CN117305303A - Method for cultivating high-fertility fine wool sheep - Google Patents
Method for cultivating high-fertility fine wool sheep Download PDFInfo
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- CN117305303A CN117305303A CN202311225209.3A CN202311225209A CN117305303A CN 117305303 A CN117305303 A CN 117305303A CN 202311225209 A CN202311225209 A CN 202311225209A CN 117305303 A CN117305303 A CN 117305303A
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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
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
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- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
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Abstract
The invention provides a method for cultivating high-fertility fine wool sheep, and belongs to the technical field of genetic engineering. The invention utilizes CRISPR/Cas9 mediated HDR to obtain FecB gene editing fine wool sheep (marked as F) 0 Generation), and obtaining F by propagation 1 Editing the filigree sheep by the substitution gene; by F 0 FecB gene editing ram of generation homozygous genotype (BB) is used as a breeding ram, and F is used as the breeding ram 1 FecB heterozygous gene editing ewe and/or BB homozygous genotype FecB gene editing ewe in the generation gene editing fine wool sheep is used as a mating ewe for artificial insemination or natural mating to obtain F 2 Substitution gene editingSheep with fine wool; the F is 2 The average reproduction rate of the generation gene editing fine wool sheep reaches 171 percent, which is equivalent to the lambing rate of the naturally mutated high-fertility FecB heterozygote, greatly shortens the breeding time and the generation interval, improves the reproduction performance of the fine wool sheep, and quickens the breeding progress of the fine wool sheep.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a method for cultivating high-fertility fine wool sheep.
Background
The gene editing technique refers to a technique for performing precise modification and directed editing of genetic material by artificially designing to achieve insertion, deletion or substitution of a specific gene or genomic target site. At present, the third generation gene editing technology represented by CRISPR/Cas9 has been widely used for research in the fields of livestock genome editing, biomedicine, human disease model construction and the like. CRISPR/Cas9 systems induce Double Strand Breaks (DSBs) that can be repaired by non-homologous end joining (NHEJ) or Homology Directed Repair (HDR) pathways. The NHEJ and the HDR act simultaneously, the HDR efficiency is improved due to the fact that the activity of the NHEJ is reduced, and small-molecule compounds act on the selection of the HDR channel and the NHEJ channel of DNA repair, so that the efficiency of the NHEJ in the DNA repair channel can be inhibited, and the HDR efficiency is improved. Many Single Nucleotide Polymorphisms (SNPs) are reported to be associated with important economic traits in livestock species. Thus, the addition of small molecule compounds to introduce precise single nucleotide substitutions via HDR is critical for the overall analysis of animal trait phenotypes and gene functions.
Reproductive traits of sheep are important economic traits of sheep. Among nearly 700 varieties in the sheep variety warehouse, most of the sheep varieties belong to seasonal oestrus, annual birth of one embryo and single lamb per embryo except for few sheep varieties with high lambing rate, premature sexual maturity and perennial oestrus. Therefore, how to effectively improve sheep fertility has become a very interesting research direction. The FecB (Fecundity booroola) gene is the first multiple-fetal major gene found in sheep. The FecB gene is located on autosomal sheep and has the biological effects of improving ovulation rate, lambing number and the like. The research shows that the FecB gene is obtained by mutating the base of the BMPR-IB gene A746G, and analysis of the amino acid sequence of the mutation site shows that partial functional inactivation of the gene finally leads to the reduction of the inhibition effect of ligand on the secretion of progesterone by granulosa cells, thereby promoting follicle maturation and the differentiation of granulosa cells and increasing the ovulation number, and further leading to the appearance of sheep high fertility phenotype.
At present, for single sheep, the propagation trait belongs to a low genetic trait, and obvious genetic progress is difficult to obtain in a short time through traditional phenotype breeding.
Disclosure of Invention
The invention aims to provide a method for cultivating high-fertility fine wool sheep, which can be used for cultivating high-fertility fine wool sheep efficiently.
The invention provides a CRISPR/Cas9 mediated homologous directional repair editing sheep FecB gene ssodN, wherein the nucleotide sequence of the ssodN is shown as SEQ ID NO. 2.
The invention also provides a CRISPR/Cas9 mediated reagent for homologous directional repair editing of sheep FecB genes, which comprises the following components: sgRNA, ssODN, cas9 mRNA and DNA ligase IV inhibitor SCR7; the nucleotide sequence of the sgRNA is shown as SEQ ID NO. 1; the nucleotide sequence of the ssODN is shown in SEQ ID No. 2; the nucleotide sequence of the Cas9 mRNA is shown as SEQ ID NO. 3.
The invention also provides a method for cultivating the high-fertility fine wool sheep, which comprises the following steps:
introducing sgRNA, ssODN, cas mRNA and a DNA ligase IV inhibitor SCR7 into fertilized eggs of the fine-wool sheep to obtain FecB gene editing fine-wool sheep;
FecB gene edited fine wool sheep as F 0 Instead of F 0 FecB gene editing ram of BB homozygous genotype is used as a ram for mating, F 0 The female sheep edited by the FecB gene and the wild female sheep are respectively used as mating female sheep for artificial insemination or natural mating to obtain F 1 Editing the filigree sheep by the substitution gene;
by F 0 FecB gene editing ram of BB homozygous genotype is used as a ram for mating, and F is used as the gene 1 FecB heterozygous gene editing ewe and/or BB homozygous genotype FecB gene editing ewe in the generation gene editing fine wool sheep is used as a mating ewe for artificial insemination or natural mating to obtain F 2 Editing the filigree sheep by the substitution gene; the F is 2 The generation gene editing fine wool sheep is high-fertility fine wool sheep.
The invention also provides application of the ssODN or the reagent or the method in breeding high-fertility fine wool sheep and/or creating multiple-embryo fine wool sheep varieties.
Preferably, the nap sheep comprises a wild type nap sheep with the base at position 746 of exon 8 of the FecB gene being A.
Preferably, the fine wool sheep comprises merino fine wool sheep.
Preferably, the merino fine wool sheep includes merino fine wool sheep (Xinjiang type).
The invention provides a CRISPR/Cas9 mediated homozygous directional repair editing sheep FecB gene ssODN. The homologous recombination template single-stranded oligonucleotide ssODN provided by the invention is characterized in that the base at 746 th position of exon 8 of FecB gene is mutated from A to G (A746G) during recombination repair of the FecB gene. The ssODN has higher specificity and can target-modify the sheep FecB gene, so that the efficient and accurate mutation of the sheep FecB gene is realized, and the combination of the two can realize the purpose of improving sheep fertility and the efficient breeding of sheep with high reproductive rate.
The invention also provides a method for cultivating the high-fertility fine wool sheep, which utilizes CRISPR/Cas9 mediated homologousSource-oriented repair (HDR) to obtain FecB gene editing fine wool sheep, and propagating to obtain F 1 Editing the filigree sheep by the substitution gene; by F 0 FecB gene editing ram of BB homozygous genotype is used as a ram for mating, and F is used as the gene 1 FecB heterozygous gene editing ewe and/or BB homozygous genotype FecB gene editing ewe in the generation gene editing fine wool sheep is used as a mating ewe for artificial insemination or natural mating to obtain F 2 And editing the lanugo by the substitution gene. F obtained with the invention 2 The average reproduction rate of the generation gene editing fine wool sheep reaches 171 percent, which is equivalent to the lambing rate of the naturally mutated high-fertility FecB heterozygote, greatly shortens the breeding time and the generation interval, improves the reproduction performance of the fine wool sheep, and quickens the breeding progress of the fine wool sheep. The invention realizes the creation of genetic resources of the multi-embryo merino fine wool sheep in a short time through gene editing, and establishes a high-efficiency and rapid method for breeding the fine wool sheep and cultivating other new varieties of high-fertility sheep.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions of the prior art, the drawings that are needed in the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of CRISPR/Cas9 mediated HDR precision editing FecB genes;
FIG. 2 is a gel electrophoresis diagram of an in vitro transcription product of FecB sgRNA, lanes FecB-sgRNA, in vitro transcription product, lane M, 100bp DNAMaroer;
FIG. 3 is a gel electrophoresis diagram of an in vitro transcription product of Cas9 mRNA, with the in vitro transcription purified mRNA product of Cas9 being taken as the Cas9 mRNA and 1kb DNAMaroer being taken as the lane M;
FIG. 4 is a gel electrophoresis diagram of gene amplification of a sheep tail tissue FecB edited by a gene, lanes 1 to 19 are PCR amplification products of a target gene FecB, and lane M is 100bp DNAMmarker;
FIG. 5 is F 0 Sanger sequencing of sheep PCR product edited by FecB geneResults;
FIG. 6 is F 0 Statistics results of different editing types of sheep are compiled by the FecB gene generation; the WT sequences shown in the figures are marked red for sgRNA sequences and green for PAM sequences; blue letters are single bases where mutations occur; the letter "a" represents the insertion, the letter "red" is the inserted base;
FIG. 7 is a flow chart of a method for selectively breeding high fertility Chinese merino naps sheep (Xinjiang type) based on an edited genotype.
Detailed Description
The invention provides a CRISPR/Cas9 mediated homologous directional repair editing sheep FecB gene ssodN, wherein the nucleotide sequence of the ssodN is shown as SEQ ID NO. 2.
The source of the ssODN is not particularly limited in the present invention, and the ssODN may be artificially synthesized by delivering it to a DNA synthesis company according to the sequence information. The HDR homologous repair template ssODN of the invention can mutate the base at 746 th position of 8 th exon of FecB gene from A to G (A746G) to achieve the purpose of accurate editing. Wherein the mutation of A746G results in the amino acid at position 249 of the FecB protein being changed from glutamine to arginine. Meanwhile, homologous arms with lengths of 67bp and 52bp are respectively arranged at the left side and the right side of the mutant base sequence. The nucleotide sequence shown in the full length 120bp,SEQ ID NO.2 of the ssODN sequence is specifically as follows: aaggtagctgtgaaagtgttcttcactacagaggaggccagctggttccgagagacagaaatatatcggacggtgttgatgaggcatgaaaacatcttgggtgagtataagtctgtatta.
The invention also provides a CRISPR/Cas9 mediated reagent for homologous directional repair editing of sheep FecB genes, which comprises the following components: sgRNA, ssODN, cas9 mRNA and DNA ligase IV inhibitor SCR7; the nucleotide sequence of the sgRNA is shown as SEQ ID NO. 1; the nucleotide sequence of the ssODN is shown in SEQ ID No. 2; the nucleotide sequence of the Cas9 mRNA is shown as SEQ ID NO. 3.
In the present invention, the nucleotide sequence shown in SEQ ID NO.1 is specifically: 5' -agagacagaaatatatcaga-3.
The sgRNA of the invention is designed by taking the 729-748 nucleotides of the 8 th exon at the 5' end of the FecB gene as the sgRNA template sequence.
In the invention, the FecB gene sequence is shown as SEQ ID NO.4, and specifically comprises the following steps:
atcaaggacg ttctacactt tggttatcag cagcctgttt atctggttca aacttctgct gaatcacaag cattttccgt tgagctatga caagagagga tacaaaaagt taaacaagca agcctgtcat acgtagaagc aaacttcctt gataacatgc ttttgcgaag ttcaggaaaattaagtgtgg gcaccaagaa agaggatggt gagagtacag cccccacccc tcgtccaaag atcttgcgat gtaaatgcca ccaccattgt ccagaagact cggtcaacaa tatttgcagc acagatggat attgtttcac gatgatagaa gaagatgact ctgggatgcc tgtggtcact tctggatgtc taggactaga aggctcagat tttcagtgtc gggacactcc cattcctcat cagagaagat ccattgaatg ctgcacagaa cggaatgaat gtaataaaga tctgcacccc acacttcctc cactgaaaaa cagagatttt gttgacggac ctatacacca caaagcttta cttatatctg tgactgtgtg tagtttgctc ttggtcctca tcattttatt ctgttacttc aggtataaaa gacaagaagc cagacctcgg tacagcattg ggttagaaca ggacgaaact tacattcctc ctggagaatc cctgagagac ttaattgagc agtcgcagag ctcagggagc ggatcaggcc tccctctgct ggtccagagg acaatagcaa agcaaattca gatggtgaaa cagattggaa aaggtcgcta tggggaagtt tggatgggaa agtggcgtgg cgaaaaggtagctgtgaaag tgttcttcac tacagaggag gccagctggt tccgagagac agaaatatatcagacggtgt tgatgaggca tgaaaacatc ttgggcttca ttgctgcaga tatcaaaggg acggggtcct ggacacaact gtacctaatc acagattatc atgaaaatgg ttccctctat gattacctga agtccaccac cctagacact aagtcgatgt tgaagctagc ctattccgca gtcagtggcc tctgtcactt acacactgaa atctttagca ctcaaggcaa accagcaatt gcccatcgag atctgaaaag taagaacatc ctggtgaaga aaaatggaac ttgctgtata gctgacctgg gcttggctgt taagtttatt agtgacacga atgaagttga cataccaccc aacactcgcg ttggcaccaa gcgctacatg cctccagaag tgttggatga gagcttgaac agaaatcact ttcagtctta catcatggcc gacatgtaca gttttggact catcctttgggaggtcgcta ggagatgtgt gtcaggaggt atagtggaag aatatcagct cccctatcat gacctggtgc ccagtgaccc ctcttacgag gacatgagag agatcgtgtg tatcaagaag ctgcggccct ccttccccaa ccggtggagc agtgacgagt gtctcaggca gatggggaaa ctcatgacgg aatgctgggc tcacaatcct gcctcaagac tgacagccct acgggttaag aaaacccttg ccaaaatgtc agagtcccag gacattaagc tctgaggcaa gagtaagtgt ctctggacaa agccagtaga tatcctcctg tttgtgggca gagcaaaaga tgttccagcagcatccaccg tcccagcctc gaacatcctc ctgctcccca gagggtgtat tcttacatct cagggagcag cctggacaaa cagaagtttc cagaagcacg gattcctcat gtctgtctgtaggcgggaga aactgcctgg gtaatttgtt caagatatga tgcatgttgc tttctaagaa agcccggtat tttgggattg cctttttttt cctcaaaaga taattttgcc aaaataaaac aaaaatgtag atgtttcaag gtatatacta ttttagttta aagaatgaca actaaagttc ttcccagaaa ctctgctgga aggtaaatta aaatatgtat ttccattggt aaaatgttgt tgcactctac caaccaaaag acaatcgttg aagctggaga accgaatgga acccatctta aaagcccccg acgtgcccct gcctcctcag accactctgg ccagccctcc actggggcag ccgcagcaat gtgaacgcat ctggggaccg gcaccgcctg tgtgggacca cctctgggga ttcccaccca tgaccttctg cagcttcaga ggatgtggga caaatgaagg ttttacgaga ctctactaga agtaaatgtt aacactgttc ttcagaacca ctttgtattt ctgattctgt taggtttttt ctttcattaa acacaaacaa agcttttgtg tgtgtaggaa gttgaacccc tgcaggcttt tgatctctca aatgaaagga tcaatattaa gtaaagtggc tgtcagctgg gtcgaagaca aggcgcttta aaatagagat aatttgctct tgagctgtaa gaagatggtg tcaaaaaggt aggcggtgag gatggaggta cacgtggctt gtgtcttagg tatgcggaag agacctcctc ggccgcatga ggggaaaagt gtgcacaact tactgtgtac aaagagggtt tcttttagtt attatctgcc ttttctctgt ctgagtttag aggagaggaa acgtatcagg ttaattgaac taattttaat tttaaattag gtgactgtaa cctccaaatg gaacagcaga ggaatgctag tggagaccac aagatatggc tgtcgggact gattctacaa taggtacagatggaggatgt aagtcttagg tgatagtgtc acgtcttagg agtgctgact cagtttattt ttattttcat gacgttcagc tcacttttta atttattatt gttttcttct ctgcagcgct tgcgcagaac atctctccac ctgttcagtt atgtaggcac acacacttct gagcagcagg agtcaaatca ttgacagggc cagtattcct tagcaacagg agcataataa agggtacaat tgtacacatc cctctcaacc ttattcattt acttcctgac tgtggctttc ttatgctgct tagaattctc tagtgtgtag caaagaattg ggaagcccct cttctttgcc cttgaaaaaa aaaaaaaaaa aaaaa。
the sgRNA and ssODN of the invention have higher specificity and can target and modify sheep FecB genes, thereby realizing accurate editing.
In the present invention, cas9 mRNA is the coding region from nucleotide 7 to 4278 at the 5' end. In the present invention, the Cas9 mRNA sequence shown in SEQ ID No.3 is specifically:
gggagaaugg acuauaagga ccacgacgga gacuacaagg aucaugauau ugauuacaaa gacgaugacg auaagauggc cccaaagaag aagcggaagg ucgguaucca cggaguccca gcagccgaca agaaguacag caucggccug gacaucggca ccaacucugu gggcugggcc gugaucaccg acgaguacaa ggugcccagc aagaaauuca aggugcuggg caacaccgac cggcacagca ucaagaagaa ccugaucgga gcccugcugu ucgacagcgg cgaaacagcc gaggccaccc ggcugaagag aaccgccaga agaagauaca ccagacggaa gaaccggauc ugcuaucugc aagagaucuu cagcaacgag auggccaagg uggacgacag cuucuuccac agacuggaag aguccuuccu gguggaagag gauaagaagc acgagcggca ccccaucuuc ggcaacaucg uggacgaggu ggccuaccac gagaaguacc ccaccaucua ccaccugagaaagaaacugg uggacagcac cgacaaggcc gaccugcggc ugaucuaucu ggcccuggcc cacaugauca aguuccgggg ccacuuccug aucgagggcg accugaaccc cgacaacagc gacguggaca agcuguucau ccagcuggug cagaccuaca accagcuguu cgaggaaaac cccaucaacg ccagcggcgu ggacgccaag gccauccugu cugccagacu gagcaagagc agacggcugg aaaaucugau cgcccagcug cccggcgaga agaagaaugg ccuguucgga aaccugauug cccugagccu gggccugacc cccaacuuca agagcaacuu cgaccuggcc gaggaugcca aacugcagcu gagcaaggac accuacgacg acgaccugga caaccugcug gcccagaucg gcgaccagua cgccgaccug uuucuggccg ccaagaaccu guccgacgcc auccugcuga gcgacauccu gagagugaac accgagauca ccaaggcccc ccugagcgcc ucuaugauca agagauacga cgagcaccac caggaccuga cccugcugaa agcucucgug cggcagcagc ugccugagaa guacaaagag auuuucuucg accagagcaa gaacggcuacgccggcuaca uugacggcgg agccagccag gaagaguucu acaaguucau caagcccauc cuggaaaaga uggacggcac cgaggaacug cucgugaagc ugaacagaga ggaccugcug cggaagcagc ggaccuucga caacggcagc aucccccacc agauccaccu gggagagcug cacgccauuc ugcggcggca ggaagauuuu uacccauucc ugaaggacaa ccgggaaaag aucgagaaga uccugaccuu ccgcaucccc uacuacgugg gcccucuggc caggggaaac agcagauucg ccuggaugac cagaaagagc gaggaaacca ucacccccug gaacuucgag gaaguggugg acaagggcgc uuccgcccag agcuucaucg agcggaugac caacuucgau aagaaccugc ccaacgagaa ggugcugccc aagcacagcc ugcuguacga guacuucacc guguauaacg agcugaccaa agugaaauac gugaccgagg gaaugagaaa gcccgccuuc cugagcggcg agcagaaaaa ggccaucgug gaccugcugu ucaagaccaa ccggaaagug accgugaagc agcugaaaga ggacuacuuc aagaaaaucg agugcuucga cuccguggaa aucuccggcg uggaagaucg guucaacgcc ucccugggca cauaccacga ucugcugaaa auuaucaagg acaaggacuu ccuggacaau gaggaaaacg aggacauucu ggaagauauc gugcugaccc ugacacuguu ugaggacaga gagaugaucg aggaacggcu gaaaaccuau gcccaccugu ucgacgacaa agugaugaag cagcugaagc ggcggagaua caccggcugg ggcaggcuga gccggaagcu gaucaacggc auccgggaca agcaguccgg caagacaauccuggauuucc ugaaguccga cggcuucgcc aacagaaacu ucaugcagcu gauccacgac gacagccuga ccuuuaaaga ggacauccag aaagcccagg uguccggcca gggcgauagc cugcacgagc acauugccaa ucuggccggc agccccgcca uuaagaaggg cauccugcag acagugaagg ugguggacga gcucgugaaa gugaugggcc ggcacaagcc cgagaacauc gugaucgaaa uggccagaga gaaccagacc acccagaagg gacagaagaa cagccgcgag agaaugaagc ggaucgaaga gggcaucaaa gagcugggca gccagauccu gaaagaacac cccguggaaa acacccagcu gcagaacgag aagcuguacc uguacuaccu gcagaauggg cgggauaugu acguggacca ggaacuggac aucaaccggc uguccgacua cgauguggac cauaucgugc cucagagcuu ucugaaggac gacuccaucg acaacaaggu gcugaccaga agcgacaaga accggggcaa gagcgacaac gugcccuccg aagaggucgu gaagaagaug aagaacuacu ggcggcagcu gcugaacgcc aagcugauua cccagagaaa guucgacaau cugaccaagg ccgagagagg cggccugagc gaacuggaua aggccggcuu caucaagaga cagcuggugg aaacccggca gaucacaaag cacguggcac agauccugga cucccggaug aacacuaagu acgacgagaa ugacaagcug auccgggaag ugaaagugau cacccugaag uccaagcugg uguccgauuu ccggaaggau uuccaguuuu acaaagugcg cgagaucaac aacuaccacc acgcccacga cgccuaccug aacgccgucg ugggaaccgc ccugaucaaa aaguacccua agcuggaaag cgaguucgug uacggcgacu acaaggugua cgacgugcgg aagaugaucg ccaagagcga gcaggaaauc ggcaaggcua ccgccaagua cuucuucuac agcaacauca ugaacuuuuu caagaccgag auuacccugg ccaacggcga gauccggaag cggccucuga ucgagacaaa cggcgaaacc ggggagaucg ugugggauaa gggccgggau uuugccaccg ugcggaaagu gcugagcaug ccccaaguga auaucgugaa aaagaccgag gugcagacag gcggcuucag caaagagucu auccugccca agaggaacag cgauaagcug aucgccagaa agaaggacug ggacccuaag aaguacggcg gcuucgacag ccccaccgug gccuauucug ugcugguggu ggccaaagug gaaaagggca aguccaagaa acugaagagu gugaaagagc ugcuggggau caccaucaug gaaagaagca gcuucgagaa gaaucccauc gacuuucugg aagccaaggg cuacaaagaa gugaaaaagg accugaucau caagcugccu aaguacuccc uguucgagcu ggaaaacggc cggaagagaa ugcuggccuc ugccggcgaa cugcagaagg gaaacgaacu ggcccugccc uccaaauaug ugaacuuccu guaccuggcc agccacuaug agaagcugaa gggcuccccc gaggauaaug agcagaaaca gcuguuugug gaacagcaca agcacuaccu ggacgagauc aucgagcaga ucagcgaguu cuccaagaga gugauccugg ccgacgcuaa ucuggacaaa gugcuguccg ccuacaacaa gcaccgggau aagcccauca gagagcaggc cgagaauauc auccaccugu uuacccugac caaucuggga gccccugccg ccuucaagua cuuugacacc accaucgacc ggaagaggua caccagcacc aaagaggugc uggacgccac ccugauccac cagagcauca ccggccugua cgagacacggaucgaccugu cucagcuggg aggcgacaaa aggccggcgg ccacgaaaaa ggccggccag gcaaaaaaga aaaaguaaga auuccuagag cucgc。
the invention also provides an expression vector for cultivating the high-fertility sheep based on the HDR gene editing method, wherein the nucleotide sequence of the sgRNA is shown as SEQ ID NO.1, and a skeleton vector of the expression vector comprises pX330. The construction method of the expression vector preferably comprises the following steps: the pX330 plasmid is subjected to BbsI enzyme digestion to obtain an enzyme digestion vector, and the annealed sgRNA is connected with the enzyme digestion vector to obtain a recombinant plasmid; and (3) taking the recombinant plasmid as a template, and obtaining the sgRNA through in vitro transcription and purification, wherein the obtained sgRNA can be used for subsequent microinjection.
In the present invention, the reagent preferably further comprises nuclease-free water.
In the present invention, the concentration of Cas9 mRNA in the reagent is preferably 100ng/μl; the concentration of sgRNA (FecB-sgRNA) in the reagent is preferably 50 ng/. Mu.L; the concentration of ssODN in the agent is preferably 50 ng/. Mu.l; the concentration of the DNA ligase IV inhibitor SCR7 in the reagent is preferably 1. Mu. Mol/L.
In the CRISPR/Cas 9-mediated homology-directed repair editing system, the Cas9 nuclease formed after the translation of the Cas9 mRNA has an active domain for cutting double-stranded DNA, so that the double-stranded DNA is broken; the sgRNA targets and recruits the Cas9/sgRNA complex to a target gene through RNA-DNA base complementation pairing, and the Cas9 performs site-directed double-stranded DNA cutting through recognition of a PAM sequence on the target gene, so that an editing effect is achieved on the target gene. After Cas9 protein makes DNA undergo Double-strand breaks (DSBs), a homologous repair template ssODN is introduced into a homologous sequence-mediated Double-strand DNA repair (HDR) -based mechanism to achieve the purpose of precise editing. The addition of SCR7 can effectively improve the HDR gene editing efficiency.
The invention also provides a method for cultivating the high-fertility fine wool sheep, which comprises the following steps:
introducing sgRNA, ssODN, cas mRNA and a DNA ligase IV inhibitor SCR7 into fertilized eggs of the fine-wool sheep to obtain FecB gene editing fine-wool sheep;
FecB gene edited fine wool sheep as F 0 Instead of F 0 FecB gene editing ram of BB homozygous genotype is used as a ram for mating, F 0 The female sheep edited by the FecB gene and the wild female sheep are respectively used as mating female sheep for artificial insemination or natural mating to obtain F 1 Editing the filigree sheep by the substitution gene;
by F 0 FecB gene editing ram of BB homozygous genotype is used as a ram for mating, and F is used as the gene 1 FecB heterozygous gene editing ewe and/or BB homozygous genotype FecB gene editing ewe in the generation gene editing fine wool sheep is used as a mating ewe for artificial insemination or natural mating to obtain F 2 Editing the filigree sheep by the substitution gene; the F is 2 The generation gene editing fine wool sheep is high-fertility fine wool sheep.
According to the invention, sgRNA, ssODN, cas mRNA and a DNA ligase IV inhibitor SCR7 are firstly introduced into fertilized eggs of the fine wool sheep to obtain FecB gene editing fine wool sheep.
In the present invention, the final concentration of the sgRNA is preferably 50 ng/. Mu.L; the final concentration of the ssODN is preferably 50 ng/. Mu.l; the final concentration of Cas9 mRNA is preferably 100ng/μl; the final concentration of the DNA ligase IV inhibitor SCR7 is preferably 1. Mu. Mol/L.
In the present invention, the sgRNA, ssODN, cas mRNA and DNA ligase IV inhibitor SCR7 are preferably dissolved in nuclease-free water to obtain a mixed solution, and then introduced into fertilized eggs, and the volume of the mixed solution introduced into each fertilized egg is preferably 80 to 100pL. In the present invention, the means of introduction preferably includes microinjection; the device used for the microinjection is preferably a microinjection instrument available from NIKON corporation.
In the present invention, the fertilized egg preferably includes a single cell stage embryo.
In one embodiment of the invention, sgRNA, ssODN, cas mRNA and a DNA ligase IV inhibitor SCR7 are introduced into fertilized eggs of a donor ewe, the introduced fertilized eggs are cultured, embryos which develop to 2-4 cell phases are transplanted into oviducts of a recipient ewe with the same period of estrus, and after birth sheep are detected and identified, so that the FecB gene editing sheep with the A-G mutation at 746 of a coding region is obtained; the time of the culture is preferably 24-48 hours; the number of fertilized eggs transplanted in a single oviduct is preferably 1-2; the recipient ewe is an aletai sheep.
After FecB gene edited nap sheep is obtained, the invention takes FecB gene edited nap sheep as F 0 Instead of F 0 FecB gene editing ram of BB homozygous genotype is used as a ram for mating, F 0 The female sheep edited by the FecB gene and the wild female sheep are respectively used as mating female sheep for artificial insemination or natural mating to obtain F 1 And editing the lanugo by the substitution gene. In the present invention, BB homozygous genotype is GG genotype.
In the present invention, the F 0 FecB gene-edited ram of generation BB homozygous genotype is preferably F of the year old 0 Editing ram by using the FecB homozygous gene; the sperm motility of the ram is preferably more than or equal to 0.8.
Obtaining F 1 After the filigree sheep is edited by the substitution gene, the invention uses F 0 FecB gene editing ram of BB homozygous genotype is used as a ram for mating, and F is used as the gene 1 FecB heterozygous gene editing ewe and/or BB homozygous genotype FecB gene editing ewe in the generation gene editing fine wool sheep is used as a mating ewe for artificial insemination or natural mating to obtain F 2 Editing the filigree sheep by the substitution gene; the F is 2 The generation gene editing fine wool sheep is high-fertility fine wool sheep.
According to the invention, gene transfer is realized through propagation, the offspring carries FecB gene editing mutation, the population quantity is increased, and finally, a gene edited high-fertility fine wool sheep breeding resource group is formed, so that more offspring of BB homozygous genotypes are obtained, and the offspring has higher fertility than heterozygous ewes.
F obtained by the invention 2 The average reproduction rate of the generation gene editing fine wool sheep reaches 171 percent, which is equivalent to the lambing rate of the naturally mutated high-fertility FecB heterozygote, greatly shortens the breeding time and the generation interval, improves the reproduction performance of the fine wool sheep, and quickens the breeding progress of the fine wool sheep. The invention realizes the creation of genetic resources of the multi-embryo merino fine wool sheep in a short time through gene editing, and establishes a high-efficiency and rapid method for breeding the fine wool sheep and cultivating other new varieties of high-fertility sheep.
The invention also provides application of the ssODN or the reagent or the method in breeding high-fertility fine wool sheep and/or creating multiple-embryo fine wool sheep varieties.
In the present invention, the multiple tires are preferably two tires or three tires.
In the present invention, the fine wool sheep preferably includes a wild type FecB gene, in which the base at position 746 is A.
In the present invention, the fine wool sheep preferably includes merino fine wool sheep.
In the present invention, the merino fine wool sheep preferably includes merino fine wool sheep (Xinjiang type).
For further explanation of the present invention, a method for raising high-fertility fine wool sheep provided by the present invention will be described in detail with reference to the accompanying drawings and examples, which should not be construed as limiting the scope of the present invention.
EXAMPLE 1 construction of sgRNA expression vector and preparation of sgRNA, cas9 mRNA, ssODN
1. Designing sgRNA and ssODN for identifying sheep FecB gene target sequence
The sheep FecB gene sequence is shown as SEQ ID NO.4, and the 729-748 nucleotides in the 5' -terminal coding region of the FecB gene are selected as sgRNA template sequences (reference genome: NM_001009431, BMPRIB (FecB) mRNA sequences) to synthesize DNAOligos with complementary sequences.
By adding the HDR homologous repair template ssODN, the base at the 746 th position of the 8 th exon of the FecB gene can be mutated from A to G (A746G), so that the purpose of accurate editing is achieved (the editing position of the FecB gene and the sgRNA sequence are shown in figure 1). Wherein the mutation of A746G results in the amino acid at position 249 of the FecB protein being changed from glutamine to arginine (Q249R). Meanwhile, homologous arms with lengths of 67bp and 52bp are respectively arranged on the left side and the right side of the mutant sequence. The total length of the ssODN sequence is 120bp, and specific sequence information is shown in table 1, and long-chain DNA synthesis is performed by the division of bioengineering (Shanghai) corporation. The synthesized ssODN was dissolved in rnase-free water and the concentration was adjusted according to experimental requirements.
TABLE 1 oligonucleotide sequences for cloning sgRNA expression vectors, ssODN and making in vitro transcription templates
2. Preparation of sgRNA
(1) The px330 plasmid was cut singly with the restriction enzyme BbsI in the following manner: 10 XFastDigestBuffer 2. Mu.L, px330 plasmid 5. Mu.L (2. Mu.g), fastDigest enzyme. Mu.L, sterile deionized water was added to 20. Mu.L, digested for 2h at 37℃and the digested product was detected by 1% agarose gel electrophoresis, and then digested and purified.
(2) Dephosphorylation of the purified product of step (1) is performed as follows: pX330 was digested with 27. Mu.l of purified product, 3. Mu.l of Buffer 3, 0.5. Mu.l of 5' phosphatase was removed by CIP, and digested for 90min at 37 ℃.
(3) 1. Mu.l of each forward and reverse primer FecB-CF/CR (100. Mu.M), 2. Mu.l of 10 XBuffer A Buffer, 2. Mu.l of ATP (10 mM), 1. Mu.l of T4 Polynucleotide Kinase and 13. Mu.l of sterilized deionized water are uniformly mixed, the temperature is 30min at 37 ℃, the temperature is 5min at 95 ℃ and naturally cooled to room temperature for primer annealing, and double-stranded DNA molecules with sticky ends at both ends are obtained.
(4) And (3) connecting the product of the step (2) with the double-stranded DNA molecule obtained in the step (3) to obtain the recombinant plasmid with correct sequencing. The sgRNA PCR amplification is carried out by taking the recombinant plasmid as a template, and the system is as follows: 0.5. Mu.l (10. Mu. Mol/L) of each of the upstream and downstream primers (FecB sgRNA-TF/TR) containing the T7 promoter, 0.5. Mu.l of template DNA (50 ng plasmid), primeSTAR Max Premix. Mu.L of high-fidelity enzyme, and 50. Mu.L of sterilized deionized water; PCR reaction procedure: pre-denaturation at 95 ℃ for 5min; denaturation at 98℃for 10s, annealing at 55℃for 15s, elongation at 72℃for 5s,35 cycles; finally, the PCR amplification product is extended for 10min at 72 ℃, and is purified by a PCR product purification kit after detection by 3% agarose gel electrophoresis, and the PCR amplification product is shown as SEQ ID NO. 1.
(5) Taking the purified PCR amplification product obtained in the step (4) and utilizing an in vitro transcription kitT7 Kit, life Technologies, cat# AM 1354) and then purified and recovered by using an RNA purification Kit to obtain FecB-sgRNA. The gel electrophoresis pattern of FecB-sgRNA is shown in FIG. 2.
3. Preparation of Cas9 mRNA
(1) PCR amplification was performed using the px330 plasmid as a template and Cas9-F/R (underlined with T7 promoter) as a primer to obtain a PCR amplification product (4312 bp). The PCR amplified product is shown as SEQ ID NO.13 through sequencing, and specifically comprises the following steps:
taatacgactcactatagggagaatggactataaggaccacgacggagactacaaggatcatgatattgattacaaagacgatgacgataagatggccccaaagaagaagcggaaggtcggtatccacggagtcccagcagccgacaagaagtacagcatcggcctggacatcggcaccaactctgtggg ctgggccgtg atcaccgacg agtacaaggt gcccagcaag aaattcaaggtgctgggcaacaccgaccggcacagcatcaagaagaacctgatcggagcc ctgctgttcg acagcggcga aacagccgag gccacccggctgaagagaaccgccagaagaagatacaccagacggaagaaccggatctgctatctgcaagagatcttcagcaacgagatggccaaggtggacgacagcttcttccacagactggaagagtccttcctggtggaagaggat aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggcctaccacgagaagtaccccaccatctaccacctgagaaagaaactggtgg acagcaccga caaggccgac ctgcggctgatctatctggc cctggcccac atgatcaagt tccggggcca cttcctgatcgagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaaccccatcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga cggctggaaa atctgatcgc ccagctgcccggcgagaaga agaatggcct gttcggaaac ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgaggatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc cgacctgtttctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctctatgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta caaagagattttcttcgacc agagcaagaa cggctacgcc ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctggaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg aagcagcggaccttcgacaacggcagcatcccccaccagatccacctggg agagctgcac gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatcgagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgaccag aaagagcgaggaaaccatca ccccctggaa cttcgaggaa gtggtggaca agggcgcttc cgcccagagc ttcatcgagcggatgaccaa cttcgataagaacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg tataacgagc tgaccaaagt gaaatacgtgaccgagggaa tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc catcgtggac ctgctgttcaagaccaaccg gaaagtgaccgtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt caacgcctccctgggcacat accacgatct gctgaaaatt atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtgctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc cacctgttcg acgacaaagt gatgaagcagctgaagcggc ggagatacac cggctggggc aggctgagccggaagctgatcaacggcatccgggacaagcagtccggcaa gacaatcctggatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac agcctgacct ttaaagagga catccagaaagcccaggtgt ccggccaggg cgatagcctg cacgagcaca ttgccaatct ggccggcagc cccgccattaagaagggcat cctgcagacagtgaaggtgg tggacgagct cgtgaaagtgatgggccggcacaagcccga gaacatcgtg atcgaaatgg ccagagagaa ccagaccacccagaagggacagaagaacagccgcgagagaatgaagcggatcgaagaggg catcaaagag ctgggcagccagatcctgaa agaacaccccgtggaaaaca cccagctgcagaacgagaag ctgtacctgt actacctgca gaatgggcgg gatatgtacg tggaccagga actggacatcaaccggctgt ccgactacga tgtggaccat atcgtgcctc agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagcgacaagaacc ggggcaagagcgacaacgtgccctccgaagaggtcgtgaa gaagatgaag aactactggc ggcagctgct gaacgccaagctgattacccagagaaagtt cgacaatctg accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacagctggtggaaacccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac actaagtacg acgagaatga caagctgatccgggaagtga aagtgatcac cctgaagtcc aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaactaccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc tggaaagcga gttcgtgtacggcgactaca aggtgtacga cgtgcggaag atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagcaacatcatga actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg cctctgatcg agacaaacggcgaaaccggggagatcgtgtgggataaggg ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaagaccgaggtg cagacaggcg gcttcagcaa agagtctatc ctgcccaagaggaacagcgataagctgatc gccagaaagaaggactgggaccctaagaag tacggcggct tcgacagccc caccgtggcc tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaactgaagagtgtg aaagagctgc tggggatcac catcatggaa agaagcagcttcgagaagaa tcccatcgac tttctggaag ccaagggctacaaagaagtg aaaaaggacc tgatcatcaa gctgcctaag tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgccggcgaactg cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc cactatgaga agctgaagggctcccccgag gataatgagc agaaacagct gtttgtggaa cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctccaagagagtg atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag cccatcagag agcaggccgagaatatcatc cacctgttta ccctgaccaa tctgggagcc cctgccgcct tcaagtactt tgacaccacc atcgaccgga agaggtacaccagcaccaaa gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc gacctgtctc agctgggaggcgacaaaagg ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa agtaagaatt cctagagctc gc。
in the nucleotide sequence shown in SEQ ID No.13, 24 th to 4295 th nucleotides from the 5' end are open reading frames of Cas 9.
Cas9-F：5’-taatacgactcactatagggagaatggactataaggaccacgac-3’(SEQ ID NO.5)；
Cas9-R：5’-gcgagctctaggaattcttac-3’(SEQ ID NO.6)。
(2) Taking the PCR amplified product obtained in the step (1)Using in vitro transcription kit (mMESSAGE from Life Technologies Co.)T7Ultra Kit, cat No. AM 1345) followed by purification recovery using an RNA purification Kit to obtain Cas9 mRNA. Gel electrophoresis of Cas9 mRNA is shown in figure 3.Cas9 mRNA is shown in SEQ ID NO. 3.Cas9 mRNA is the coding region from nucleotide 7 to 4278 from the 5' end.
Example 2 production and identification of FecB Gene-edited sheep
1. Experimental sheep selection
Donor ewes: selecting Xinjiang fine wool sheep with excellent body condition, no reproductive disease and 2-4 years old; recipient ewes: the Arseltai sheep with weight of more than 50kg and age of 2-4 years, good fat and no reproductive disease are selected. Semen collection ram: selecting Xinjiang fine wool sheep with weight of 70-85 kg, fine semen detection and 1-3 years old.
2. Synchronous estrus and superovulation
The CIDR method is used to induce sheep to have synchronous estrus. The specific operation is as follows: the CIDR suppository (vaginal progesterone suppository) is put into the vagina of a donor ewe, and follicle stimulating hormone is injected in a decreasing manner every 12h for 3 consecutive days from the 10 th day of the putting, wherein the total injection dose is 240 units/unit; CIDR plug was removed in the morning of day 12, donor ewe vagina was purged and prostaglandin was injected intramuscularly at 0.1 mg/dose. After 12h of thrombus removal, each test is carried out in the morning and evening, and 200IU luteinizing hormone/female sheep of an oestrus donor is injected.
The recipient ewe and the donor ewe are simultaneously embedded with CIDR, the CIDR is removed 12 hours before the thrombus of the donor ewe is removed, 330IU of pregnant mare serum gonadotropin is injected each time, and the estrus of 3 goats is tested in the morning and evening every day after the thrombus is removed for 12 hours, so that the estrus time of the goats is recorded in detail.
3. Artificial insemination
Semen of ram is collected and microscopic examination is carried out, and sperm motility is more than 0.8, so that the ram can be used for insemination. And artificially inseminating the donor ewes with estrus time of 12-19 h by adopting a laparoscope deep insemination method.
4. Acquisition of single cell stage embryos
The operation method washes out the prokaryotic embryo from the oviduct 19-21 hours after insemination of the donor ewe. And selecting single-cell-stage embryo with uniform cytoplasm, regular morphology, integrity and compactness and no cleavage.
5. Fertilized egg microinjection and in vitro culture
Cas9 mRNA, fecB-sgRNA, ssODN and a DNA ligase IV inhibitor SCR7 prepared in the above example 1 are mixed with water without nuclease according to the final concentration of 100 ng/. Mu.L, 50 ng/. Mu.L and 1 mu.mol/L respectively, the mixed solution is injected into cytoplasm of single-cell-stage embryo obtained in the step 4 by using microinjection instrument of NIKON company (injection dosage: 80-100 pL mixed solution is injected into each embryo), fertilized eggs after injection are washed and then transferred into four-hole culture plates filled with culture solution according to the density of 50-60 pieces/hole, and are placed into a carbon dioxide incubator for culturing for 48 hours to count cleavage rate, and the non-injection group replaces the mixed solution with equal volume of nuclease-free water.
6. Embryo transfer and pregnancy detection
Embryo developed to 2-4 cell stage is transplanted into oviduct of recipient ewe treated by synchronous estrus, 1-2 embryo is transplanted in single oviduct, and B ultrasonic diagnosis of gestation condition of recipient ewe is carried out 60 days after embryo transplantation (Table 2). The results show that: the experiment shows that 68 injected embryos are transplanted, 57 recipients are transplanted, 18 recipients are pregnant, and the pregnancy rate is 31.58%, and the result is shown in Table 2.
TABLE 2FecB Gene edited sheep production
7. Identification of FecB Gene-edited sheep
(1) Design of specific PCR amplification primers
Specific primers FecB-F and FecB-R are designed for the sheep FecB gene sequence (accession number: NM_ 001009431) and for the sheep FecB gene exon 8 sequence by using Oligo 7.0 software for amplifying a coding region of FecB gene 335bp in length, and the relevant information of the primers is shown in Table 1.
(2) Lamb ear tissue DNA extraction
The lamb tail tissue samples of the test group and the control group are collected, the genome DNA is extracted by a genome DNA extraction kit (Beijing Tiangen, product number DP 304), and the experimental operation is strictly carried out according to the specification. The extracted DNA is detected by agarose gel electrophoresis with the concentration of 0.8%, the concentration of the DNA is measured by a Nanodrop ultra-micro spectrophotometer, and the DNA which is qualified by detection is diluted to 100 ng/. Mu.L for PCR amplification of the template.
(3) PCR amplification and sequencing identification
The PCR amplification system is as follows: 2 XPCRASTERMASTERM mix 25. Mu.L, 100ng of template DNA, 1. Mu.L of FecB-F/R primers (concentration 10. Mu. Mol/L) each, and sterilized ultrapure water was made up to 50. Mu.L. The PCR amplification procedure was: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s, annealing at 58℃for 30s, elongation at 72℃for 30s,35 cycles; finally, the PCR products were subjected to agarose gel electrophoresis at 72℃for 7min, and the result is shown in FIG. 4, wherein the target band amplification length was 335bp, and the single PCR product which meets the target band length was sent to Sanger sequencing by Bio-company. Sequencing results were analyzed by sequence alignment using Chromas and Clustalx software to identify genotypes. Samples with consecutive peaks and disordered sequence in Sanger sequencing were cloned into pMD-19T vector, respectively, and 10 monoclonal sequences were randomly picked for further determination of edit type.
Sanger sequencing results (see FIGS. 5, 6) indicate that: at birth 19F 0 In the lambs, 9 individuals (4 females and 5 males) had replaced, deleted and inserted the FecB gene A746G site with an editing efficiency of 47.4% (see Table 2). Wherein the sequencing peak diagram of the PCR product of 5 individuals (2 individuals with 3 parents) only shows a G single peak at the target site, double allele A/G substitution occurs, and 5 individuals are homozygous genotypes (BB) (FIG. 5, # 1-5); sequencing peaks of PCR products of 1 individual (male) showed only A/G set peaks at the target site, which were heterozygous genotypes (B+) (FIGS. 5, # 6); in addition to the appearance of a cap peak at the target site, a continuous cap peak and disordered sequence at the sgRNA sequence of the PCR product sequencing peak map of 3 individuals (2 females and 1 males) need to be subjected to TA cloning to further determine the editing type. The TA clone sequencing result shows that the three areThe body has different types of edit modifications (fig. 5, #7, #8, #9-1 and # 9-2). FIG. 6 shows the results of insertion of single base G at positions 747-748 with the addition of an effective target mutation in which the A/G exact substitution occurred in individuals # 7; inserting 4bp base (TATC) at 745-746 position while the# 8 individual is subjected to A/G accurate replacement; the #9 individual has two editing types, namely, insertion of a single base G at 747-748 with accurate A/G substitution (same #7 individual), single base T/A substitution at 744, and deletion of a single base C at 745. CRISPR/Cas9 induced precise replacement efficiency is as high as 31.6% (6/19), demonstrating that Cas9 mediated HDR repair is effective in sheep production.
Example 3FecB Gene-edited sheep breeding and offspring lambing behavior
Because sheep individuals carrying FecB genes have higher fertility, the individuals carrying FecB genes, particularly the individuals containing homozygous mutation, can be selected and remained according to genotypes in the cultivation of offspring fine wool sheep, so that the aim of rapidly improving the fertility of the fine wool sheep population is fulfilled. A flow chart of a method for selectively breeding high-fertility Chinese merino fine wool sheep (Xinjiang type) based on the edited genotype is shown in fig. 7. The method comprises the following steps:
1. hybrid mating
1. Selection of FecB Gene-edited ram and Gene-edited ewe and wild Fine wool ewe
Selecting Sanger sequencing result as the year F of BB homozygous genotype 0 The ram is adopted as a breeding for the FecB gene editing ram, the weight of the ram reaches more than 55kg, the ram reaches more than 60cm, the single ejaculation amount is more than 2.0mL, and the semen detection is excellent (the sperm motility is more than 0.8). The ram is fed with carrot eggs every day before the breeding, and the ram moves for 1h in the morning and evening.
Respectively selecting 48 wild Chinese merino fine wool sheep (Xinjiang) and 4F with weight of more than 45kg, good fat condition and no reproductive disease 0 The FecB gene editing ewe is used as a breeding ewe.
2. Synchronous estrus and deep insemination
The vaginal sponge suppository is put into the vagina of the ewe and recorded as 0 day, the vaginal sponge suppository is taken out on the 12 th day, the vagina of the ewe is cleaned, and pregnant mare blood is injected intramuscularlyClear 330IU. After the thrombus is removed for 48 hours, the deep insemination of the laparoscope is carried out, 25 mug of 3 # is promoted by intramuscular injection, and the insemination is carried out again at intervals of 6-8 hours. Obtained after the birth of pregnant ewe 1 Replacing individuals.
3. Natural mating
B ultrasonic examination of pregnancy is carried out about 45 days after the artificial insemination and the mating, and the female sheep which is not pregnant is picked out and F 0 The FecB homozygous gene is edited into a ram mixed circle for 35 days, so that the ram mixed circle can be freely mated. And producing the pregnant ewe to obtain the newborn lamb.
4. Genotyping and lambing phenotype recording
34 of the 48 wild ewes bred were lambed 37 (20 female 17 male), and 15-20 day old newborn lambs were genotype tested by sanger sequencing according to the method of example 1, which showed that 4 were wild type (++), 33 were heterozygous (b+), 19 female lambs and 14 male lambs; 4F of mating 0 The generation of gene editing female sheep is that 3 female sheep are lambed, and the female lambs are respectively 2 female sheep, namely 2 homozygous (BB) lambs and 35 heterozygous (B+) lambs are obtained by genotype detection and 2 heterozygous (BB) and 2 heterozygous (B+) lambs, so that F which is mainly FecB heterozygotes is established 1 And editing the ewe population by the generation genes.
TABLE 3F 1 Sheep genotype results edited by FecB gene generation
2. Backcross
Will F 0 FecB gene editing ram (TG 343) with BB homozygous gene and 18F with good growth condition and weight greater than 45kg 1 Natural mating of the female sheep edited by the FecB heterozygote (B+) gene, and 24F are produced by 14 female sheep 2 And (3) the lambs are edited by the gene, wherein 1 female sheep produces three lambs and 8 female sheep produces two lambs, the average breeding rate reaches 171.43% (table 4), and the lamb production rate is equivalent to that of a naturally mutated high-fertility FecB heterozygote. Through genotype detection, 12F are only (50%, 12/24) 2 The lamb substitute is FecB homozygote (BB), 12F (50%, 12/24) 2 The lamb is FecB heterozygote (B+), and the offspring genotype accords with Mendelian geneticAnd the rule is transmitted, so that the edited genotype can be inherited stably.
TABLE 4F 1 Lambing and genotype results of filigree sheep edited by FecB gene
The research results show that the genetic resource of the high-fertility FecB gene fine wool sheep is created through gene editing, so that the breeding difficulty of wool quality reduction caused by introducing multiple genes through hybridization in the past is overcome, the genetic resource of the multi-lamb fine wool sheep can be obtained in a short time, the significance on fine wool sheep breeding is great, and a high-efficiency and rapid novel method is established for breeding other novel varieties of high-fertility sheep in the future.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (7)
1. The CRISPR/Cas9 mediated homologous directional repair editing sheep FecB gene ssODN is characterized in that the nucleotide sequence of the ssODN is shown as SEQ ID NO. 2.
2. A CRISPR/Cas9 mediated agent for homologous directed repair editing of sheep FecB gene, characterized by comprising the following components: sgRNA, ssODN, cas9 mRNA and DNA ligase IV inhibitor SCR7; the nucleotide sequence of the sgRNA is shown as SEQ ID NO. 1; the nucleotide sequence of the ssODN is shown in SEQ ID No. 2; the nucleotide sequence of the Cas9 mRNA is shown as SEQ ID NO. 3.
3. A method for breeding high fertility fine wool sheep, comprising the steps of:
introducing sgRNA, ssODN, cas mRNA and a DNA ligase IV inhibitor SCR7 into fertilized eggs of the fine-wool sheep to obtain FecB gene editing fine-wool sheep;
FecB gene edited fine wool sheep as F 0 Instead of F 0 FecB gene editing ram of BB homozygous genotype is used as a ram for mating, F 0 The female sheep edited by the FecB gene and the wild female sheep are respectively used as mating female sheep for artificial insemination or natural mating to obtain F 1 Editing the filigree sheep by the substitution gene;
by F 0 FecB gene editing ram of BB homozygous genotype is used as a ram for mating, and F is used as the gene 1 FecB heterozygous gene editing ewe and/or BB homozygous genotype FecB gene editing ewe in the generation gene editing fine wool sheep is used as a mating ewe for artificial insemination or natural mating to obtain F 2 Editing the filigree sheep by the substitution gene; the F is 2 The generation gene editing fine wool sheep is high-fertility fine wool sheep.
4. Use of the ssODN of claim 1 or the agent of claim 2 or the method of claim 3 for breeding high fertility fine wool sheep and/or creating multiple-born fine wool sheep breeds.
5. The use according to claim 4, wherein the nap sheep comprises a wild type nap sheep having base a at position 746 of exon 8 of the FecB gene.
6. The use according to claim 5, wherein the nap sheep comprises merino nap sheep.
7. The use according to claim 6, wherein said merino nap sheep comprises merino nap sheep (Xinjiang type).
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