CN105925579A - sgRNA (Subgnomic Ribonucleic Acid) for specific recognition of porcine IGF2 (Lnsulin-like growth factors-2) gene intron and encoding DNA (Deoxyribose Nucleic Acid) and application of sgRNA for specific recognition of porcine IGF2 gene intron - Google Patents
sgRNA (Subgnomic Ribonucleic Acid) for specific recognition of porcine IGF2 (Lnsulin-like growth factors-2) gene intron and encoding DNA (Deoxyribose Nucleic Acid) and application of sgRNA for specific recognition of porcine IGF2 gene intron Download PDFInfo
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Abstract
The invention discloses an sgRNA (Subgnomic Ribonucleic Acid) for specific recognition of a porcine IGF2 (Lnsulin-like growth factors-2) gene intron and an encoding DNA (Deoxyribose Nucleic Acid) and application of sgRNA for the specific recognition of the porcine IGF2 gene intron. The invention provides a complete set of sgRNA which comprises an sgRNA-L-1 and an sgRNA-R-2; in the sgRNA-L-1, the nucleotide sequence for identifying fragments of a target sequence is used as the sequence I; in the sgRNA-R-2, the nucleotide sequence for identifying fragments of a target sequence is used as the sequence II. The sgRNA for the specific recognition of the porcine IGF2 gene intron disclosed by the invention can be used for knocking out a ZBED6 factor binding region in the IGF2 gene intron, so that the development of pig muscle cells can be promoted and the muscle content of a pig can be increased.
Description
Technical field
The invention belongs to gene engineering technology field, the sgRNA of concrete a pair specific recognition pig IGF2 gene intron
And coding DNA and application.
Background technology
The speed of growth of pig and the thickness of backfat are one of important breeding objectives of current pig industry, have important economic valency
Value.But the speed of growth of pig and the thickness of backfat are by the complex character of the controlled by multiple genes of different levels, it is multiple genes
And the exercising result of the regulated and control network of product formation.Have now been found that MSTN, IGF2, MC4R, JHDM1A,
The economic characters such as the thickness of backfat of pig and the speed of growth are all had significantly by multiple gene such as TEF-1, RYR1, COPB1
Impact, and said gene all contains the QTN position having a major impact the thickness of backfat and two kinds of character of the speed of growth
Point.So many gene, if carrying out breeding selection-breeding by traditional breeding technology and method, it would be desirable to decades are even
The selection-breeding time of upper a century.
IGF2 (Insulin-like growth factors-2) gene, i.e. IMA-IGF2BP3-001, be also called
Somatomedin (Somatomedin A).In pig, IGF2 gene (accession number AYO44828) is positioned at No. 2 of pig
The short arm of a chromosome, about 23.8kb, 5 ' ends are INS (insulin) genes, and 3 ' ends are h19 genes.This gene has 4
Individual promoter, 10 exons, front 7 exons (Exon1 to Exon 6 is plus Exon 4b) are not encode egg
White leading exon (Leader exon), exon 7-9 encodes pre-pro IGF2, totally 182 aminoacid.
Exons 1, is respectively arranged with a promoter before 4,5,6, can transcribe out 7 kinds of transcripts altogether.Four different promoters
Different spatial and temporal expressions make the tissue of IGF2 gene and spatial and temporal expression present high complexity.It addition, in the IGF2 of pig
The most conservative containing subsequence, especially intron 2,3,4,5 are respectively 68% with the gene comparision homology of people,
66%, 74% and 73%.The qtl analysis of pig shows that IGF2 gene intron 3 the 3072nd bit base is by G (guanine)
To A (adenine) single base mutation to the speed of growth of pig, lean meat percentage, the thickness of backfat, area of longissimus muscle and
The character such as cardiac weight have the impact of highly significant.Its main cause is that transcription factor ZBED6 can be attached to IGF2
3072 the most methylated neighbouring GCTCG sequences of gene intron 3, thus IGF2 individual after lowering birth
The expression of gene.When there is the sudden change of G to A list base, transcription factor ZBED6 not can be incorporated into this position,
Thus the expression about 3 times of IGF2 gene after improving pig birth, make the Lean mass of pig increase by 15～30%, the thickness of backfat
Reduce 10～20%.
The one that CRISPR/Cas9 technology is antibacterial and archeobacteria is formed in very long evolutionary process resist phage with
And the acquired immune system of foreign nucleic acid molecules.The most conventional CRISPR/Cas9 system is little by Cas9 albumen and one
Section single stranded RNA (sgRNA) forms, and single stranded RNA is responsible for identifying target DNA sequence, and Cas9 albumen then can be at strand
On the target site that RNA identifies, double-stranded DNA is cut, thus foreign nucleic acid molecules of degrading.Mali etc. prove
CRISPR/Cas9 system can carry out accurate targeting in the multiple mammalian cell such as 293T, K562, iPS and cut
Cut, and target practice efficiency far surpass ZFN and TALEN technology, be by gene functional research, animal-plant gene group editor,
Cultivate animals and plants new varieties, make bioreactor and the effective tool of disease model.
Summary of the invention
One purpose of the present invention is to provide complete sgRNA.
The complete sgRNA that the present invention provides, is made up of sgRNA-L-1 and sgRNA-R-2；
Described sgRNA-L-1 is responsible for identify that the nucleotides sequence of target fragment is classified as sequence 1；
Described sgRNA-R-2 is responsible for identify that the nucleotides sequence of target fragment is classified as sequence 2.
In above-mentioned complete sgRNA, the nucleotides sequence of described sgRNA-L-1 is classified as sequence 5；
The nucleotides sequence of described sgRNA-R-2 is classified as sequence 6.
Another object of the present invention is to provide complete DNA molecular.
The complete DNA molecular that the present invention provides, above-mentioned by the DNA molecular and coding encoding above-mentioned sgRNA-L-1
The DNA molecular composition of sgRNA-R-2.
The target fragment that above-mentioned sgRNA-L-1 identifies, for the nucleotide shown in sequence in sequence table 3, for pig IGF2 gene
ZBED6 binding site upstream 128bpDNA molecule on introne 3.
The target fragment that above-mentioned sgRNA-R-2 identifies, for the nucleotide shown in sequence in sequence table 4, for pig IGF2 gene
ZBED6 binding site downstream 98bpDNA molecule on introne 3.
The complete DNA molecular of above-mentioned complete sgRNA or above-mentioned turns on IGF2 gene intron 3 in pig genome
It is also the scope of protection of the invention that record factor Z BED6 recognition site carries out the application in gene editing.
The complete DNA molecular of above-mentioned complete sgRNA or above-mentioned is IGF2 gene intron 3 in preparation is to pig genome
It is also the model that the present invention protects that upper transcription factor ZBED6 recognition site carries out the application in gene editing product.
The 3rd purpose of the present invention is to provide a kind of to transcription factor ZBED6 on IGF2 gene intron 3 in pig genome
Recognition site carries out the method for gene editing.
The method that the present invention provides, comprises the steps: to import above-mentioned complete sgRNA and Cas9 protein mRNA
In pig, it is achieved to the gene editing of transcription factor ZBED6 recognition site on IGF2 gene intron 3 in pig genome.
In above-mentioned, on described IGF2 gene intron 3, the nucleotides sequence of transcription factor ZBED6 recognition site is classified as sequence
8 306-310 positions.
The experiment proves that, the invention provides on a pair specific recognition pig IGF2 gene intron 3
The sgRNA of ZBED6 binding site, high specificity.When carrying out sequence knockouts, can effectively reduce CRISPR/Cas9
Phenomenon that what system caused miss the target, reduces the adverse effect that genome is brought by Non-specific cleavage.What the present invention provided should
To sgRNA, the intron of pig gene can be modified at cell or individual level by/Cas9 system, can not only
For cultivating high lean meat percentage new product boar, and the research of pig IGF2 gene function can be used for.
Accompanying drawing explanation
Fig. 1 is that sgRNA-L-1 and sgRNA-R-2 jointly knocks out ZBED6 land on IGF2 gene intron 3 and shows
It is intended to.
Fig. 2 is that agarose gel electrophoresis detection sgRNA-L-1 Yu sgRNA-R-2 knocks out efficiency.
Detailed description of the invention
Experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
In following embodiment, complete sgRNA, is made up of sgRNA-L-1 and sgRNA-R-2.
The nucleotides sequence of sgRNA-L-1 is classified as sequence 5, and it includes that the nucleotides sequence of sgRNA-L, sgRNA-L is classified as sequence
Row 1, its target sequence is sequence 3, and sequence 3 is pig IGF2 gene intron 3 (introne 3 partial sequence is sequence 8)
On ZBED6 binding site (sequence 8 306-310 position) upstream 128bpDNA molecule；
The nucleotides sequence of sgRNA-R-2 is classified as sequence 6, and it includes that the nucleotides sequence of sgRNA-R, sgRNA-R is classified as sequence
Row 2, its target sequence is sequence 4, and sequence 4 is the ZBED6 binding site downstream 98 on pig IGF2 gene intron 3
BpDNA molecule.
Fig. 1 is that sgRNA-L-1 and sgRNA-R-2 jointly knocks out ZBED6 land on IGF2 gene intron 3 and shows
It is intended to.
Embodiment 1, complete sgRNA and application thereof
One, in the IGF2 gene intron 3 during Cas9 System-mediated deletes Pig embryos, ZBED6 binding site is complete
The preparation of sgRNA
1, in vitro transcription vector construction
According to the IGF2 gene intron 3 in Pig embryos as target practice sequential design oligonucleotide (Oligo) DNA
Sequence, send reliable business primer Synesis Company to carry out synthesizing (every synthesis 1OD, way of purification selects PAGE).
Particular sequence is as follows:
(1) IGF2-sgL2:
IGF2-sgL-up2:TAGGACTGGTTTCGCCCTCCTCCG
IGF2-sgL-dn:AAACCGGAGGAGGGCGAAACCAGT
(2) IGF2-sgR2:
IGF2-sgR-up2:TAGGAGCAGCGCCCCGACGCGCCC
IGF2-sgR-dn:AAACGGGCGCGTCGGGGCGCTGCT
Above two couples of oligo DNA are annealed respectively, forms the double chain DNA fragment IGF2-sgL2 with sticky end
And IGF2-sgR2.
With restricted enzyme BsmB I, pT7-sgRNA (addgene, Plasimd 65565) carrier is carried out enzyme action,
Reclaim the fragment after enzyme action.After IGF2-sgL2 and IGF2-sgR2 fragment is connected respectively to enzyme action recovery
In pT7-sgRNA carrier, obtain in vitro transcription carrier pT7-IGF2-sgL and pT7-IGF2-sgR.
Through order-checking, pT7-IGF2-sgL carrier is the insertion of the encoding gene by sgRNA-L shown in sequence 1
The carrier obtained between the BsmB I restriction enzyme site of pT7-sgRNA carrier, expresses with segment composition on carrier and obtains
sgRNA-L-1；
PT7-IGF2-sgR carrier is that the encoding gene of sgRNA-R shown in sequence 2 is inserted pT7-sgRNA carrier
The carrier obtained between BsmB I restriction enzyme site, expresses with segment composition on carrier and obtains sgRNA-R-2.
2, in vitro transcription sgRNA
Take in vitro transcription carrier pT7-IGF2-sgL and pT7-IGF2-sgR, XmnI in step 1 respectively restricted interior
Cut enzyme (NewEngland Biolabs, R0194V) single endonuclease digestion and carry out linearisation, and carry out glue recovery.After fetching receipts
In vitro transcription carrier 12 μ L, according in vitro transcription test kit (Ambion, Maxiscript T7Kit) explanation
The requirement of book carries out the in vitro transcription (without attaching the names of pre-determined candidates and tailing) of sgRNA, obtains the sgRNA-L-1 shown in sequence 5
With the sgRNA-R-2 shown in sequence 6.
The mRNA of synthetic Cas9, nucleotides sequence is classified as sequence 7.
Two, complete sgRNA deletes the application in the IGF2 gene intron in Pig embryos under Cas9 System-mediated
1, Pig embryos microinjection
Take the Vitro Embryo after pig internal fertilization or external fertilization, carry out kytoplasm microinjection, by an above-mentioned preparation
SgRNA-R-1, sgRNA-L-2 and Cas9 mRNA mixing, obtain mixed system, and each material be in system
Final concentration of 12.5ng/ μ LsgRNA-R-1,12.5ng/ μ LsgRNA-L-2 and 125ng/ μ LCas9's
mRNA；Mixed system, is obtained containing sgRNA during about 10pL is expelled to Pig embryos according to each embryo's injection volume
Pig embryos.
Carry out the microinjection of 32 Pig embryos altogether.
Injection concrete operations see document: Lillico, S.G., et al., Live pigs produced from
genome edited zygotes.Scientific Reports,2013.3:p.2847.
2, PCR detection
Take the single Pig embryos containing sgRNA obtained in step 1, put into unicellular lysate (sky, Beijing bounties,
Article No. 130803-1) in repeatedly blow and beat and crack several times, 10000 revs/min are centrifuged, take lysate supernatant, in order to
Under two pairs of primers carry out nest-type PRC reaction, IGF2 gene intron 3 subregion in amplification embryo.
Nest-type PRC primer 1 (amplification length 818bp):
IGF2-F:ACGAAAGGAGACGCTTGACC
IGF2-R:CTGGAAGTTAGTGCCCGAAA
Nest-type PRC primer 2 (amplification length 485bp):
IGF2-MSD-F:GAAAACGAAAGGAGACGCTTGACC
IGF2-MSD-R:CACGCTTCTCCTGCCACTGAGAG
Nest-type PRC second is taken turns amplified production and carries out agarose gel electrophoresis.
Result as in figure 2 it is shown, for the embryo not occurring ZBED6 binding site in IGF2 gene intron 3 to delete,
Expanding fragment length is 485bp；For there is the embryo that in IGF2 gene intron 3, ZBED6 binding site is deleted,
Expanding fragment length is about 225bp.
Statistics sgRNA is protein mediated lower to the ZBED6 binding site editor effect in IGF2 gene intron 3 at Cas9
Rate, it is the ratio of embryo/whole injection embryos that deletion occurs.
Result sgRNA Cas9 protein mediated lower to IGF2 gene intron 3 in ZBED6 binding site delete
Embryo's number be 6, editorial efficiency is 18.75%.
Embodiment 2, the preparation of complete sgRNA
Can also prepare complete sgRNA by the following method:
According to sgRNA target practice sequential design oligonucleotide (Oligo) DNA sequence, reliable business primer is sent to close
Cheng company carries out synthesizing (every synthesis 1OD, way of purification selects PAGE).Particular sequence is as follows:
(1) IGF2-sgL:
IGF2-sgL-up:CACCACTGGTTTCGCCCTCCTCCG
IGF2-sgL-dn:AAACCGGAGGAGGGCGAAACCAGT
(2) IGF2-sgR:
IGF2-sgR-up:CACCAGCAGCGCCCCGACGCGCCC
IGF2-sgR-dn:AAACGGGCGCGTCGGGGCGCTGCT
Above two couples of Oligo DNA are annealed respectively, forms the double chain DNA fragment IGF2-sgL with sticky end
And IGF2-sgR.
Above-mentioned double chain DNA fragment IGF2-sgL and IGF2-sgR with sticky end is connected into respectively with restricted interior
(addgene, Plasimd 42330, this carrier contains Cas9 albumen to be compiled to cut the pX330 after enzyme BbsI enzyme action reclaims
Code gene, can express Cas9 albumen) in carrier, obtain pX330-IGF2-sgL carrier and carry with pX330-IGF2-sgR
Body.
Through order-checking, pX330-IGF2-sgL carrier is the insertion of the encoding gene by sgRNA-L shown in sequence 1
The carrier obtained between the BbsI restriction enzyme site of pX330 carrier, expresses with segment composition on carrier and obtains sgRNA-L-1；
PX330-IGF2-sgR carrier is the BbsI that the encoding gene of sgRNA-R shown in sequence 2 inserts pX330 carrier
The carrier obtained between restriction enzyme site, expresses with segment composition on carrier and obtains sgRNA-R-2.
PX330-IGF2-sgL carrier and pX330-IGF2-sgR carrier are proceeded to porcine fetus fibroblasts jointly,
To the transgenic cell expressing sgRNA and Cas9 nuclease.
Claims (9)
- The most complete sgRNA, is made up of sgRNA-L-1 and sgRNA-R-2；Described sgRNA-L-1 is responsible for identify that the nucleotides sequence of target fragment is classified as sequence 1；Described sgRNA-R-2 is responsible for identify that the nucleotides sequence of target fragment is classified as sequence 2.
- Complete sgRNA the most according to claim 1, it is characterised in that:The nucleotides sequence of described sgRNA-L-1 is classified as sequence 5；The nucleotides sequence of described sgRNA-R-2 is classified as sequence 6.
- The most complete DNA molecular, DNA molecular and coding right by sgRNA-L-1 described in coding claim 1 are wanted The DNA molecular seeking sgRNA-R-2 described in 1 forms.
- 4. the target fragment that the described sgRNA-L-1 during claim 1 or 2 is arbitrary identifies, for sequence 3 in sequence table Shown nucleotide.
- 5. the target fragment that the described sgRNA-R-2 during claim 1 or 2 is arbitrary identifies, for sequence 4 in sequence table Shown nucleotide.
- 6. the complete sgRNA described in claim 1 or 2 or the complete DNA molecular described in claim 3 are to pig base Because of the application during transcription factor ZBED6 recognition site carries out gene editing on IGF2 gene intron 3 in group.
- 7. the complete sgRNA described in claim 1 or 2 or the complete DNA molecular described in claim 3 prepare right Answering during transcription factor ZBED6 recognition site carries out gene editing product on IGF2 gene intron 3 in pig genome With.
- 8. one kind carries out gene volume to transcription factor ZBED6 recognition site on IGF2 gene intron 3 in pig genome The method collected, comprises the steps: to lead complete sgRNA and the Cas9 protein mRNA described in claim 1 or 2 Enter in pig, it is achieved to the gene editing of transcription factor ZBED6 recognition site on IGF2 gene intron 3 in pig genome.
- 9. according to the application described in claim 6 or 7 or the method described in claim 8, it is characterised in that: described On IGF2 gene intron 3, the nucleotides sequence of transcription factor ZBED6 recognition site is classified as sequence 8 306-310 position.
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| CN109082439A (en) * | 2018-07-04 | 2018-12-25 | 中山大学 | A method of pig meat yield is improved using CRISPR/Cas9 |
| CN109576284A (en) * | 2018-12-21 | 2019-04-05 | 中国农业科学院北京畜牧兽医研究所 | One multi-functional myb transcription factor gene and application thereof |
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| CN112041445A (en) * | 2018-03-28 | 2020-12-04 | 中国科学院动物研究所 | Method for producing pigs with improved properties |
| CN113061609A (en) * | 2021-03-24 | 2021-07-02 | 中国农业科学院北京畜牧兽医研究所 | sgRNA for specifically recognizing porcine IGF2R locus, and coding DNA and application thereof |
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| CN105593367A (en) * | 2015-06-11 | 2016-05-18 | 深圳市第二人民医院 | CRISPR-Cas9 specificity pig SLA-1 gene knockout method and sgRNA used for specific targeting SLA-1 gene |
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| CN105593367A (en) * | 2015-06-11 | 2016-05-18 | 深圳市第二人民医院 | CRISPR-Cas9 specificity pig SLA-1 gene knockout method and sgRNA used for specific targeting SLA-1 gene |
| CN105112446A (en) * | 2015-06-25 | 2015-12-02 | 中国医学科学院基础医学研究所 | Method for high-efficiency establishment of genetically modified animal model through haploid stem cells |
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| CN112041445A (en) * | 2018-03-28 | 2020-12-04 | 中国科学院动物研究所 | Method for producing pigs with improved properties |
| CN109082439A (en) * | 2018-07-04 | 2018-12-25 | 中山大学 | A method of pig meat yield is improved using CRISPR/Cas9 |
| CN109576284A (en) * | 2018-12-21 | 2019-04-05 | 中国农业科学院北京畜牧兽医研究所 | One multi-functional myb transcription factor gene and application thereof |
| CN109913460A (en) * | 2019-03-28 | 2019-06-21 | 南京北恒生物科技有限公司 | A kind of double sites sgRNA knock out the CRISPR/Cas9 system and application of INS gene |
| CN113061609A (en) * | 2021-03-24 | 2021-07-02 | 中国农业科学院北京畜牧兽医研究所 | sgRNA for specifically recognizing porcine IGF2R locus, and coding DNA and application thereof |
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