CN105925579B - The sgRNA and its coding DNA of a pair of of specific recognition pig IGF2 gene introns and application - Google Patents
The sgRNA and its coding DNA of a pair of of specific recognition pig IGF2 gene introns and application Download PDFInfo
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Abstract
The invention discloses the sgRNA and its coding DNA of a pair of of specific recognition pig IGF2 gene introns and applications.The present invention provides complete sgRNA, is made of sgRNA L 1 and sgRNA R 2；Identify that the nucleotides sequence of the segment of target sequence is classified as sequence 1 in the sgRNA L 1；Identify that the nucleotides sequence of the segment of target sequence is classified as sequence 2 in the sgRNA R 2.The present invention can be used for knock-out pig IGF2 genes and open ZBED6 factor binding regions in introne 3, can promote the development of pig muscle cell, increase the Lean mass of pig.
Description
Technical field
The invention belongs to gene engineering technology field, the sgRNA of specific a pair of specific recognition pig IGF2 gene introns
And its coding DNA and application.
Background technology
The speed of growth and the thickness of backfat of pig are one of important breeding objectives of current pig breeding industry, have important economic valency
Value.But the speed of growth of pig and the thickness of backfat be by the complex character of the controlled by multiple genes of different levels, be multiple genes and its
The exercising result for the regulated and control network that product is formed.Have now been found that MSTN, IGF2, MC4R, JHDM1A, TEF-1, RYR1,
Multiple genes such as COPB1 all have significant impact, and said gene to economic characters such as the thickness of backfat of pig and the speeds of growth
In all contain the QTN sites that are had a major impact to two kinds of characters of the thickness of backfat and the speed of growth.So many gene, if passed through
Traditional breeding technology and method carry out breeding selection and breeding, it would be desirable to the decades even upper century-old selection and breeding time.
IGF2 (Insulin-like growth factors-2) gene, i.e. insulin-like growth factor 2, also known as makes a living
It is long to adjust plain (Somatomedin A).In pig, IGF2 genes (accession number AYO44828) positioned at pig No. 2 the short arm of a chromosome,
About 23.8kb, 5 ' ends are INS (insulin) genes, and 3 ' ends are h19 genes.The gene has 4 promoters, 10 extrons, and preceding 7
A extron (Exon1 to Exon 6 adds Exon 4b) is the leading extron (Leader exon) for not encoding albumen, outer aobvious
Sub- 7-9 encodes pre-pro IGF2, totally 182 amino acid.Exons 1, each before 4,5,6 there are one promoters, can transcribe altogether
Go out 7 kinds of transcripts.The different spatial and temporal expressions of four different promoters cause the tissue of IGF2 genes and spatial and temporal expression that height is presented
Complexity.In addition, the IGF2 intron sequences of pig are more conservative, especially introne 2,3,4,5 is more homologous with the sequence of people
Property is respectively 68%, 66%, 74% and 73%.The qtl analysis of pig shows the 3072nd bit base of IGF2 gene introns 3 by G (birds
Purine) 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 heart
The characters such as weight have the influence of highly significant.It is included the main reason is that transcription factor ZBED6 can be attached to IGF2 genes
The GCTCG sequences not being methylated near son 3072 of 3, so as to lower the expression quantity of individual IGF2 genes after birth.
When the mutation that G to A single bases occur, transcription factor ZBED6 not can be incorporated into the position, so as to improve IGF2 bases after pig birth
About 3 times of the expression quantity of cause, makes the Lean mass of pig increase by 15~30%, and the thickness of backfat reduces 10~20%.
CRISPR/Cas9 technologies be one kind that bacterium and archeobacteria are formed in very long evolutionary process resist bacteriophage with
And the acquired immune system of foreign nucleic acid molecules.Currently used CRISPR/Cas9 systems are by Cas9 albumen and a bit of list
Chain RNA (sgRNA) is formed, and single stranded RNA is responsible for identifying target DNA sequence dna, the target site that Cas9 albumen can then be identified in single stranded RNA
On double-stranded DNA is cut, so as to foreign nucleic acid molecules of degrading.Mali etc. prove CRISPR/Cas9 systems can 293T, K562,
Accurate targeting cutting, and the far super ZFN and TALEN technologies of target practice efficiency are carried out in a variety of mammalian cells such as iPS, is to carry out
Gene functional research, cultivates the effective of animals and plants new varieties, making bioreactor and disease model at animal-plant gene group editor
Tool.
Invention content
A purpose of the invention is to provide complete sgRNA.
Complete sgRNA provided by the invention, is made of sgRNA-L-1 and sgRNA-R-2；
The nucleotides sequence for being responsible for identification target segment in the sgRNA-L-1 is classified as sequence 1；
The nucleotides sequence for being responsible for identification target segment in the sgRNA-R-2 is classified as sequence 2.
In above-mentioned complete sgRNA, the nucleotides sequence of the sgRNA-L-1 is classified as sequence 5；
The nucleotides sequence of the sgRNA-R-2 is classified as sequence 6.
Another object of the present invention is to provide complete DNA molecular.
Complete DNA molecular provided by the invention, by encoding the DNA molecular of above-mentioned sgRNA-L-1 and encoding above-mentioned sgRNA-
The DNA molecular composition of R-2.
The target segment of above-mentioned sgRNA-L-1 identifications, is the nucleotide in sequence table shown in sequence 3, is in pig IGF2 genes
Containing the ZBED6 binding sites upstream 128bpDNA molecules on son 3.
The target segment of above-mentioned sgRNA-R-2 identifications, is the nucleotide in sequence table shown in sequence 4, is in pig IGF2 genes
Containing the ZBED6 binding sites downstream 98bpDNA molecules on son 3.
Above-mentioned complete sgRNA or above-mentioned complete DNA molecular are transcribed in pig genome on IGF2 gene introns 3
The application that factor Z BED6 recognition sites are carried out in gene editing is also the scope of protection of the invention.
Above-mentioned complete sgRNA or above-mentioned complete DNA molecular are in preparing to pig genome on IGF2 gene introns 3
The application that transcription factor ZBED6 recognition sites carry out in gene editing product is also the model that the present invention protects.
Third purpose of the present invention is to provide in a kind of genome to pig transcription factor ZBED6 on IGF2 gene introns 3
The method that recognition site carries out gene editing.
Method provided by the invention, includes the following steps：Above-mentioned complete sgRNA and Cas9 protein mRNAs are imported into pig
In, realize in pig genome on IGF2 gene introns 3 transcription factor ZBED6 recognition sites gene editing.
In above-mentioned, the nucleotides sequence of transcription factor ZBED6 recognition sites is classified as sequence 8 on the IGF2 gene introns 3
306-310.
The experiment proves that the present invention provides the ZBED6 on a pair of of specific recognition pig IGF2 gene introns 3
The sgRNA of binding site, high specificity.When carrying out sequence knockouts, miss the target caused by CRISPR/Cas9 systems can be effectively reduced
Phenomenon reduces the adverse effect that Non-specific cleavage brings genome.It is provided by the invention this to sgRNA ,/Cas9 can be passed through
System modifies the introne of pig gene in cell or individual level, can be used not only for cultivating high lean meat percentage new varieties
Pig, and the research of pig IGF2 gene functions can be used for.
Description of the drawings
Fig. 1 knocks out ZBED6 combined areas schematic diagram on IGF2 gene introns 3 jointly for sgRNA-L-1 and sgRNA-R-2.
Fig. 2 detects sgRNA-L-1 and sgRNA-R-2 for agarose gel electrophoresis and knocks out efficiency.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Complete sgRNA, is made of sgRNA-L-1 and sgRNA-R-2 in following embodiments.
The nucleotides sequence of sgRNA-L-1 is classified as sequence 5, and including sgRNA-L, the nucleotides sequence of sgRNA-L is classified as sequence
1, target sequence is sequence 3, and sequence 3 is the ZBED6 knots on pig IGF2 gene introns 3 (introne 3 partial sequence is sequence 8)
Close site (sequence 8 306-310) upstream 128bpDNA molecules；
The nucleotides sequence of sgRNA-R-2 is classified as sequence 6, and including sgRNA-R, the nucleotides sequence of sgRNA-R is classified as sequence
2, target sequence is sequence 4, and sequence 4 is the ZBED6 binding sites downstream 98bpDNA molecules on pig IGF2 gene introns 3.
Fig. 1 knocks out ZBED6 combined areas schematic diagram on IGF2 gene introns 3 jointly for sgRNA-L-1 and sgRNA-R-2.
Embodiment 1, complete sgRNA and its application
First, Cas9 System-mediateds delete Pig embryos in IGF2 gene introns 3 in ZBED6 binding sites it is complete
The preparation of sgRNA
1st, in-vitro transcription vector construction
IGF2 gene introns 3 in Pig embryos are used as target practice sequence design oligonucleotide (Oligo) DNA sequences
Row send reliable business primer Synesis Company to be synthesized (every synthesis 1OD, way of purification selection PAGE).Particular sequence
It 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, formed with cohesive end double chain DNA fragment IGF2-sgL2 with
And IGF2-sgR2.
Digestion is carried out to pT7-sgRNA (addgene, Plasimd 65565) carrier with restriction enzyme BsmB I, is returned
Receive the segment after digestion.The pT7-sgRNA that IGF2-sgL2 and IGF2-sgR2 segments are connected respectively to after digestion recycling is carried
In body, in-vitro transcription carrier pT7-IGF2-sgL and pT7-IGF2-sgR are obtained.
By sequencing, pT7-IGF2-sgL carriers are by the insertion pT7- of the encoding gene of sgRNA-L shown in sequence 1
The carrier obtained between the BsmB I restriction enzyme sites of sgRNA carriers, expresses to obtain sgRNA-L-1 with segment composition on carrier；
PT7-IGF2-sgR carriers are the BsmB that the encoding gene of sgRNA-R shown in sequence 2 is inserted into pT7-sgRNA carriers
The carrier obtained between I restriction enzyme sites expresses to obtain sgRNA-R-2 with segment composition on carrier.
2nd, in-vitro transcription sgRNA
The in-vitro transcription carrier pT7-IGF2-sgL and pT7-IGF2-sgR, XmnI restriction enzyme in step 1 are taken respectively
Single endonuclease digestion is linearized enzyme (NewEngland Biolabs, R0194V), and carries out glue recycling.Fetch the in-vitro transcription after receiving
Carrier 12 μ L carry out the body of sgRNA according to the requirement of in-vitro transcription kit (Ambion, Maxiscript T7Kit) specification
It is outer transcription (without attach the names of pre-determined candidates and tailing), obtain the sgRNA-L-1 shown in the sequence 5 and sgRNA-R-2 shown in sequence 6.
The mRNA of artificial synthesized Cas9, nucleotides sequence are classified as sequence 7.
2nd, complete sgRNA deletes the application in the IGF2 gene introns in Pig embryos under Cas9 System-mediateds
1st, Pig embryos microinjection
Take pig internal fertilization or it is in vitro fertilization after Vitro Embryo, carry out cytoplasm microinjection, above-mentioned one is prepared
The mRNA mixing of sgRNA-R-1, sgRNA-L-2 and Cas9, obtain mixed system, and each substance is final concentration of in system
The mRNA of 12.5ng/ μ LsgRNA-R-1,12.5ng/ μ LsgRNA-L-2 and 125ng/ μ LCas9；By mixed system according to every
A embryo's injection volume is about that 10pL is injected into Pig embryos, obtains the Pig embryos containing sgRNA.
The microinjection of 32 Pig embryos is carried out altogether.
Concrete operations are injected referring to document：Lillico,S.G.,et al.,Live pigs produced from
genome edited zygotes.Scientific Reports,2013.3:p.2847.
2nd, PCR is detected
The single Pig embryos containing sgRNA obtained in step 1 are taken, are put into unicellular lysate (Beijing day bounties, article No.
It blows and beats in 130803-1) and is cracked several times repeatedly, 10000 revs/min of centrifugations take lysate supernatant, drawn with following two Dui
Object carries out nest-type PRC reaction, expands 3 subregion of IGF2 gene introns in 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 into amplified production into row agarose gel electrophoresis.
The results are shown in Figure 2, for the embryo that ZBED6 binding sites are deleted in IGF2 gene introns 3, amplification do not occur
Fragment length is 485bp；For the embryo that ZBED6 binding sites are deleted in IGF2 gene introns 3, expanding fragment length occurs
About 225bp.
SgRNA is counted in the protein mediated lower ZBED6 binding site editorial efficiencies in IGF2 gene introns 3 of Cas9,
It is the ratio of embryo/whole injection embryo deleted.
As a result sgRNA Cas9 it is protein mediated it is lower to IGF2 gene introns 3 in the embryo deleted of ZBED6 binding sites
Tire number is 6, editorial efficiency 18.75%.
The preparation of embodiment 2, complete sgRNA
Complete sgRNA can also be prepared by the following method：
According to sgRNA target practice sequence design oligonucleotide (Oligo) DNA sequence dna, reliable business primer is sent to synthesize
Company is synthesized (every synthesis 1OD, way of purification selection 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, formed with cohesive end double chain DNA fragment IGF2-sgL and
IGF2-sgR。
Above-mentioned double chain DNA fragment IGF2-sgL and IGF2-sgR with cohesive end are connected into respectively and use restriction enzyme
(addgene, Plasimd 42330, the carrier contain Cas9 protein coding genes to pX330 after enzyme BbsI digestions recycling, can table
Up to Cas9 albumen) in carrier, obtain pX330-IGF2-sgL carriers and pX330-IGF2-sgR carriers.
By sequencing, pX330-IGF2-sgL carriers are by the insertion pX330 of the encoding gene of sgRNA-L shown in sequence 1
The carrier obtained between the BbsI restriction enzyme sites of carrier, expresses to obtain sgRNA-L-1 with segment composition on carrier；
PX330-IGF2-sgR carriers are the BbsI enzymes that the encoding gene of sgRNA-R shown in sequence 2 is inserted into pX330 carriers
The carrier obtained between enzyme site expresses to obtain sgRNA-R-2 with segment composition on carrier.
PX330-IGF2-sgL carriers and pX330-IGF2-sgR carriers are transferred to porcine fetus fibroblasts jointly, obtained
Express the transgenic cell of sgRNA and Cas9 nucleases.
Claims (11)
1. complete sgRNA, is made of sgRNA-L-1 and sgRNA-R-2；
The nucleotides sequence for being responsible for identification target segment in the sgRNA-L-1 is classified as sequence 1；
The nucleotides sequence for being responsible for identification target segment in the sgRNA-R-2 is classified as sequence 2.
2. complete sgRNA according to claim 1, it is characterised in that：
The nucleotides sequence of the sgRNA-L-1 is classified as sequence 5；
The nucleotides sequence of the sgRNA-R-2 is classified as sequence 6.
3. institute in complete DNA molecular, the DNA molecular of sgRNA-L-1 described in coding claim 1 and coding claim 1
State the DNA molecular composition of sgRNA-R-2.
4. the target segment of the sgRNA-L-1 identifications in any one of claims 1 or 2, is the core in sequence table shown in sequence 3
Thuja acid.
5. the target segment of the sgRNA-R-2 identifications in any one of claims 1 or 2, is the core in sequence table shown in sequence 4
Thuja acid.
6. the complete DNA molecular described in complete sgRNA or claim 3 described in claims 1 or 2 is in pig genome
Transcription factor ZBED6 recognition sites carry out the application in gene editing on IGF2 gene introns 3.
7. application according to claim 6, it is characterised in that：Transcription factor ZBED6 knows on the IGF2 gene introns 3
The nucleotides sequence in other site is classified as sequence 8 306-310.
8. the complete DNA molecular described in complete sgRNA or claim 3 described in claims 1 or 2 is being prepared to pig genome
Transcription factor ZBED6 recognition sites carry out the application in gene editing product on middle IGF2 gene introns 3.
9. application according to claim 8, it is characterised in that：Transcription factor ZBED6 knows on the IGF2 gene introns 3
The nucleotides sequence in other site is classified as sequence 8 306-310.
10. transcription factor ZBED6 recognition sites carry out the side of gene editing on IGF2 gene introns 3 in a kind of genome to pig
Method includes the following steps：Complete sgRNA and Cas9 protein mRNAs described in claims 1 or 2 are imported in pig, are realized to pig
In genome on IGF2 gene introns 3 transcription factor ZBED6 recognition sites gene editing.
11. according to the method described in claim 10, it is characterized in that：Transcription factor ZBED6 on the IGF2 gene introns 3
The nucleotides sequence of recognition site is classified as sequence 8 306-310.
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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 |
| CN109576284B (en) * | 2018-12-21 | 2021-09-17 | 中国农业科学院北京畜牧兽医研究所 | Multifunctional 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 |
| CN113061609B (en) * | 2021-03-24 | 2022-12-09 | 中国农业科学院北京畜牧兽医研究所 | sgRNA for specifically recognizing porcine IGF2R site 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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| 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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