purifying recycling, obtains Cas9mRNA.The gel electrophoresis figure of Cas9mRNA is shown in Fig. 4.7 institute of sequence of Cas9mRNA such as sequence tables
Show.Cas9mRNA from the nucleotide of 5 ' end the 7th to 4278 be code area.
Embodiment 2, injection sgRNA/Cas9mRNA embryo's mutation efficiencies detection
One, the acquisition of sheep zygotes
1, the maturation of egg mother cell
From slaughterhouse acquisition Sheep Ovary (ovary come from Kazakh sheep), with physiological saline sterilizing and washing 3-4 time, extraction ovum
Mother cell is washed 3-4 times with ripe liquid, and (volume of ripe drop is 75-78 μ l to the ripe drop that then instillation has balanced, often
Drop instills 25-30 pieces of egg mother cell), it is put into containing 5%CO238.6 DEG C of incubators in culture (following incubator culture is this
The same terms).
The ripe liquid of balance：Ripe drop is placed into 2h in the incubator.Ripe liquid：TCM199 culture solutions+volume basis contains
The FBS+0.05IU/ml FSH+0.05IU/ml LH+1 μ g/ml estradiol+24.2 μ g/ml Sodium Pyruvates that amount is 10%+
0.1mM/L cysteine+10ng/ml EGF+100IU/ml penicillin+100IU/ml streptomysins.
2, egg mother cell is in vitro fertilization
(1) egg mother cell of maturation in vitro 24-26h is taken out, is gently blown and beaten with 0.1% hyaluronidase to remove degranulation
Cell, then with by semen washing 3 times, be then placed in the fertilization drop balanced (each fertilization drop be 50-70 μ l in vitro by
Sperm is put into 20-30 pieces of egg mother cell).
Balance is by sperm：Fertilization drop is placed into 3-4h in the incubator.By sperm：SOF liquid+volumn concentration is
20% heat sheep blood serum+6IU/ml heparin sodium+100IU/ml gentamicins.SOF liquid：NaCl containing 6.29mg/ml, 0.534mg/
ml KCl、0.162mg/ml KH2PO4, 0.6 μ l/ml sodium lactates, 0.089mg/ml MgSO4、2.1mg/ml NaHCO3、
0.0357mg/ml Sodium Pyruvates and 0.299mg/ml CaCL2·2H2O, solvent are water.
(2) sperm (sperm come from Kazakh sheep) of freezing, water-bath is taken to thaw, move into balanced in by sperm, be put into
Then 20-25min (great-hearted sperm can be upstream) in incubator draws the sperm on top, centrifuged with the rotating speed of 1500rpm
4-5min discards supernatant liquid, obtains Sperm pellets (carrying out sperm count).
(3) sperm for obtaining step (2) is added in the fertilization drop for completing step (1), makes a concentration of (2-4) of sperm
×106A/ml, 38.6 DEG C of stationary incubation 12-18h, with the culture solution balanced pressure-vaccum fertilized eggs repeatedly, then press 50-70 pieces/
The density in hole moves into four well culture plates.
The preparation method of the culture solution balanced：In the incubator by culture solution, 3-4h is placed.Culture solution：SOF liquid+
3mg/ml BSA。
Two, the microinjection of single-cell zygotes
1, ASIP-sgRNA and the Cas9mRNA mixing prepared embodiment 1 is (eventually with Nuclease-free Water adjustment
Concentration is respectively 50ng/ μ L and 100ng/ μ L).
2, test process：The mixed liquor that step 1 obtains step 1 is injected into using the microinjection instrument of NIKON companies to obtain
In the cytoplasm of the fertilized eggs in one cell stage arrived (each fertilized eggs injection 80-100pL mixed liquors), it is placed in incubator and trains
It supports.Control treatment：Nuclease-free Water are injected into what step 1 obtained using the microinjection instrument of NIKON companies
In cytuloplasm in one cell stage, it is placed in incubator and cultivates.
3, embryo's target gene editorial efficiency detects
(1) embryo samples are collected
In step 2 cultivate 7d after, take embryo, washed 2 times with PBS buffer solution, is subsequently placed in 5 μ L lysates, instantaneously from
37 DEG C of incubation 3h after the heart.Lysate：Solvent is Tris-HCl (50mM, pH8.0), containing 0.5% (V/V) Triton X-100 and
1mg/mL Proteinase K。
(2) PCR amplification
Nest-type PRC is carried out by template of the pyrolysis product of step (1).First round PCR amplification uses ASIP-S and ASIP-AS
The primer pair (pcr amplified fragment length is 426bp) of composition, the second wheel PCR amplification use ASIP-in-S and ASIP-in-AS groups
At primer pair (pcr amplified fragment length be 322bp).
(3) T7 endonucleases (T7EN1) and sequencing identification
The control group and test group PCR product mixed in equal amounts that step (2) is obtained respectively carry out denaturation annealing, are formed different
Source heteroduplex.Cycle of annealing：95℃10min；85 DEG C, 75 DEG C, 65 DEG C, 55 DEG C, 45 DEG C, 35 DEG C, 25 DEG C of each 1min (cooling speed
0.3 DEG C/s of rate)；10℃Pause.
T7EN1 enzymes (NEB), 37 DEG C of incubation 30min are added in annealed product, the agarose that digestion products carry out 2% coagulates
Gel electrophoresis.
T7EN1 digestion results are shown in Fig. 5, the sample that mutates in test group occur two kinds of segments (about 183bp and
139bp), and in test group only a kind of segment (322bp) of the sample and control group not mutated.
The pcr amplification product that above-mentioned steps (2) obtain is sequenced.The sequencing comparison result of part pcr amplification product
As shown in Figure 6.PAM sequences are adjoined in the position that ASIP gene mutations occur, and mutation type includes the deletion of 2-10bp bases, 2-
The forms such as the insertion of 8bp bases and base replacement.
The sequencing result of whole pcr amplification products shows：ASIP gene elminations/insertion mutation efficiency occurs for sheep embryo
55.96% (61/109), and gene knockout develops without lethal sheep embryo, it was demonstrated that DNA molecular is special by Cas9 and sgRNA
Property editor.
The production of embodiment 3, gene editing sheep
1, experiment sheep selection
Selection body condition is excellent, does meat sheep used as donor without reproductive diseases and in 2-4 Sui Xinjiang Merino.Weight is chosen in 50kg
More than, the age is 2-4 Sui, and Altai Sheeps in good condition, without reproductive diseases do acceptor ewe.Weight is chosen in 70-85kg, sperm
It detects excellent and does semen collection ram in 1-3 Sui Xinjiang Merino.
2, estrus synchronization and superfecundation
In the sheep oestrous cycle, meat sheep used as donor vagina is put into CIDR vaginal plugs, and be put into CIDR vaginal plugs the 10th day starts
FSH (ningbo of china three lives company) is continuously injected in a manner of successively decreasing, primary every 12h injections, totally 3 days, accumulated dose was 240 single
Position/only, CIDR bolts were taken out the 12nd day morning, clean vagina, and intramuscular injection PG 0.1mg (ningbo of china three lives company).It removes
Start to try feelings with ram after bolt 12h, each examination feelings are primary sooner or later, are spaced 12h, when meat sheep used as donor heat LH injection 200IU/ only (in
State Ningbo three lives company).
Acceptor ewe heeling-in CIDR synchronous with meat sheep used as donor, 12h removes CIDR, every injection before meat sheep used as donor removes bolt
330IU PMSG (ningbo of china three lives company) are removed after bolt 12h daily and are tried feelings, in detail record hair with examination feelings ram each 2 times sooner or later
The feelings time.
3, artificial insemination
The sperm of artificial semenpicking ram, microscopy, motility rate can be used for semen deposition up to 0.8 or more.To the donor of heat 12-19h
Ewe carries out artificial insemination.
4, the acquisition of protokaryon embryo
19-21h after meat sheep used as donor semen deposition, modus operandi rush from fallopian tubal and take protokaryon embryo.Pick out cytoplasm uniformly, form rule
Then, the protokaryon embryo (one cell stage) of the complete and fine and close non-spilting of an egg.
5, ASIP-sgRNA and the Cas9mRNA mixing prepared embodiment 1 is (eventually with Nuclease-free Water adjustment
Concentration is respectively 50ng/ μ L and 100ng/ μ L).
6, test process：The mixed liquor that step 5 obtains step 4 is injected into using the microinjection instrument of NIKON companies to obtain
In the cytoplasm of the fertilized eggs in one cell stage arrived (each fertilized eggs injection 80-100pL mixed liquors), it is placed in incubator and trains
It supports.Non-injection group replaces the mixed liquor with isometric Nuclease-free Water.
7, embryo transfer and gestation detection
When protokaryon ovigerm is split to 2-4 cells, by the fallopian tubal of the acceptor ewe of embryo transfer to Estrus synchronization, often
Oviductus lateralis transplants 1-2 pieces of embryo, and transplanting carries out B ultrasound cyesiognosis after 60 days to acceptor ewe.
8, the identification of ASIP gene sheep is deleted in targeting
6 lambs are obtained after the production of test process group acceptor ewe, are respectively designated as GM081 (hair color is black), GM105
(hair color is black-and-white flower), GM106 (hair color is black), GM108 (hair color is white), GM109 (hair color is black-and-white flower), GM110
(hair color is brown white flower).It finds that trait expression is apparent after lamb birth, four kinds of different hair colors is substantially presented：Black, black and white, palm fibre
White and white, are partly shown in Fig. 7.
6 lambs are obtained after non-injection group of acceptor ewe production, are white.
(1) DNA extractions and PCR amplification
It is born or so latter week, acquisition test process group lamb ear tissue sample (while acquiring the non-note of same time birth
A group lamb ear tissue is penetrated to compare), genomic DNA is extracted, PCR amplification is carried out with the ASIP-S and ASIP-AS primer pairs formed
(the PCR product fragment length of amplification is 426bp).The electrophoretogram of PCR product is shown in Fig. 8.
(2) TA cloning and sequencings compare
Above-mentioned lamb ear tissue PCR product is cloned into pMD-19T carriers respectively, 20-30 monoclonal of random picking is surveyed
Sequence is accurately positioned mutational site.The edit format of the lamb of mutation is shown in that (annotation form after sequence is " n/m ", n to Fig. 9
Indicate that the edit format quantity, m indicate the monoclonal sum of institute's picking).The total 144 monoclonals sequencing of 6 lambs of picking, is compiled
Types results (being shown in Table 2) are collected to show：It is wild type that GM108 lambs, which survey 19 monoclonals, and 125 lists of remaining lamb
PAM sequences are adjoined in the position that ASIP gene mutations occur for clone, including 10 kinds of mutant forms, mutation type is mainly with 4bp
It based on the deletion of 2bp bases, is secondly deleted for 27bp bases, longest deletes segment as 27bp.Delete the lamb of 4bp and 2bp bases
Black or black and white is presented in wool color more, and coloring is deeper, and the GM108 lamb hair colors not mutated are white.
Table 2 injects ASIP-Cas9mRNA gene editing sheep difference editing types statistics
Comprehensive PCR sequencings and TA cloning and sequencings are deleted as a result, there is 5 lambs that ASIP genes occur in 6 lambs of production
Except/insertion mutation, positive rate is up to 83.33% (being shown in Table 3), and grows fine at present.
ASIP gene mRNA microinjection producer gene editor's sheep results statistics is deleted in table 3 CRISPR/Cas9 targetings
Claims (8)
1. the sgRNA of targeting modification sheep ASIP genes that can be special is RNA shown in the sequence 4 of sequence table.
2. encoding the DNA molecular of sgRNA described in claim 1.
3. a kind of complete nucleic acid molecules of specific knockdown sheep ASIP genes, including sgRNA described in claim 1.
4. a kind of complete nucleic acid molecules of specific knockdown sheep ASIP genes, including sgRNA described in claim 1 and
Cas9mRNA。
5. a kind of complete nucleic acid molecules of specific knockdown sheep ASIP genes, including the DNA molecular described in claim 2.
6. a kind of complete nucleic acid molecules of specific knockdown sheep ASIP genes, including the DNA described in claim 2 and coding
The DNA molecular of Cas9mRNA.
7. a kind of method of specific knockdown sheep ASIP genes, includes the following steps：By sgRNA described in claim 1 and
Cas9mRNA cotransfection ovine cells, to knock out sheep ASIP genes.
8. a kind of method obtaining the sheep that hair color changes, includes the following steps：By sgRNA described in claim 1 and
Cas9mRNA cotransfection ovine cells obtain the sheep of hair color change to knock out sheep ASIP genes.
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